InteractiveFly: GeneBrief

Dynactin 1, p150 subunit: Biological Overview | References

BIOLOGICAL OVERVIEW

Parkinsonian Perry syndrome, involving mutations in the dynein motor component dynactin or p150^Glued, is characterized by TDP-43 pathology in affected brain regions, including the substantia nigra. However, the molecular relationship between p150^Glued and TDP-43 is largely unknown. This study reports that a reduction in TDP-43 protein levels alleviates the synaptic defects of neurons expressing the Perry mutant p150^G50R in Drosophila. Dopaminergic expression of p150^G50R, which decreases dopamine release, disrupts motor ability and reduces the lifespan of Drosophila. p150^G50R expression also causes aggregation of dense core vesicles (DCVs), which contain monoamines and neuropeptides, and disrupts the axonal flow of DCVs, thus decreasing synaptic strength. The above phenotypes associated with Perry syndrome are improved by the removal of a copy of Drosophila TDP-43, TBPH, thus suggesting that the stagnation of axonal transport by dynactin mutations promotes TDP-43 aggregation and interferes with the dynamics of DCVs and synaptic activities (Hosaka, 2017)

Perry syndrome (PS) is an autosomal dominant disorder characterized by parkinsonism with depression, sleep disturbance, weight loss, and central hypoventilation. Genome-wide linkage analysis has identified disease-segregating missense mutations located in the dynactin (DCTN1) gene. The gene product of dynactin, p150^Glued, forms a complex with dynein, the microtubule-dependent retrograde motor. Disease-associated missense mutations (G71R, G71E, G71A, T72P, Q74P) are located in the cytoskeleton-associated protein Gly-rich (CAP-Gly) domain of p150^Glued, which has been implicated in binding to microtubules recruiting dynein (Farrer, 2009, Ayloo, 2014, Tacik, 2014) and stabilizing the plus-end of microtubules (Lazarus, 2013). A glycine to serine substitution at residue 59 (G59S), which causes distal hereditary motor neuropathy 7B (HMN7B), appears to affect the structure of the CAP-Gly domain and produces severe synaptic phenotypes, including p150^Glued aggregation, dynein accumulation at nerve terminals and disruption of axonal transport. Mutations associated with PS show milder synaptic phenotypes but cause impaired retrograde flux (Hosaka, 2017)

The TAR DNA-binding protein of 43 kDa (TDP-43) is a highly conserved heterogeneous ribonucleoprotein (hnRNP) involved in the transcription, splicing, stabilization and transport of specific mRNAs. TDP-43 has been identified as the key component of intracellular ubiquitin-positive inclusions observed in affected brain areas of patients with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and Alzheimer's disease. A pathological feature of PS is the accumulation of TDP-43 in affected areas. Increased TDP-43 is toxic to neurons, possibly because of its proneness to aggregation and conversion to an abnormal protein structure similar to that of prion and α-Synuclein. On the other hand, TDP-43 is indispensable to mouse development and Drosophila survival , thus suggesting that the control of appropriate protein levels is critical for TDP-43 function (Hosaka, 2017)

The molecular relationship between p150^Glued and TDP-43 is largely unknown. To determine whether TDP-43 contributes to PS phenotypes, TDP-43 protein levels wetr manipulated in a Drosophila PS model, and reduced TDP-43 was found to improve defects in axonal transport and the synaptic activity of central dopaminergic neurons, as well as motor neurons, caused by the neuron-specific expression of a PS-associated p150^Glued mutation (Hosaka, 2017)

Whereas mutations of p150^Glued cause PS, in which TDP-43 pathology has been reported in the basal ganglia, including the substantia nigra, whether TDP-43 contributes to neurodegeneration in PS remained unknown. This study analyzed the molecular relationship between p150^Glued and TDP-43 in a Drosophila PS model (Hosaka, 2017)

Neuronal expression of the p150^G50R mutation, located in the CAP-Gly domain, led to swelling of distal boutons and the prominent aggregation of DCVs but not mitochondria in axons and synapses. DCVs travel between proximal axons and terminal boutons via axonal transport, which efficiently delivers neurotransmitters to synaptic boutons. Although p150^G50R disrupts both the anterograde and retrograde flow of DCVs, the anterograde/retrograde ratios of moving DCVs suggested that the retrograde flow is especially affected by p150^G50R, thus leading to accumulation of organelles and materials at nerve terminals. Consistently with the morphological abnormalities in synaptic phenotypes, synaptic strength, dopamine release and motor ability were decreased by p150^G50R expression without detectable neuron loss. Although p150^WT expression also affected the retrograde flow of DCVs and the DCV distribution at the terminal boutons, the phenotypes were milder than those of p150^G50R. Given that p150^WT protein was expressed at higher levels than p150^G50R in transgenic flies, in which both p150^WT and p150^G50R transgenes were inserted in the same genomic locus and both transcript levels were similar, p150^G50R exerts highly deleterious effects on neuronal activity despite its unstable expression. Thus, the data obtained with p150^G50R expression would reflect synaptic dysfunction as an early neurodegeneration event in PS (Hosaka, 2017)

Altered mitochondrial distribution at the boutons by p150^G50R was not significantly rescued by TBPH reduction, thus suggesting that the improvement of DCV phenotypes markedly contributes to the rescue effects in neuronal functions and survival. However, it cannot be excluded that mitochondrial functions may be affected in p150^G50R flies, because the fluorescence intensity of mitoGFP (cytochrome C oxidase subunit VIII-GFP fusion protein), which inserts in the mitochondrial inner membrane in a membrane potential-dependent manner, was somewhat reduced by p150^G50R. Ultrastructural analysis of synaptic mitochondria also suggested that mitochondrial cristae were partly damaged in p150^G50R flies. Because mitochondrial shuttling between cell bodies and nerve terminals in neurons would be important to maintain mitochondrial proteins derived from the nuclear genome, which include the respiratory complex I, III, IV and V subunits, the effects of PS mutations on mitochondrial functions at nerve terminals should be examined in future studies (Hosaka, 2017)

TDP-43 accumulation in affected regions is a prominent feature of PS pathology. TDP-43 forms cytoplasmic messenger ribonucleoprotein (mRNP) granules, which also move via axonal transport to synaptic boutons to deliver mRNA for synaptic activities. The discovery in Drosophila that the ablation of a copy of the TBPH gene improves the axonal aggregation of DCVs and synaptic defects provides three possible molecular mechanisms: First, a reduced concentration of TBPH in axons and nerve terminals improves axonal flow, suppressing the aggregation of TBPH. Second, TBPH reduction alters the expression of proteins regulated by TBPH at transcript levels, thus alleviating synaptic dysfunctions. Third, the above two mechanisms contribute to rescue effects. The third mechanism is preferred for the reasons listed below (Hosaka, 2017)

The DCV aggregates in axons and synapses were suppressed by TBPH reduction without improving the velocity of axonal transport, thus suggesting that aggregation-prone TBPH promotes DCV aggregation when the axonal flow stagnates. However, the numbers of DCVs and SVs were increased in the synaptic boutons of TBPH^+/− flies, thus suggesting that TBPH negatively regulates DCV and SV production. A variety of TBPH target mRNAs have been reported in Drosophila, and further studies may reveal a target(s) to regulate DCVs and SVs. Regarding synaptic stabilization, microtubule-associated MAP1B/Futsch is an evolutionarily conserved target of TDP-43/TBPH, which negatively or positively regulates synaptic MAP1B/Futsch expression and might maintain axonal transport through regulating microtubule dynamics. Although no obvious changes were detected in the levels of Futsch protein in TBPH^+/− flies, the downregulation of Futsch/MAP1B may explain the observation that some phenotypes in TBPH^+/− flies were reversed by p150^G50R expression. Because p150^Glued stabilizes the plus ends of microtubules, the ectopic expression of p150^G50R may partially alleviate the microtubule destabilization caused by Futsch/MAP1B downregulation. Alternatively, the decreased axonal flow resulting from p150^G50R expression may enable efficient synaptic capture of TDP-43-mRNP granules, which regulate local protein translation for synaptic activity including Futsch/MAP1B (Hosaka, 2017).

Autoregulation of TDP-43 mRNA has been demonstrated in mammals and has been suggested in Drosophila. Consistently with these reports, the decrease in TBPH protein was at most 30% in TBPH^+/− flies. Although the genetic ablation of a copy of the TBPH gene in itself produced some synaptic phenotypes and decreased the lifespan in flies, the transient knockdown of TDP-43 would be a suitable strategy for therapeutic intervention in PS. It has been demonstrated that the transient inhibition of a truncated N-terminal huntingtin with an abnormal polyQ stretch improves the neuropathology and the motor phenotype in a Huntington's disease mouse model. Thus, disease phenotypes caused by TDP-43 proteinopathies may be reversible under appropriate levels of TDP-43 control (Hosaka, 2017).

Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In the Drosophila female germline, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar messenger RNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. This study shows through in vitro reconstitution that Tm1-I/C, a tropomyosin-1 isoform, links kinesin-1 in a strongly inhibited state to DDBE-associated oskar mRNA. Nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing competition with dynein until kinesin-1 is activated in the oocyte. Thus work reveals a new strategy for ensuring sequential activity of microtubule motors (Heber, 2024).

oskar (osk) mRNA localization in the Drosophila egg chamber is an attractive system for studying dual motor transport. Delivery of osk to the posterior pole of the developing oocyte, which drives abdominal patterning and germline formation in the embryo, is driven by the successive activities of dynein and kinesin-1. In early oogenesis, osk mRNA that is synthesized in the nurse cells is transported into the interconnected oocyte by dynein in complex with dynactin and the activating adaptor Bicaudal D (BicD), which is linked to double-stranded mRNA localization signals by the RNA-binding protein Egalitarian (Egl). Association of Egl with BicD and consequent dynein activation are enhanced by binding of Egl to RNA, indicating a role for the cargo in promoting dynein activity. In early oogenesis, microtubule minus ends are nucleated in the oocyte, consistent with the dynein-based delivery of mRNAs into this cell. During mid-oogenesis, the polarity of the microtubule network shifts dramatically, with plus ends pointing towards the oocyte posterior. At this stage, Khc translocates osk to the posterior pole. This process is independent of Klc, raising the question of how Khc is linked to osk and how its motor activity is regulated (Heber, 2024).

Transport of osk RNA by Khc requires the unique I/C isoform of tropomyosin-1, Tm1-I/C (hereafter Tm1). Tm1 binds to a noncanonical but conserved cargo-binding region in the Khc tail and stabilizes interaction of the motor with RNA, suggesting a function as an adaptor. Both Khc and Tm1 are loaded onto osk ribonucleoprotein particles (RNPs) shortly after their export from the nurse cell nuclei, although the motor only appears to become active in the mid-oogenesis oocyte. Similarly, dynein remains associated with osk RNPs during Khc-mediated transport within the oocyte, but is inactivated by displacement of Egl by Staufen. How the two motors are linked simultaneously to osk RNPs, and how Khc is inhibited during dynein-mediated transport into the oocyte, is not known (Heber, 2024).

This study shows that Tm1 inhibits Khc by stabilizing its autoinhibited conformation through a new mechanism involving the motor’s regulatory tail domain and stalk. Tm1 also links Khc to the dynein-transported osk RNP, thereby allowing cotransport of inactive Khc on osk RNA by dynein. In vivo, such a mechanism would avoid competition between the two motors during delivery of osk RNPs to the oocyte by dynein, while ensuring that Khc is available on these structures to mediate their delivery to the oocyte posterior in mid-oogenesis. With its cargo-binding and motor regulatory functions, it is proposed that Tm1 is a noncanonical light chain for kinesin-1 (Heber, 2024).

Tm1 was recently implicated as an RNA adaptor for kinesin-1. The current study reveals a previously unknown role of Tm1 in osk transport. Tm1 is shown to negatively regulates Khc activity, which is proposed to occurs a conformational change in the Khc stalk that stabilizes the Khc motor-tail interaction and thereby enhances autoinhibition (Heber, 2024).

With its functions in cargo binding and motor regulation, it is speculated that Tm1 is an alternative Klc in the Drosophila female germline. Because precise osk RNA localization during oogenesis is critical for development, positive regulators of Khc function such as PAT1 and negative regulators such as Tm1 may have replaced Klc to provide nuanced control of Khc activity. If this hypothesis is correct, Klc should also be dispensable for Tm1-dependent RNA localization in somatic tissues (Heber, 2024).

Tm1 also stimulates association of Khc in a strongly inhibited state with dynein-associated osk RNPs. This mechanism would allow dynein-mediated transport of osk RNPs from the nurse cells to the oocyte to proceed without competition with Khc, while positioning the plus-end directed motor on the RNPs for their posteriorward transport within the ooplasm in mid-oogenesis (Heber, 2024).

Kinesin-1 autoinhibition is not fully understood, in part because of a paucity of structural information for the full-length molecule. It has been proposed that interaction of the tail’s IAK motif with the motor domain plays a key role in kinesin-1 autoinhibition. Consistent with this notion, strongly enhanced motility was obserced of Drosophila Khc when the IAK motif was deleted. However, it was recently shown that Klc inhibits Khc activity independently of the IAK-motor interaction. It was also found that the IAK motif is not needed for inhibition of Khc by Tm1. Instead, it was found that Tm1-mediated inhibition occurs via the Khc AMB domain, a region adjacent to the IAK motif that is essential for osk localization (Heber, 2024).

Structural analyses suggest that Tm1 stabilizes the autoinhibited folded conformation of Khc by inducing rearrangement of the Khc coiled-coil stalk. Therefore, a model is proposed in which both the motor-IAK interaction and interactions within the Tm1-bound stalk that include the AMB domain act synergistically to achieve the stable inhibited conformation of Khc (Heber, 2024).

Two recent studies have proposed compact structural arrangements for mammalian kinesin-1. Those studies used chemical crosslinking and cryo-EM, which are likely to enrich for a homogeneous population of compact conformations of Khc and Khc–Klc tetramers, to provide detailed static structural information. By contrast, in the current study of Drosophila Khc and Khc–Tm1 complexes, NMR and SAXS were employed providing insight into the different conformational states, and thus flexibility, of kinesin-1 by enabling analysis of structures in solution. A previous study observed the compact, inhibited conformation in the isolated Khc, showing that Klc-binding does not induce a new fold of Khc but rather stabilizes the inhibited conformation. This is in agreement with the current model, in which Tm1, a putative alternative Klc, shifts the structural equilibrium of Khc towards the autoinhibited state by stabilizing its compact conformation (Heber, 2024).

Although it is known that many cargo types are transported by the concerted action of dynein and kinesins, the underlying regulatory mechanisms have been elusive. This study has identified one of very few examples of a factor that not only links dynein and kinesin-mediated transport, but also modulates transport through differential motor regulation. Linkage of dynein and kinesin-3 by the dynein-activating adaptor Hook3 has been demonstrated, but the cellular events for which this is relevant are still emerging. Recently, reconstituted coupling of dynein and kinesin-1 by TRAK1 and TRAK2 has provided insight into how the motors are recruited and regulated for mitochondrial transport. However, unlike this study's integration of DDBE and Khc in reconstituted osk RNPs, these systems lacked intact native cargoes and thereby excluded potential cargo-directed positioning and modulation of motor complexes (Heber, 2024).

Several studies have reported that active dynein and kinesin motors engage in a tug-of-war when artificially coupled. This study also observe motor opposition in reconstituted RNPs containing DDBE and Khc, presumably because of the stochastic engagement of autoinhibited Khc with microtubules. However, the observation that Tm1 supports efficient dynein-mediated RNA transport through robust inhibition of Khc highlights the importance of regulatory factors in addition to mechanical coupling in native transport complexes. Supporting the in vivo relevance of negative regulation of Khc during bidirectional transport, kinesin-1-activating IAK mutations were recently shown to impair dynein-mediated transport processes in Aspergillus nidulans Collectively, these observations point to complex interplay between opposite-polarity motors that are bound simultaneously to cargoes. Further reconstitutions of dual motor systems on native cargoes should reveal generalities of dynein-kinesin crosstalk, as well as any cargo-specific regulatory mechanisms (Heber, 2024).

This study provides mechanistic insight into two critical aspects of osk mRNA transport—assembly of the dual motor complex and how Khc activity is suppressed during dynein-mediated delivery of the transcript from the nurse cells to the oocyte. However, it is not understood how Khc takes over from dynein after osk RNPs arrive in the oocyte. Although recent work has shown that inactivation of dynein by the RNA-binding protein Stau is part of this process, how Tm1- and IAK-mediated inhibition of Khc is alleviated to allow delivery of the mRNA to the oocyte posterior is an open question. One candidate to fulfill this role is Ensconsin, which is required for posterior osk localization and is enriched in the oocyte relative to the nurse cells. Strikingly, the human counterpart of Ensconsin (MAP7) was recently shown to stimulate activity of mammalian kinesin-1 in vitro. Other candidate Khc activators include the exon junction complex, which, together with the SOLE RNA structure, is essential for transport of osk to the oocyte posterior. Because Tm1 needs to remain bound to the osk RNP throughout its posterior translocation, it is likely that the activating factor(s) induces a conformational change in the Khc–Tm1 complex rather than dissociation of Tm1. Future investigations of these regulatory mechanisms are likely to elucidate how kinesin-1 activity is orchestrated in other systems (Heber, 2024).

Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles

Synaptic neuropeptide and neurotrophin stores are maintained by constitutive bidirectional capture of dense-core vesicles (DCVs) as they circulate in and out of the nerve terminal. Activity increases DCV capture to rapidly replenish synaptic neuropeptide stores following release. However, it is not known whether this is due to enhanced bidirectional capture. Experiments at the Drosophila neuromuscular junction, where DCVs contain neuropeptides and a bone morphogenic protein, show that activity-dependent replenishment of synaptic neuropeptides following release is evident after inhibiting the retrograde transport with the dynactin disruptor mycalolide B or photobleaching DCVs entering a synaptic bouton by retrograde transport. In contrast, photobleaching anterograde transport vesicles entering a bouton inhibits neuropeptide replenishment after activity. Furthermore, tracking of individual DCVs moving through boutons shows that activity selectively increases capture of DCVs undergoing anterograde transport. Finally, upregulating fragile X mental retardation 1 protein (Fmr1, also called FMRP) acts independently of futsch/MAP-1B to abolish activity-dependent, but not constitutive, capture. Fmr1 also reduces presynaptic neuropeptide stores without affecting activity-independent delivery and evoked release. Therefore, presynaptic motoneuron neuropeptide storage is increased by a vesicle capture mechanism that is distinguished from constitutive bidirectional capture by activity dependence, anterograde selectivity, and Fmr1 sensitivity. These results show that activity recruits a separate mechanism than used at rest to stimulate additional synaptic capture of DCVs for future release of neuropeptides and neurotrophins (Cavolo, 2016).

Synapses are supplied by anterograde axonal transport from the soma, the site of synthesis of synaptic vesicle proteins and dense-core vesicles (DCVs) that contain neuropeptides and neurotrophins. Delivery to synapses was thought to be based on a one-way anterograde trip until it was discovered that DCVs are subject to sporadic capture while traveling bidirectionally through en passant boutons as part of long-distance vesicle circulation (Wong, 2012). Interestingly, constitutive DCV capture occurs both during fast anterograde and retrograde transport, which are mediated by different motors (i.e., the kinesin 3 family member unc-104/Kif1A and the dynein/dynactin complex, respectively). Balanced capture in both directions is advantageous because DCVs are distributed equally among en passant boutons (Wong, 2012). In principle, bidirectional capture could occur by parallel regulation of anterograde and retrograde motors or by modification of the microtubules that both anterograde and retrograde DCV motors travel on (Cavolo, 2016).

Before the discovery of bidirectional capture of circulating vesicles, activity was shown to replenish the presynaptic neuropeptide pool following release by inducing Ca2+-dependent capture of DCVs being transported through boutons. This result and subsequent experiments (Bulgari, 2014) established that capture, rather than delivery or DCV turnover, limits synaptic neuropeptide stores. Activity-dependent capture was first described with a GFP-tagged neuropeptide in the Drosophila neuromuscular junction (NMJ), but also occurs with neurotrypsin, wnt/wingless, and brain-derived neurotrophic factor. Mechanistically, activity-dependent capture was characterized in terms of the rebound in presynaptic GFP-tagged peptide content following release and correlated with decreased retrograde transport. However, it is now evident that the reduction in retrograde flux could be caused by enhanced bidirectional capture as DCVs travel back and forth through the terminal as part of vesicle circulation (Wong, 2012). Therefore, prior studies support the hypothesis that there is only one synaptic capture mechanism, which is bidirectional and facilitated by activity (Cavolo, 2016).

This study tested the above hypothesis by investigating the directionality of activity-dependent capture. Experiments were performed with multiple approaches, including inhibiting retrograde transport, particle tracking, and simultaneous photobleaching and imaging (SPAIM; Wong, 2012). Furthermore, the effect of fragile X retardation protein (Fmr1, also called FMRP) was examined because it is known to affect bouton size and neuropeptide release. Together, these studies establish that different mechanisms mediate synaptic capture at rest and in response to activity (Cavolo, 2016).

Until recently, it was thought that presynaptic neuropeptide stores were set by controlling synthesis and delivery by fast one-way axonal transport of DCVs. However, studies of the Drosophila NMJ have shown that there is an excess of DCVs delivered to type-I boutons by long-distance vesicle circulation. Therefore, because DCV delivery is not limiting, the presynaptic neuropeptide pool is determined by capture, which was found to be bidirectional (Wong, 2012). However, in addition to constitutive capture, activity induces Ca2+-dependent capture. This is advantageous because tapping into the circulating vesicle pool removes delays associated with synthesis and transport, which can take days in humans, to rapidly replace released peptides. Surprisingly, experiments presented in this study demonstrate that activity-dependent capture is unidirectional and selectively sensitive to a genetic perturbation (i.e., Fmr1 overexpression). Therefore, activity does not simply enhance constitutive bidirectional capture that operates at rest, but instead stimulates an independent synaptic capture mechanism (Cavolo, 2016).

Previously, it was not possible to genetically block activity-dependent capture to determine its contribution to steady-state presynaptic stores. However, this study documented inhibition of activity-dependent capture by Fmr1 overexpression. As this was accompanied by a dramatic decrease in presynaptic DCV number, it is concluded that activity-dependent capture makes a large contribution to steady-state presynaptic peptide stores and hence the capacity for future release. At the Drosophila NMJ, DCVs contain a bone morphogenic protein and neuropeptides. Thus, it is possible that activity-dependent capture affects development and acute synaptic function (Cavolo, 2016).

Capture efficiency measurements revealed that the previously detected decrease in retrograde traffic following activity was an indirect effect of vesicle circulation; activity-induced capture of only anterograde DCVs at each en passant bouton simply leaves fewer DCVs for the retrograde trip back into the axon without changing retrograde capture. Of interest, anterograde selectivity for activity-induced capture rules out mechanisms that would perturb transport in both directions (e.g., microtubule breaks). DCV anterograde transport is mediated by the unc-104/Kif1A motor, which also transports SSV proteins and is required for formation of boutons. Therefore, activity-dependent capture may regulate unc-104/Kif1A to affect synaptic release of both small-molecule transmitters and peptides. However, alternative targets could be involved, including proteins that mediate DCV interaction with this anterograde motor or alter the DCV itself (e.g., its phosphoinositides, which may bind to the unc-104/Kif1A pleckstrin homology domain) (Cavolo, 2016).

Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles

Axonal transport is critical for maintaining synaptic transmission. Of interest, anterograde and retrograde axonal transport appear to be interdependent, as perturbing one directional motor often impairs movement in the opposite direction. In this study live imaging of Drosophila and hippocampal neuron dense-core vesicles (DCVs) containing a neuropeptide or brain-derived neurotrophic factor shows that the F-actin depolymerizing macrolide toxin mycalolide B (MB) rapidly and selectively abolishes retrograde, but not anterograde, transport in the axon and the nerve terminal. Latrunculin A does not mimic MB, demonstrating that F-actin depolymerization is not responsible for unidirectional transport inhibition. Given that dynactin initiates retrograde transport and that amino acid sequences implicated in macrolide toxin binding are found in the dynactin component Actin-related protein 1 (Arp1), this study examined dynactin integrity. Remarkably, cell extract and purified protein experiments show that MB induces disassembly of the dynactin complex. Thus imaging selective retrograde transport inhibition led to the discovery of a small-molecule dynactin disruptor. The rapid unidirectional inhibition by MB suggests that dynactin is absolutely required for retrograde DCV transport but does not directly facilitate ongoing anterograde DCV transport in the axon or nerve terminal. More generally, MB's effects bolster the conclusion that anterograde and retrograde axonal transport are not necessarily interdependent (Cavolo, 2015).

The biochemical and live-neuron imaging studies presented in this study lead to several new insights into dynactin and axonal transport. First, MB is a novel dynactin inhibitor that potently and directly disrupts the dynactin complex. The only other condition reported to have this effect is high concentration of the chaotropic salt potassium iodide. The recently published dynactin structure (Urnavicius, 2015) provides additional insights into how MB may trigger subunit release. The predicted MB-binding site on Arp1, based on interactions of similar macrolide toxins with actin, is immediately adjacent to the site where the dynamitin N-terminus binds Arp1. MB binding may thus dislodge dynamitin and the associated p150^Glued and p24 components, in addition to depolymerizing the Arp1 filament itself. The identification of a rapidly acting small-molecule inhibitor will facilitate future in vitro studies of dynactin activity. MB is also potentially useful for in vivo analysis of dynactin's multiple functions (e.g., in mitosis and subcellular transport), although its effect on F-actin must of course be taken into account. Finally, MB may also be used to abolish dynactin-dependent retrograde transport to detect more easily the fate of DCVs and other organelles undergoing anterograde transport (Cavolo, 2015).

Second, the simplest interpretation of MB experiments is that intact dynactin is absolutely required for initiation of retrograde transport of DCVs in axons of hippocampal neurons and a Drosophila sensory neuron, as well as in the synaptic terminal of Drosophila motor neurons. This requirement (as opposed to facilitation) was not evident in living cells previously because dynactin inhibition by antibodies and genetic perturbations was incomplete. Indeed, there is no other case in which retrograde axonal transport can be inhibited so effectively by targeting dynactin without detectable reduction of anterograde transport. The essentially complete inhibition of retrograde axonal transport of DCVs by MB suggests that no alternative mechanism exists for bypassing the requirement for dynactin in retrograde transport in the nerve terminal and axon. Even though DCVs contain bioactive peptides for release at the nerve terminal, dynactin-dependent retrograde transport is required for uniform delivery to en passant boutons. Therefore the results suggest that dynactin's only contribution to the maintenance of nerve terminal anterograde cargoes (e.g., neuropeptide stores) derives from its requirement for retrograde transport (Cavolo, 2015).

This study also shows that anterograde DCV transport in the axon and terminal is not inhibited by dynactin complex disruption. This finding was not anticipated, because anterograde transport in intact axons and axoplasm is inhibited by genetic perturbation of dynactin and a dynactin antibody, respectively. However, MB acts rapidly, which obviates the slow, indirect effects that can occur in genetic experiments, and without introducing the steric hindrance of antibody binding. A more subtle effect of dynactin on anterograde transport is suggested by the finding that MB, but not LatA, increased anterograde run length. This further demonstrates the effectiveness of MB and suggests that dynactin has an attenuating effect on anterograde transport, which is very different from previous models. However, as no statistically significant effect was seen on anterograde flux, the physiological importance of this observation is unclear, especially when compared with the accompanying dramatic inhibition of retrograde transport. Overall MB experiments suggest that dynactin's function in axonal transport of DCVs should be redefined from bidirectional facilitator to being strictly required for retrograde transport without a direct requirement in anterograde transport (Cavolo, 2015).

The independent nature of anterograde and retrograde axonal transport revealed by MB treatment is reminiscent of what is seen when the dynein regulators Nudel and LIS1 are inhibited. In these cases, retrograde axonal transport of other (but not all) organelles is preferentially inhibited. Therefore there are now multiple examples in which perturbation of dynein accessory components (dynactin, LIS1, or Nudel) does not also block plus end-directed axonal transport. The current work is unique in that MB is the first rapidly acting small-molecule inhibitor of retrograde transport to yield clear evidence that retrograde and anterograde transport in the axon and nerve terminal are not mechanistically linked (Cavolo, 2015).

Drosophila Strip serves as a platform for early endosome organization during axon elongation

Early endosomes are essential for regulating cell signalling and controlling the amount of cell surface molecules during neuronal morphogenesis. Early endosomes undergo retrograde transport (clustering) before their homotypic fusion. Small GTPase Rab5 is known to promote early endosomal fusion, but the mechanism linking the transport/clustering with Rab5 activity is unclear. This study showed that Drosophila Strip is a key regulator for neuronal morphogenesis. Strip knockdown disturbs the early endosome clustering, and Rab5-positive early endosomes become smaller and scattered. Strip genetically and biochemically interacts with both Glued (the regulator of dynein-dependent transport) and Sprint (the guanine nucleotide exchange factor for Rab5), suggesting that Strip is a molecular linker between retrograde transport and Rab5 activation. Overexpression of an active form of Rab5 in strip-mutant neurons suppresses the axon elongation defects. Thus, Strip acts as a molecular platform for the early endosome organization that has important roles in neuronal morphogenesis (Sakuma, 2014).

A forward genetic mosaic screen was performed to identify genes for their cell autonomous functions in dendrite and axonal development, and an evolutionarily conserved protein, Striatin-interacting protein (Strip), was identified that works as a molecular linker between retrograde transport and Rab5 activity in early endosome organization. Strip and its orthologues were reported to be a component of Striatin-interacting phosphatase and kinase (STRIPAK) complex; however, the function of Strip has not yet been reported. Interestingly, this study found that Strip forms the protein complex with both Glued, the orthologue of mammalian p150Glued, and Sprint, the orthologue of RIN-1. Glued is an essential component of the dynactin complex and regulates the initiation of retrograde transport on microtubule, and Sprint is supposed to be a guanine nucleotide exchange factor (GEF) for Rab5. This study found that Strip affects the transport of early endosomes by forming complex with Glued in developing neurons. Furthermore, clustering of early endosomes is defective in strip-knockdown Drosophila S2 cells and HeLa cells; early endosomes become smaller and scattered as reported in dynein-inhibited HeLa cells. Moreover, similar to other GEFs, Sprint and Strip have a higher affinity for guanosine diphosphate (GDP)-bound forms of Rab5 than for guanosine triphosphate (GTP)-bound form. In addition, Strip seems to stabilize protein level of Sprint. Finally, the expression of constitutively active form of Rab5 in strip-mutant neurons suppresses the axon elongation defect. These data demonstrate that Strip coordinates dynein-dependent transport and Rab5 activation at the clustering and fusion of early endosomes, which are required for axon elongation (Sakuma, 2014).

During neural development, axon termini are exposed to a constantly changing surrounding environment. Thus, the spatio-temporal regulation of the amount of adhesion molecules and receptors at the axon growth cone is essential. This study identified an uncharacterized protein Strip that forms complexes with Glued and Sprint, and positively regulates clustering (retrograde transport along microtubule) of early endosomes. To form mature early endosomes from internalized small early endosomes, the coordination between clustering and fusion of early endosomes is crucial. For this, Strip serves as a molecular linker by interacting with Glued in clustering and with Sprint, Rab5 and probably with Vps45 in fusion. Vps45 is a Sec-1/Munc18 (SM) protein that targets Avl (Syntaxin 7) to promote fusion of early endosomes, which seems to function with Strip in the wing development. As this study also observed genetic interaction between sprint and Glued in axon elongation, Strip seems to be a platform for early endosome organization by forming a dynein-Glued-Strip-Sprint-Rab5 complex. The following model to explain the physiological role of Strip in the organization of early endosomes. During axon elongation, the strength of the elongation signal could be properly regulated by the dynein-Glued-Strip-Sprint-Rab5 complex by affecting the organization of mature early endosomes. Mature early endosomes are essential vesicular structures for cell signalling, serving both as signalling centres and as sorting centres to degrade or recycle cargoes. In vertebrate neurons, target-derived nerve growth factor (NGF) mediates axon elongation via retrograde axon transport of TrkA-containing signalling endosomes locally or to the cell body to stimulate the expression of downstream target genes essential for long-term axon growth and target innervation. Furthermore, the amount of cell adhesion molecules might also be regulated by Strip-mediated early endosome organization. It has been reported that the regulation of the amount and localization of molecules such as L1 and integrin in the axon growth cone is essential for growth cone motility (Sakuma, 2014).

Further investigations are still needed to identify which Strip domains are important for the formation of the dynein-Glued-Strip-Sprint-Rab5 complex. As Glued and Sprint were identified using a yeast two-hybrid screen, it seems that the binding between Glued and Strip, and that between Sprint and Strip, are likely to be direct. Furthermore, it is thought that Glued and Sprint bind to Strip simultaneously and form a tertiary complex for two reasons; (1) Sprint and Glued showed genetic interaction in PNs and exhibited a phenotype similar to that of stip^dogi PNs and (2) Vps45 and Glued exhibited genetic interaction at the wing vein formation. Thus, Strip seems to serve as a molecular linker between early endosome clustering and fusion by simultaneously forming a complex with Glued and Sprint. In addition, Strip seems to stabilize Sprint protein level. Unfortunately, it was not possible to produce a usable anti-Sprint antibody; therefore, Sprint localization could not be studied. However, it is speculated that Sprint stabilized by Strip could determine the spatio-temporal localization and the activity of Rab5. Regarding the binding between Strip and Glued, it is hypothesized that Strip may act as an adaptor between early endosomes and dynein. Unlike the highly diverse kinesin superfamily proteins (KIFs), which encompass 45 genes, cytoplasmic dyneins have only two heavy-chain family members in mammals. Thus, adaptor proteins for the dynein motor are thought to be important to determine the cargo selectivity, and some adaptor proteins have already been reported. Although Kif16b links Rab5 and microtubules, no specific molecule linking the dynein heavy chain with Rab5-positive early endosomes has been reported yet. Therefore, it is speculated that Strip could serve as an adaptor protein between Rab5-positive early endosomes and the dynein/dynactin complex by binding to Glued, similar to RILP and ORP1L that serve as adaptors between late endosomes and the dynein/dynactin complex (Sakuma, 2014).

This study has provided genetic evidences that Strip, Glued, Sprint and Rab5 are required for both axon elongation and dendrite branching in PNs. However, the expression of a constitutively active form of Rab5 partially suppressed axon elongation and did not suppress dendrite branching defects of strip^dogi PNs. It is considered that the level and the spatio-temporal pattern of Rab5 activity are crucial. Axon elongation and dendrite branching may require different levels or patterns of Rab5 activity. Furthermore, the failure to suppress the dendrite phenotype could also be explained by the fact that not only formation but also localization of mature early endosomes is required for dendrite branching. In Drosophila dendritic arborization (da) neurons, the proximal branching phenotype has been observed in the dendrite of Rab5², dynein light intermediate chain (dlic) or the kinesin heavy chain (khc) mutant. Consistent with this, dendrite proximal branching phenotype was also observed in Rab5² or strip^dogi PNs. The additional dendrite branched out from the dendrite stalk at a more proximal side in Rab5² or strip^dogi PNs than in wild-type PNs. Therefore, it is hypothesized that the constitutively active form of Rab5 could not suppress the dendrite phenotype in strip^dogi PNs, as the expression of the constitutively active form of Rab5 in strip^dogi PNs could promote early endosome maturation in axon and dendrites, but could not adjust early endosome subcellular localization, which is crucial for dendrite branching (Sakuma, 2014).

Strip is structurally and functionally conserved. Branching and elongation defects in strip^dogi PNs were rescued by the expression of the mouse homologue, Strip1. In addition, Strip1 was found to be involved in neuronal migration in the mouse developing cerebral cortex. In the mouse developing cerebral cortex, the Rab5-dependent endosomal trafficking pathway is required for neuronal migration, rather than for neurite extension, probably because it occurs before dendrite formation and axon elongation in vivo. Electroporation of Strip1 shRNA into the cerebral cortex at E14 caused migration defects, similar to those observed when Rab5 shRNAs were used. Thus, strip/Strip1 is involved in Rab5-dependent neural development including neuronal morphogenesis and migration events in both Drosophila and the mouse (Sakuma, 2014).

During this study of Strip function, Strip was reported as a member of the STRIPAK complex. The core component of the PP2A phosphatase complex shows phosphatase activity, whereas the regulatory subunit determines the substrate specificity. Thus, PP2A phosphatase targets specific molecules by changing the regulatory subunit. When Strip is included in the PP2A complex, Cka serves as a regulatory subunit and targets Hippo and probably RAF that belongs to the RAS-MAPK pathway. As early endosome organization is important for many signal-transduction pathways, it might be possible that axon elongation and dendrite targeting require both the dynein-Glued-Strip-Sprint-Rab5 complex and the Strip-STRIPAK (Cka)-Hippo or the Strip-STRIPAK (Cka)-RAS-MAPK complex, but this does not seem possible, at least, in neuronal morphogenesis of PNs. When PNs homozygous for Cka were generated, no defects were found in axon elongation or dendrite branching. Thus, it is thought that the Strip-STRIPAK complex might not have a major role in neuronal morphogenesis (Sakuma, 2014).

The disruption of dynein or dynactin function causes neurodegeneration in many species, and this has been suggested to reflect impaired transport of signalling endosomes. In humans, G59S and G59A missense mutations of p150^Glued have been discovered in motor neuropathy 7B and Perry syndrome, respectively. Both mutations are located within the p150^Glued cytoskeleton-associated protein glycine-rich (CAP-Gly) domain, which is an MT-binding domain required for efficient retrograde transport. Progressive motor neuron degeneration is observed in p150^Glued transgenic mice, with pathological similarities to amyotrophic lateral sclerosis (ALS), including muscle atrophy, axonal swelling, disrupted intracellular trafficking and proliferation of degenerative organelles such as lysosomes and autophagosomes. Furthermore, motor neurons in Als2 (juvenile ALS-associated gene, Rab5GEF)-deficient mice show axonal degeneration. Moreover, the primary neurons in this mouse model show disturbed endosomal trafficking of the insulin-like growth factor 1 (IGF-1) and brain-derived neurotrophic factor (BDNF) receptors. These observations suggest that retrograde transport and Rab5GEF may play a particularly important role in axonal health by transmitting information about the local environment from the axon to the soma. As Strip affects the early endosome organization by forming a complex with Gl and Spri, studying the functions of Strip may provide new insights into both developmental and pathological processes (Sakuma, 2014).

Dynactin subunit p150^Glued is a neuron-specific anti-catastrophe factor

Regulation of microtubule dynamics in neurons is critical, as defects in the microtubule-based transport of axonal organelles lead to neurodegenerative disease. The microtubule motor cytoplasmic dynein and its partner complex dynactin drive retrograde transport from the distal axon. It has been shown that the p150^Glued subunit of dynactin promotes the initiation of dynein-driven cargo motility from the microtubule plus-end. Because plus end-localized microtubule-associated proteins like p150^Glued may also modulate the dynamics of microtubules, it was hypothesized that p150^Glued might promote cargo initiation by stabilizing the microtubule track. This study demonstrates in vitro using assembly assays and TIRF microscopy, and in primary neurons using live-cell imaging, that p150^Glued is a potent anti-catastrophe factor for microtubules. p150^Glued alters microtubule dynamics by binding both to microtubules and to tubulin dimers; both the N-terminal CAP-Gly and basic domains of p150^Glued are required in tandem for this activity. p150^Glued is alternatively spliced in vivo, with the full-length isoform including these two domains expressed primarily in neurons. Accordingly, this study found that RNAi of p150^Glued in nonpolarized cells does not alter microtubule dynamics, while depletion of p150^Glued in neurons leads to a dramatic increase in microtubule catastrophe. Strikingly, a mutation in p150^Glued causal for the lethal neurodegenerative disorder Perry syndrome abrogates this anti-catastrophe activity. Thus, this study found that dynactin has multiple functions in neurons, both activating dynein-mediated retrograde axonal transport and enhancing microtubule stability through a novel anti-catastrophe mechanism regulated by tissue-specific isoform expression; disruption of either or both of these functions may contribute to neurodegenerative disease (Lazarus, 2013).

Microtubules are dynamic, polarized polymers of tubulin that serve as tracks for long-distance transport in eukaryotic cells. In neurons, transport along microtubules is especially important yet particularly vulnerable to disruption, as these cells are long-lived and postmitotic with elongated axonal processes that can extend up to a meter. There is accumulating evidence that axonal transport is disrupted in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington's disease. In neurodegeneration, defects in microtubule dynamics may precede transport defects (Lazarus, 2013).

The rate-limiting step in microtubule formation is nucleation from soluble tubulin, which in the canonical pathway is catalyzed by γ-TuRC enriched at the centrosome. However, in large, postmitotic cells like neurons, noncentrosomal nucleation may be particularly important. Following nucleation, the dynamics of polymerization and depolymerization are strongly influenced by microtubule-associated proteins (MAPs). In particular, a spatially specialized group of MAPs that localize to the microtubule plus end, the plus end-tracking proteins (+TIPs), are ideally poised to modulate dynamics in cells (Lazarus, 2013).

One of these +TIPs is dynactin, a large complex that binds and activates cytoplasmic dynein and also associates with microtubules through its dimeric p150^Glued subunit. p150^Glued has two alternatively spliced microtubule-binding domains at its N-terminus: a cytoskeleton associated protein glycine-rich (CAP-Gly) domain, followed by a serine-rich basic domain. The p150^Glued microtubule-binding N-terminus is dispensable for most dynein-mediated organelle transport. However, it has been recently shown that it Glued specifically required for efficient transport initiation from the distal axon in neurons (Lloyd, 2012; Moughamian, 2012; Lazarus, 2013 and references therein).

Because p150^Glued is specifically enriched at the microtubule plus end, it was hypothesized that it might modify microtubule dynamics. Using solution assays and direct visualization of microtubule dynamics using TIRF microscopy, it was shown that p150^Glued promotes microtubule formation by binding both to microtubules and to soluble tubulin. Both the CAP-Gly and basic domains are required for tubulin-binding in vitro. The full-length isoform encoding both these domains in tandem is primarily expressed in neurons, so it is hypothesized that this pro-polymerization activity might be a neuron-specific function of p150^Glued. Accordingly, it was found that in epithelial cells depleted of p150^Glued there is no effect on microtubule dynamics, while in primary neurons, a significant increase was observed in catastrophe upon depletion of p150^Glued that was specifically rescued by expression of the neuronal isoform. Finally, it was found that a mutation in p150^Glued causative for Perry syndrome, a lethal Parkinson's syndrome, inhibits the anti-catastrophe activity. Thus, the novel neuron-specific anti-catastrophe activity described here may facilitate microtubule stabilization in neurons. It is speculated that disruption of this function may contribute to neurodegeneration (Lazarus, 2013).

This study shows that p150^Glued promotes microtubule formation in vitro by catalyzing nucleation, increasing the polymerization rate, and inhibiting catastrophe. These activities require dimerization and are dependent on the ability of p150^Glued to form a stable complex with tubulin through interactions with both the N-terminal CAP-Gly and basic domains. In primary neurons, it was observed that the dominant effect of p150^Glued on microtubule dynamics is the suppression of catastrophe. Finally, it was determined that a single point mutation within the CAP-Gly domain of p150^Glued causative for a fatal familial form of Parkinson disease, known as Perry Syndrome, leaves p150^Glued unable to promote microtubule assembly either in vitro or in neurons (Lazarus, 2013).

Dynactin was originally identified as a large protein complex that supported dynein-mediated vesicle transport. The N-terminus of the 150 kDa subunit binds microtubules independently of dynein, and increases the processivity of dynein in vitro. Recently, it has been demonstrated that the microtubule-binding N-terminus of p150^Glued is dispensable for organelle localization and vesicular motility in nonneuronal cells. However, the microtubule-binding CAP-Gly domain of dynactin is required for efficient transport initiation from the distal axon in neurons. A plus end-localized pool of p150^Glued may serve to load dynein onto the microtubule. However, biochemical analyses and immunolocalization suggest that a large proportion of dynactin may in fact not be in complex with dynein. Enriched at the plus end, this population of dynactin would be perfectly poised to affect microtubule dynamics (Lazarus, 2013).

The data suggest a mechanism whereby p150^Glued could modify microtubule dynamics. Recently, it has been suggested that the kinetics of tubulin association and dissociation with the microtubule plus end may be much faster than previously appreciated. This makes it increasingly plausible that one mode whereby MAPs alter microtubule dynamics is by modulating the off-rate of tubulin subunits from microtubule plus ends. Since p150^Glued can bind both to microtubules and to soluble tubulin, and because dimerization appears necessary for p150^Glued to robustly modify dynamics, it is speculated that p150^Glued may be acting in this capacity by binding to both microtubules and tubulin at the same time, decreasing the off-rate and inhibiting catastrophe, enabling efficient initiation of dynein-mediated retrograde runs. Interestingly, this mechanism is distinct from the mode by which cytoplasmic dynein independently functions to inhibit catastrophe. Areas of the distal neuron where both cytoplasmic and dynactin are localized could be sites of particularly robust microtubule stabilization (Lazarus, 2013).

The regulation of these microtubule-modifying abilities of p150^Glued may be multifactorial. The basic region is necessary for the modification of microtubule dynamics by p150^Glued, likely by ensuring a stable complex with the distributed acidic nature of tubulin. The basic region is also serine- and threonine-rich, and has been shown to be the target of phosphorylation by regulatory kinases, which might further modulate the p150-tubulin interaction during mitosis, or during development. p150^Glued also binds to CLIP-170 (Ligon, 2003; Hayashi, 2005; Honnappa, 2006; Goodson 2003), which could further modify the behavior of p150^Glued in the cell (Lazarus, 2013).

In vivo, only the p150^Glued isoform expressed in neurons includes both the full CAP-Gly and basic domains that are necessary to modify microtubule assembly dynamics. It has been recently shown that young, developing neurons depleted of p150^Glued are morphologically normal (Moughamian, 2012. In fact, profound depletion of both EB1 and EB3, which should effectively disrupt plus-end targeting, has no gross effects on neurite outgrowth. It may be that, as recent evidence suggests, only as neurons age, and their processes lengthen and elaborate, does the centrosome lose its function as a microtubule organizing center and microtubule dynamics become particularly reliant on plus-end regulation. It is perhaps telling that human patients with the Q74P p150^Glued mutation do not show disease onset until the fifth decade of life. More broadly, microtubule dynamics may alter in aging or degenerating neurons, as suggested from studies of cells from patients with sporadic Parkinson's and Alzheimer's disease (Lazarus, 2013).

In summary, this study has identified and characterized a novel role for p150^Glued in the tissue-specific stabilization of microtubules, and implicated defects in neurodegeneration. Further studies to disentangle the effects of the mutation on axonal transport and microtubule stability in neurons will be required to clarify the pathogenesis involved (Lazarus, 2013).

Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs

Microtubule-based transport mediates the sorting and dispersal of many cellular components and pathogens. However, the mechanisms by which motor complexes are recruited to and regulated on different cargos remain poorly understood. This study describes a large-scale biochemical screen for novel factors associated with RNA localization signals mediating minus end-directed mRNA transport during Drosophila development. The protein Lissencephaly-1 (Lis1) was identified, and minus-end travel distances of localizing transcripts were found to be dramatically reduced in lis1 mutant embryos. Surprisingly, given its well-documented role in regulating dynein mechanochemistry, this study has uncovered an important requirement for Lis1 in promoting the recruitment of dynein and its accessory complex dynactin to RNA localization complexes. Furthermore, evidence is proided that Lis1 levels regulate the overall association of dynein with dynactin. These data therefore reveal a critical role for Lis1 within the mRNA localization machinery and suggest a model in which Lis1 facilitates motor complex association with cargos by promoting the interaction of dynein with dynactin (Dix, 2013).

Normal dynactin complex function during synapse growth in Drosophila requires membrane binding by Arfaptin

Mutations in DCTN1, a component of the dynactin complex, are linked to neurodegenerative diseases characterized by a broad collection of neuropathologies. Because of the pleiotropic nature of dynactin complex function within the neuron, defining the causes of neuropathology in DCTN1 mutants has been difficult. This study combined a genetic screen with cellular assays of dynactin complex function to identify genes that are critical for dynactin complex function in the nervous system. This approach identified the Drosophila homologue of Arfaptin, a multifunctional protein that has been implicated in membrane trafficking. Arfaptin and the Drosophila DCTN1 homologue, Glued, function in the same pathway during synapse growth but not during axonal transport or synapse stabilization. Arfaptin physically associates with Glued and other dynactin complex components in the nervous system of both flies and mice and colocalizes with Glued at the Golgi in motor neurons. Mechanistically, membrane binding by Arfaptin mediates membrane association of the dynactin complex in motor neurons and is required for normal synapse growth. Arfaptin represents a novel dynactin complex-binding protein that specifies dynactin complex function during synapse growth (Chang, 2013).

The p150^Glued CAP-Gly domain regulates initiation of retrograde transport at synaptic termini

p150^Glued is the major subunit of dynactin, a complex that functions with dynein in minus-end-directed microtubule transport. Mutations within the p150^Glued CAP-Gly microtubule-binding domain cause neurodegenerative diseases through an unclear mechanism. A p150^Glued motor neuron degenerative disease-associated mutation introduced into the Drosophila Glued locus generates a partial loss-of-function allele (Gl^G38S) with impaired neurotransmitter release and adult-onset locomotor dysfunction. Disruption of the p150^Glued CAP-Gly domain in neurons causes a specific disruption of vesicle trafficking at terminal boutons (TBs), the distal-most ends of synapses. Gl^G38S larvae accumulate endosomes along with dynein and kinesin motor proteins within swollen TBs, and genetic analyses show that kinesin and p150^Glued function cooperatively at TBs to coordinate transport. Therefore, the p150^Glued CAP-Gly domain regulates dynein-mediated retrograde transport at synaptic termini, and this function of dynactin is disrupted by a mutation that causes motor neuron disease (Lloyd, 2012).

Dynactin is required for transport initiation from the distal axon

Dynactin is a required cofactor for the minus-end-directed microtubule motor cytoplasmic dynein. Mutations within the highly conserved CAP-Gly domain of dynactin cause neurodegenerative disease. This study shows that the CAP-Gly domain is necessary to enrich dynactin at the distal end of primary neurons. While the CAP-Gly domain is not required for sustained transport along the axon, the distal accumulation facilitates the efficient initiation of retrograde vesicular transport from the neurite tip. Neurodegenerative disease mutations in the CAP-Gly domain prevent the distal enrichment of dynactin thereby inhibiting the initiation of retrograde transport. Thus, a model is proposed in which distal dynactin is a key mediator in promoting the interaction among the microtubule, dynein motor, and cargo for the efficient initiation of transport. Mutations in the CAP-Gly domain disrupt the formation of the motor-cargo complex, highlighting the specific defects in axonal transport that may lead to neurodegeneration (Moughamian, 2012).

Structural dynamics and multiregion interactions in dynein-dynactin recognition

Cytoplasmic dynein is a 1.2-MDa multisubunit motor protein complex that, together with its activator dynactin, is responsible for the majority of minus end microtubule-based motility. Dynactin targets dynein to specific cellular locations, links dynein to cargo, and increases dynein processivity. These two macromolecular complexes are connected by a direct interaction between dynactin's largest subunit, p150^Glued, and dynein intermediate chain (IC) subunit. This study demonstrated using NMR spectroscopy and isothermal titration calorimetry that the binding footprint of p150^Glued on IC involves two noncontiguous recognition regions, and both are required for full binding affinity. In apo-IC, the helical structure of region 1, the nascent helix of region 2, and the disorder in the rest of the chain are determined from coupling constants, amide-amide sequential NOEs, secondary chemical shifts, and various dynamics measurements. When bound to p150^Glued, different patterns of spectral exchange broadening suggest that region 1 forms a coiled-coil and region 2 a packed stable helix, with the intervening residues remaining disordered. In the 150-kDa complex of p150^Glued, IC, and two light chains, the noninterface segments remain disordered. The multiregion IC binding interface, the partial disorder of region 2 and its potential for post-translational modification, and the modulation of the length of the longer linker by alternative splicing may provide a basis for elegant and multifaceted regulation of binding between IC and p150^Glued. The long disordered linker between the p150^Glued binding segments and the dynein light chain consensus sequences could also provide an attractive recognition platform for diverse cargoes (Morgan, 2011).

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. The role of Dynein and Dynein-interacting proteins were studied during Drosophila muscle development, and several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, were found to contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function (Folker, 2012).

Single-molecule assays reveal that RNA localization signals regulate dynein-dynactin copy number on individual transcript cargoes

Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA-motor complexes on the cytoskeleton is orchestrated. This study has reconstituted transport of mRNPs containing specific RNAs in vitro. It was shown directly that mRNAs that are either apically localized or non-localized in Drosophila embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. It was found that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro, and evidence is provided that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. These findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules (Amrute-Nayak, 2012).

This study has reconstituted transport of specific mRNA species along individual microtubules in vitro and employed single-molecule-resolution measurements to shed light on the composition of transport complexes. An in vivo study of oskar mRNA transport in Drosophila oocytes has demonstrated that asymmetric RNA localization can be achieved by a random walk of a single motor species along a weakly polarized microtubule cytoskeleton. The current findings provide direct evidence for an additional mechanism for RNA targeting in which localization signals control sorting by regulating the net directionality of bidirectional motor complexes on individual microtubules. It is proposed that this is associated with modest differences in the number of motors assembled on individual mRNA molecules. These findings raise fascinating questions about how dynein-dynactin and the unidentified plus-end motor(s) are bound to localizing and non-localizing mRNA molecules and how their activities are orchestrated in time and space (Amrute-Nayak, 2012).

Aurora A contributes to p150^glued phosphorylation and function during mitosis

Aurora A is a spindle pole-associated protein kinase required for mitotic spindle assembly and chromosome segregation. This study shows that Drosophila melanogaster Aurora A phosphorylates the dynactin subunit p150^glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150^glued. Although wild-type p150^glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by Aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150^glued. These results suggest that Aurora A kinase participates in vivo to the phosphoregulation of the p150^glued MBD to limit the microtubule binding of the dynein-dynactin complex and thus regulates spindle assembly (Rome, 2012).

A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation

The Glued gene of Drosophila encodes the homologue of the vertebrate p150Glued subunit of dynactin. The Glued¹ mutation compromises the dynein-dynactin retrograde motor complex and causes disruptions to the adult eye and the CNS, including sensory neurons and the formation of the giant fiber system neural circuit. A 2-stage genetic screen was performed to identify mutations that modified phenotypes caused by over-expression of a dominant-negative Glued protein. Over 34,000 flies were screened and 41 mutations were isolated that enhanced or suppress an eye phenotype. Of these, 12 were assayed for interactions in the giant fiber system by which they altered a giant fiber morphological phenotype and/or altered synaptic function between the giant fiber and the tergotrochanteral muscle motorneuron. Six showed interactions including a new allele of atypical protein kinase C (aPKC). This cell polarity regulator interacts with Glued during central synapse formation. The five other interacting mutations were mapped to discrete chromosomal regions. This study has used a novel approach to screen for genes involved in central synapse formation by performing a primary screen, using a sensitized background, on the adult eye and then a secondary screen, on the isolated mutations, for synaptic phenotypes. This study shows that forward genetic screens are powerful tools for identifying genes with roles in CNS development. This has highlighted a role for aPKC in the formation of an identified central synapse (Ma, 2009).

The success of the two-stage screening approach may have been facilitated by the fact that Glued has a plethora of distinct roles during eye development, including organizing optic neural architecture and an involvement in the formation of sensory neuronal circuits. Therefore an eye phenotype was available on which to base the screen. However, this does not preclude such a method being used for identifying genes involved in other aspects of neural differentiation. It was found that 50% (6/12) of the isolated mutation-containing chromosomes that altered the eye phenotype also altered GFS phenotypes when tested (Ma, 2009).

The over-expression of the truncated Glued protein caused strong phenotypes in both the eye and GF neurons, greater than those caused by heterozygosity for the dominant Gl¹ allele. This is likely to be due to the GAL4-UAS system producing many more molecules of the truncated product than Gl¹/+ cells in which, theoretically, a maximum of half of the Glued molecules will be truncated. Consistent with this observation, both the suppressors and enhancers isolated during this screen showed stronger effects on Gl^DN eye phenotypes than on those produced by Gl¹. Determining interactions with the Gl¹allele also allowed confirmation of GAL4-independent interactions with the Glued locus. For all of the mutations (with the exception of EG162), the alterations of the weaker Gl¹/+ eye phenotype were not obvious, however, SEM and sectioning was performed to show interactions with two of the mutations (EG37 and SG13) (Ma, 2009).

Two different disruptions of Glued function, one strong and the other weaker, were used to assay successfully the effects of both enhancer and suppressor mutations in the giant fiber system (GFS) using both morphological and electrophysiological criteria. The severe disruptions of GF morphology and synaptic function enabled the effects of suppressor mutations to be clearly observed. This was less reliable when assaying the effects of mutations isolated as enhancers as either no increase of the already severe phenotype was seen or the interaction was lethal. For the enhancers, therefore, double heterozygotes were generated with Gl¹/+. As was the case in the eye, interactions were less pronounced and only two enhancers, EG37/+ and EG162/+ showed enhancement of the Gl¹/+ electrophysiological phenotype. Indeed, the subtlety of some interactions with Gl¹/+ may have resulted in the analyses being unable to detect some positive interacting loci in the GFS that altered the eye phenotype caused by Gl^DN (Ma, 2009).

Some EMS alleles were generated, two of which were mapped to known genetic loci and four of which were mapped to discrete chromosomal locations. However, these four complement all the available lethal alleles in these regions indicating that the mutations lie in loci for which there are few or no lethal alleles available. Identification of the location of these new alleles will require either new rounds of mutagenesis, such as via P-element excision in the mapped regions, finer mapping using SNPs or custom made deficiencies using stocks from the DrosDel project. Completion of the BDGP Gene Disruption Project may also enable mapping of the lesions along with more recent approaches using other transposable elements that may disrupt genes refractory to P-element disruption. Interestingly, no mutations were isolated in genes that encode known components of the retrograde motor complex including any further alleles of Glued. During some of the early genetic analysis of the Glued locus, dominant second-site suppressors of the Gl¹ eye phenotype were isolated and reported. Of these, two were mapped to the X chromosome (Su [Gl]27 and Su [Gl]57, and the others, Su(Gl)77 and Su(Gl)102 are alleles of Dynein heavy chain 64C (Ma, 2009).

Two new alleles of known genes, Su(H) and aPKC were isolated. Of the two, this study showned that alleles of aPKC genetically interact with Glued in the GFS and suppress the abnormalities in GF-TTMn synapse formation seen when the retrograde motor complex is compromised by Gl^DN. These abnormalities are: a lack of the presynaptic 'bends'; a branching event that takes place after the two neurons have met; swollen axon tips and a weak or absent functional synaps. aPKC is part of a protein complex, with PAR-3 (Bazooka in Drosophila) and PAR6 that regulates cell polarity in a number of different tissues/cells of Drosophila and vertebrates including neurons. So what is the role of aPKC in the GF neuron? In vertebrate neurons aPKC is needed for neurite outgrowth. In contrast, aPKC in flies is an essential part of the machinery that polarizes dividing neuroblasts but is not needed postmitotically for outgrowth. The data also indicate that aPKC is not needed for neurite extension since the introduction of aPKC mutations into the sensitized background has no effect on GF outgrowth. aPKC is involved in memory formation in Drosophila and at the developing larval NMJ it regulates microtubules (MTs) both pre- and postsynaptically during synapse formation. Indeed MTs are one of the major targets of the PAR-3/PAR-6/aPKC complex in several contexts. aPKC regulates MT orientation in fibroblasts and MT organization in the early embryo. At the NMJ it controls MT stability with a reduction in aPKC activity causing a decreased association of MTs with the microtubule associated protein Futsch and MT fragmentation. Dynein-dynactin is known to be involved in MT organization during growth cone remodeling as well as polarizing MTs in axons. The data indicate that dynein-dynactin and aPKC are acting antagonistically during formation of the GF presynaptic structure and suggest that both are needed to control microtubule organization and dynamics in synapse formation but have opposing roles. One simple explanation is that one of the roles of dynein-dynactin in the GF is to alter MT dynamics at the tip of the axon, when it has reached its post-synaptic target, so that they are more mobile enabling the presynaptic bend to be formed. aPKC regulates the stability of MTs thereby confining axon branching to a single bend. Blocking dynein-dynactin function prevents the MT re-organization needed for formation of the bends and this is ameliorated when aPKC function is reduced (Ma, 2009).

Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts

Mitotic spindle orientation in polarized cells determines whether they divide symmetrically or asymmetrically. Moreover, regulated spindle orientation may be important for embryonic development, stem cell biology, and tumor growth. Drosophila neuroblasts align their spindle along an apical/basal cortical polarity axis to self-renew an apical neuroblast and generate a basal differentiating cell. It is unknown whether spindle alignment requires both apical and basal cues, nor have molecular motors been identified that regulate spindle movement. Using live imaging of neuroblasts within intact larval brains, independent movement of both apical and basal spindle poles is detected, suggesting that forces act on both poles. Reducing astral microtubules decreases the frequency of spindle movement, but not its maximum velocity, suggesting that one or few microtubules can move the spindle. Mutants in the Lis1/dynactin complex strongly decrease maximum and average spindle velocity, consistent with this motor complex mediating spindle/cortex forces. Loss of either astral microtubules or Lis1/dynactin leads to spindle/cortical polarity alignment defects at metaphase, but these are rescued by telophase. It is proposed that an early Lis1/dynactin-dependent pathway and a late Lis1/dynactin-independent pathway regulate neuroblast spindle orientation (Siller, 2008).

This study shows that spindle/cortical polarity alignment is established at prophase in Drosophila larval neuroblasts, and that both apical and basal spindle poles move independently, as if spindle/cortex forces are applied to both poles. Reducing astral microtubule number reduces the frequency of spindle pole movements, but that maximum spindle pole velocity is unaffected, suggesting that maximum velocity may occur when only one or a few microtubules are simultaneously contacting the cortex. Yhe Lis1/dynactin complex is required for spindle pole movement; reducing Lis1/dynactin complex activity reduces the maximum and average spindle velocity, even though astral microtubules still contact the cortex. This suggests that Lis1/dynactin is required to translate microtubule-cortex contact into spindle movement. Finally, this study shows that Lis1/dynactin is required for spindle orientation at metaphase but not at telophase (Siller, 2008).

Lis1-dependent dynamic microtubule-cortex interactions were observed at both apical and basal spindle poles, as well as asynchronous movements of apical and basal spindle poles. What are the candidate apical or basal cortical proteins that might regulate spindle pole movement? Insight into the role of cortical proteins in regulating spindle movement has been made in both C. elegans and mammals, and can be used to model spindle dynamics in Drosophila. Apical proteins in neuroblasts known to regulate spindle force in C. elegans include Gαi, Pins and Mud. During the first division of the C. elegans zygote, enrichment of the Gα-binding and activating Pins-related GPR1/2 proteins at the posterior cortex leads to increased Gα activity, resulting in higher cortex-spindle force generation, spindle pole rocking, and posterior spindle displacement. This suggests that Gαi/Pins/Mud may promote movement of the apical spindle pole in Drosophila neuroblasts, which is supported by the finding that reducing Gαi can decrease spindle rocking (Siller, 2008).

In C. elegans, Lin-5 mediates the physical interaction of Lis1/dynein/dynactin with the cortical Gα and the Pins-related GPR1/2 proteins, and reduction of dynein or Lis1 function also reduces spindle pole rocking and posterior spindle displacement. Furthermore, in mammalian tissue culture cells Gαi overexpression can induce robust spindle rocking that requires LGN (a Pins/GPR-related protein) and NuMA (a Mud/Lin-5-related protein that binds dynein/dynactin). An attractive model is that Gαi/LGN activates NuMA, which interacts with dynein/dynactin/Lis1-loaded astral microtubules. In Drosophila neuroblasts, Gαi, Pins (LGN-related) and Mud (NuMA-related Pins-binding protein) are all enriched at the apical cortex and required for proper metaphase spindle orientation. Thus, it is tempting to propose that apical Gαi/Pins/Mud interacts with dynein/dynactin/Lis1-loaded astral microtubules to center the apical spindle pole with the apical cortical domain. Identifying a physical link between Mud and dynein/dynactin/Lis1, and determining its functional importance in spindle orientation, would be a good test of this model (Siller, 2008).

Surprisingly, it was found that third instar larval neuroblasts have more vigorous basal spindle pole rocking than apical spindle pole rocking, revealing a Gαi-independent spindle force generation mechanism at the basal cortex. Basal cortical proteins include Armadillo, DE-cadherin, β-catenin, APC2, and Mud. Components of the APC2/DE-cadherin/α-catenin/β-catenin complex physically interact with the dynein complex in mammalian cells, and are required for spindle positioning in the Drosophila pre-cellular embryo, epithelial cells, and germline stem cells. Previous studies indicated no spindle positioning defects in neuroblasts after reduction of APC2 function, however these findings do not rule out a role for APC2 in spindle orientation because it may function redundantly with an apical cue, such as the Gαi/Pins/Mud pathway (Siller, 2008).

This study has demonstrated that both apical and basal spindle pole movements are greatly diminished in Lis1 mutant larval neuroblasts (even in those with well-formed bipolar spindles and asters), providing first evidence that Lis1/dynactin is a critical component in the regulation of both apical and basal cortex-spindle forces. How does Lis1 regulate cortex-spindle forces? One possibility is that translocation of cortically associated motor proteins towards microtubule minus-ends results in movement of the microtubule towards the cortex. Consistent with this hypothesis, Lis1 colocalizes with and binds the microtubule minus-end motor dynein/dynactin complex. Specifically, the budding yeast Lis1 homologue (Pac1) targets dynein to astral microtubule plus-ends where it promotes movement of astral microtubules towards the cortex, resulting in translocation of the spindle apparatus through the bud neck. By analogy, Lis1 may regulate spindle pole movement in neuroblasts by promoting dynein-dependent movement of astral microtubules towards the cortex. Alternatively, Lis1 may modulate the polymerization/depolymerization cycle (dynamic instability) of cortically-attached astral microtubules or the duration of astral microtubule-cortex interactions. In support of this latter hypothesis, loss of Lis1 or dynein function in Aspergillus nidulans or budding yeast results in reduced microtubule catastrophe and/or decreased shrinkage rates, thereby promoting assembly of overly long microtubules. Currently, it was not possible to visualize astral microtubule plus-ends with sufficient spatial and temporal resolution to distinguish between these models for Lis1 function (Siller, 2008).

Both models for Lis1 function described above would require association of Lis1 protein with astral microtubules and/or the neuroblast cortex. Indeed, Lis1/dynactin complex proteins have been detected on astral microtubule plus-ends or at the cortex in mammalian, nematode, and yeast cells. The localization of HA-tagged Lis1, GFP-tagged Lis1, endogenous Lis1, and endogenous dynactin protein distribution were analyzed using various fixation and live imaging methods in embryonic and larval neuroblasts, but no enrichment of Lis1/dynactin at the cortex or at astral microtubule plus-ends was found. The most likely explanation is that Lis1/dynactin at these sites is masked by the high level of cytoplasmic protein present in neuroblasts (Siller, 2008).

The Lis1/dynactin complex is required for reliable spindle orientation with the apical/basal polarity axis in metaphase neuroblasts. These spindle orientation defects may be due in part to failure in anchoring one centrosome at the apical cortex during interphase, as reported for wild type neuroblasts; this study observed mis-positioned interphase centrosomes in Lis1 mutants, but this this phenotype was not analyzed in detail. It was surprising to find that spindle orientation was essentially normal at telophase in Lis1 and dynactin (Gl) mutant neuroblasts, despite severe defects at metaphase. This indicates that there are two pathways for regulating spindle orientation: an early Lis1/dynactin-dependent pathway (prophase/metaphase), and a late Lis1/dynactin-independent pathway (anaphase/telophase). There are several models consistent with these findings: (1) Lis1 and dynactin mutants have a delay in anaphase onset which allows sufficient time for 'telophase rescue' to occur. (2) A spindle orientation checkpoint -- analogous to the yeast spindle orientation checkpoint -- may delay cytokinesis until proper spindle orientation has occurred. These first two hypotheses are disproven by the finding that Lis1 rod double mutants have normal metaphase progression but still show metaphase defects and 'telophase rescue' of spindle orientation. (3) The cleavage furrow may be positioned by cortical polarity cues, such that cell elongation at early anaphase may mechanically re-orient the spindle along the long axis of the neuroblast. This model is unlikely because it is commonly accepted that the position of the cleavage furrow is determined by the position of the mitotic spindle and not by cortical cues. (4) Additional microtubule-cortex regulators unrelated to Lis1/dynactin promote telophase spindle orientation (Siller, 2008).

The fourth model is the most likely, except that microtubule-cortex regulators unrelated to Lis1/dynactin have not yet been identified in Drosophila neuroblasts. Help may come from analysis of budding yeast spindle orientation pathways, where Lis1/dynactin-dependent and -independent pathways have been identified. Several components of the yeast Lis1/dynactin-independent pathway are evolutionarily conserved, including the microtubule plus-end binding protein Bim1p, called EB1 in Drosophila. It is tempting to speculate that these proteins may regulate the Lis1/dynactin-independent pathway in Drosophila neuroblasts (Siller, 2008).

Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam

Many membrane proteins, including Drosophila Dscam, are enriched in dendrites or axons within neurons. However, little is known about how the differential distribution is established and maintained. Dscam isoforms carrying exon 17.1 (Dscam[TM1]) are largely restricted to dendrites, while Dscam isoforms with exon 17.2 (Dscam[TM2]) are enriched in axons. This study investigated the mechanisms underlying the dendritic targeting of Dscam[TM1]. Through forward genetic mosaic screens and by silencing specific genes via targeted RNAi, it was found that several genes, encoding various components of the dynein-dynactin complex, are required for restricting Dscam[TM1] to the mushroom body dendrites. In contrast, compromising dynein/dynactin function did not affect dendritic targeting of two other dendritic markers, Nod and Rdl. Tracing newly synthesized Dscam[TM1] further revealed that compromising dynein/dynactin function did not affect the initial dendritic targeting of Dscam[TM1], but disrupted the maintenance of its restriction to dendrites. The results of this study suggest multiple mechanisms of dendritic protein targeting. Notably, dynein-dynactin plays a role in excluding dendritic Dscam, but not Rdl, from axons by retrograde transport (Yang, 2008).

Multiple lines of evidence indicate that the dynein/dynactin complex has an important function in maintaining proper distribution of dendritic Dscam in MB neurons. First, mutations in three components (Lis1, Dmn and p24) of the dynein/dynactin complex were recovered based on mislocalization of dendritic Dscam through a MARCM-based genetic mosaic screen. Second, silencing other components of the complex with RNAi also resulted in mistargeting of dendritic Dscam to axons. Third, disrupting dynein/dynactin function with dominant-negative Glued reproduced the mislocalization phenotype. Further, newly synthesized Dscam[TM1] was preferentially targeted to dendrites. Interestingly, compromising dynein/dynactin function did not affect the targeting from cell bodies to dendrites but disrupted the continuous exclusion of dendritic Dscam from axons. Altogether, these findings show that dynein/dynactin normally acts to prevent Dscam[TM1] from entering axons by retrograde axonal transport (Yang, 2008).

Acute induction by TARGET, in which GAL4-dependent expression of UAS-transgene is acutely controlled by a temperature-sensitive GAL4 repressor, GAL80ts, revealed two mechanisms underlying the dendritic distribution of Dscam[TM1]. Newly synthesized Dscam[TM1] was largely excluded from axons, suggesting directed dendritic targeting and the involvement of selective transport in the dendritic distribution of Dscam[TM1]. Though dynein/dynactin is essential for restricting Dscam[TM1] to dendrites, knocking down dynein/dynactin function did not disrupt the directed dendritic targeting. This leads to the belief that dynein/dynactin is required for preventing dendritic Dscam from misdistributing into axons. When dynein/dynaction function was compromised, newly synthesized Dscam[TM1] remained consistently targeted to dendrites but later leaked into axons. Dendritic Dscam gradually filled the axons; and it took about six hours for Dscam[TM1] to reach the axon termini. This protracted process of mislocalization suggests that dendritic Dscam passively leaks into the axons, and that dynein/dynactin-mediated retrograde axonal transport normally acts to rapidly move leaked Dscam[TM1]-containing vesicles out of the axons. In summary, these phenomena not only demonstrate a dynein-dynactin-independent mechanism of selective transport that preferentially targets Dscam[TM1]-containing vesicles to dendrites, but also implicate the involvement of retrograde axonal transport in preventing accumulation of Dscam[TM1] in axons. These two independent mechanisms act together to ensure restriction of dendritic Dscam to the dendrites (Yang, 2008).

Although the dynein/dynactin complex is essential for maintaining dendritic distribution of Dscam[TM1], the results do not reveal whether mislocalized Dscam[TM1] is on the plasma membrane or in vesicles inside the cytoplasm. It is possible that dendritic Dscam passively leaks into axons either through membrane diffusion or mistargeting of vesicles. Since blocking endocytosis with temperature-sensitive shibire mutant showed no obvious effect on Dscam dendritic distribution, the model is favored that dynein/dynactin acts to prevent axonal accumulation of Dscam[TM1] by actively moving mistargeted Dscam[TM1]-containing vesicles out of axons by retrograde axonal transport (Yang, 2008).

Dscam[TM1]-containing cargos are primarily targeted to dendrites via a dynein/dynactin-independent process. In addition, they are effectively excluded from the axons by dynein/dynactin-mediated retrograde axonal transport. However, dynein/dynactin is not routinely needed for excluding dendritic proteins from the axons. Since no biological process can be carried out with absolute fidelity, it is conceivable that dendritic molecules of most kinds may accidentally leak into the axons. Some salvage mechanism(s) should exist for actively clearing mislocalized molecules to prevent any significant accumulation in the wrong places. One of the possibilities is that dynein/dynactin mediates retrograde axonal transport and can serve as a general mechanism for removing dendritic molecules out of axons. This hypothesis remains to be tested thoroughly. Nonetheless, blocking dynein/dynactin function did not affect the distribution of two other dendritic markers checked. Nod-β-gal is a reliable minus-end reporter of microtubules, and misdistribution of Nod-β-gal in MB axons has been shown in short stop mutant clones, in which microtubule polarity is perturbed. Absence of Nod-β-gal from the axons of dynein/dynactin mutant neurons demonstrates that the microtubules in axons remained uniformly polarized with minus ends pointing toward cell bodies, and rules out the possibility that dendritic Dscam became mislocalized due to abnormal microtubule organization. As to Rdl-HA, which, like Dscam[TM1], is a membrane protein, a lack of effect on its somatodendritic distribution indicates that dynein/dynactin is selectively involved in preventing dendritic Dscam from leaking into the axons. Diverse mechanisms may be utilized to efficiently clear different dendritic proteins in axons (Yang, 2008).

Regarding the mechanism(s) of selective transport, directed dendritic targeting apparently requires motor proteins that selectively move cargos toward the dendrites. Since dendrites, but not axons, carry microtubules with minus ends pointing away from cell bodies, potential candidates that underlie directed dendritic targeting include all minus-end-directed microtubule motors. Notably, dynein/dynactin is dispensable to the initial dendritic targeting of Dscam[TM1] or the continuous dendritic restriction of Rdl, arguing against any critical role for minus-end-directed dynein/dynactin in transporting cargos into the dendrites. Other microtubule motors that might support such directional movement include dendrite-specific plus-end-directed motors (e.g., KIF17 and KIF21B), though it remains mysterious how a plus-end-directed motor can be well restricted to dendrites. In theory, forward genetic mosaic screens will ultimately allow uncovering of the diverse mechanisms of dendritic protein targeting. Encouragingly, mutants have been obtained that exhibit different mislocalization phenotypes, further characterization of which should shed additional light on neuron polarity and its underlying cellular/molecular mechanisms. Notably, in DC-B9 mutant clones, mistargeted Dscam[TM1]::GFP existed abundantly in the MB peduncle, preferentially accumulated at the end of the peduncle, but never extended into the axon lobes. This intriguing phenotype suggests presence of distribution barriers not only in the beginning of axons but also at the junction between the proximal axon domain (peduncle) and the distal axon segment (lobe), and implies another possible mechanism for restricting Dscam[TM1] to the dendritic membrane (Yang, 2008).

Furthermore, the functional roles of each subunit of the dynein/dynactin complex have not been fully determined. Although several studies of the dynein light chains in mammalian cells indicate that dynein subunits can be functionally specialized, studies in Drosophila show that strong loss-of-function mutations in different dynein/dynactin subunits show extensive overlap in the resulting mutant phenotypes. The current data indicate that Lis1, Dmn, Glued, p24, p25, Dhc64C, Dhc62B, and Dlc90F all participate in the complete function of dynein/dynactin complex in maintaining dendritic distribution of Dscam. This result supports the idea that all the dynein/dynactin subunits work together to fulfill its diverse functions, and loss of any subunits may result in different degrees of similar dynein/dynactin-dysfunctional phenotypes (Yang, 2008).

With respect to Dscam targeting motifs, the cytoplasmic juxtamembrane domain of Dscam may dictate its TM-dependent subcellular localization. However, further structure-distribution analysis only allowed location of an axonal targeting motif to the cytoplasmic juxtamembrane region of TM2, leaving its dendritic targeting motif(s) still undetermined. In addition, using the same system it could not be determined whether any of the mutants recovered here also affects the axonal targeting of Dscam[TM2], since transgenic Dscam[TM2] becomes uniformly distributed upon overexpression following an analogous induction. The involvement of multiple mechanisms in targeting specific Dscams to specific neuronal domains further supports the notion that Dscam isoform compositions in the dendrites versus axons of the same neurons need to be independently regulated, elucidation of the physiological significance of which promises to shed new light on how the brain develops and operates (Yang, 2008).

In summary, this study has uncovered a scavenger mechanism for maintaining dendritic distribution of Dscam[TM1] and provide an in vivo model to study neuron polarity and differential protein targeting. On top of the many known functions of dynein/dynactin (including mitosis, vesicular transport, retrograde signaling, neuronal migration), dynein/dynactin helps restrict certain dendritic proteins to the somatodendritic domain of neurons by preventing them from spreading into the axons. Notably, multiple independent mechanisms act together to locate Dscam[TM1] to dendrites; and diverse mechanisms are utilized to target different dendritic proteins to the dendrites (Yang, 2008).

Microtubule binding by dynactin is required for microtubule organization but not cargo transport

Dynactin links cytoplasmic dynein and other motors to cargo and is involved in organizing radial microtubule arrays. The largest subunit of dynactin, p150^glued, binds the dynein intermediate chain and has an N-terminal microtubule-binding domain. To examine the role of microtubule binding by p150^glued, this study replaced the wild-type p150^glued in Drosophila melanogaster S2 cells with mutant DeltaN-p150 lacking residues 1-200, which is unable to bind microtubules. Cells treated with cytochalasin D were used for analysis of cargo movement along microtubules. Strikingly, although the movement of both membranous organelles and messenger ribonucleoprotein complexes by dynein and kinesin-1 requires dynactin, the substitution of full-length p150^glued with DeltaN-p150^glued has no effect on the rate, processivity, or step size of transport. However, truncation of the microtubule-binding domain of p150^glued has a dramatic effect on cell division, resulting in the generation of multipolar spindles and free microtubule-organizing centers. Thus, dynactin binding to microtubules is required for organizing spindle microtubule arrays but not cargo motility in vivo (Kim, 2007).

Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/Dynactin in spindle assembly and mitotic checkpoint control

Lis1 is required for nuclear migration in fungi, cell cycle progression in mammals, and the formation of a folded cerebral cortex in humans. Lis1 binds dynactin and the dynein motor complex, but the role of Lis1 in many dynein/dynactin-dependent processes is not clearly understood. This study generated and/or characterized mutants for Drosophila Lis1 and a dynactin subunit, Glued, to investigate the role of Lis1/dynactin in mitotic checkpoint function. In addition, an improved time-lapse video microscopy technique was developed that allows live imaging of GFP-Lis1, GFP-Rod checkpoint protein, GFP-labeled chromosomes, or GFP-labeled mitotic spindle dynamics in neuroblasts within whole larval brain explants. Mutant analyses show that Lis1/dynactin have at least two independent functions during mitosis: initially promoting centrosome separation and bipolar spindle assembly during prophase/prometaphase, and subsequently generating interkinetochore tension and transporting checkpoint proteins off kinetochores during metaphase, thus promoting timely anaphase onset. Furthermore, Lis1/dynactin/dynein physically associate and colocalize on centrosomes, spindle MTs, and kinetochores, and regulation of Lis1/dynactin kinetochore localization in Drosophila differs from both C. elegans and mammals. It is concluded that Lis1/dynactin act together to regulate multiple, independent functions in mitotic cells, including spindle formation and cell cycle checkpoint release (Siller, 2005).

This study shows that both Lis1 and Gl are enriched on centrosomes/spindle poles in wild type neuroblasts, and Lis1/Gl are required for centrosome separation in prophase neuroblasts. A role for centrosome separation has been reported for dynein in Drosophila embryos, dynein in mammalian cells, and dynein/dynactin/Lis1 in C. elegans blastomeres. However, the exact mechanism by which they promote centrosome separation is unclear. One proposed model suggests that dynein may promote centrosome separation by generating pulling forces on astral MTs attached to the cortex or cytoplasmic structures. Alternatively, dynein associated with the nuclear envelope may exert pulling forces on astral MTs to promote centrosome separation. No GFP-Lis1 was detected on the nuclear envelope or at the neuroblast cortex, although it is possible that high cytoplasmic levels mask low levels of Lis1/dynactin at these sites. Thus, it remains unclear how Lis1/Gl promotes centrosome separation in neuroblasts. Centrosome separation is not completely blocked in Lis1 or Gl mutant neuroblasts, either due to residual amounts of maternal protein or due to the presence of a Lis1/dynactin/dynein-independent pathway. Interestingly, cortical non-muscle myosin II has been shown to contribute to centrosome separation in some cell types, raising the possibility that Lis1/dynactin/dynein and myosin II play partially redundant roles in neuroblast centrosome separation (Siller, 2005).

These observations further support a role for centrosomal/spindle pole-associated Lis1/Gl in spindle assembly, spindle pole focusing, and centrosome attachment in prometaphase and metaphase neuroblasts. Detachment of centrosomes from the spindle has been observed in dynein mutants in Drosophila, and in mammalian cells with reduced dynein or dynactin function. These findings show that Lis1 and dynactin act as cofactors for dynein-dependent focusing of spindle poles and attachment of spindle MTs minusends to centrosomes. In vertebrate cells dynein/dynactin is thought to contribute to focusing of spindle poles and attaching MT-minus ends to centrosomes by transporting pericentriolar proteins and MT-binding proteins, such as NuMA, to centrosomes. Although no clear NuMA orthologue is encoded in the Drosophila genome, a dynein/dynactin/Lis1 complex may contribute to spindle pole focusing by concentrating other MT cross-linking proteins with NuMA-like function at spindle MT minus ends (Siller, 2005).

Gl and Lis1 mutant neuroblasts occasionally form multipolar spindles and have more than two centrosome-like Centrosomin/gamma-tubulin structures. Due to the lack of Drosophila centriolar markers it was not possible to determine whether these extra centrosomelike structures contained centrioles. Multipolar spindles have also been observed in mammalian cells overexpressing Lis1 protein or in which Lis1 function was reduced. Time-lapse analysis of Lis1 mutant neuroblasts reveal occasional co-segregation of both centrosomes into the neuroblast as a consequence of incomplete centrosome separation and centrosome detachment from the spindle. Such a mis-segregation event may be followed by duplication of both centrosomes during the next cell cycle leading to supernumerary centrosomes. Alternatively, extra centrosomes in Lis1 and Gl mutant neuroblasts may be due to uncoupling of centrosome duplication from the cell cycle or centrosome fragmentation (Siller, 2005).

Time-lapse imaging experiments show that loss of Lis1/Gl in neuroblasts results in extension of both prometaphase and metaphase. Prometaphase in Lis1 mutant neuroblasts is characterized by delayed congression of chromosomes to the equatorial plate: this is likely to be largely due to inefficient kinetochore capturing as an indirect result of spindle assembly defects. Importantly, in Lis1 mutant neuroblasts congression of all chromosomes into a tight metaphase plate eventually occurs, suggesting that Lis1/Gl are not absolutely critical for MT/kinetochore attachment per se (Siller, 2005).

In addition, severe delays were observed in metaphase-to-anaphase transition. A few of these neuroblasts showed individual chromosomes that were transiently lost from and recongressed to the metaphase plate. Thus, consistent with findings in mammalian cells, Lis1 appears to play some role in maintaining stable chromosome alignment in metaphase neuroblasts. However, in contrast to mammalian studies, it was found that loss of Lis1 function causes delays in metaphase-to-anaphase transition even when all chromosomes stay aligned in a tight metaphase plate. Thus, mitotic checkpoint activity remains high even after apparent bipolar kinetochore attachment. Two defects appear to contribute to prolonged checkpoint activity in Lis1 mutant metaphase neuroblasts: reduced inter-kinetochore tension and failure to transport checkpoint proteins (e.g., Rod) off kinetochores. Reduced inter-kinetochore tension may be due to lack of Lis1/dynactin on kinetochores or on spindle pole/MTs (which may affect forces acting on kinetochore pairs as a consequence of altered spindle morphology or MT dynamics). Defects in Rod checkpoint protein transport off kinetochores can be explained as a direct consequence of depletion of kinetochore-associated Lis1/dynactin/dynein motor complex, which in wild type cells is loaded with Rod at kinetochores. However, previous studies have indicated that Rod and Zw10 are removed from kinetochores in response to inter-kinetochore tension not MT-attachment. Therefore, in addition to its direct role as a 'carrier', Lis1/dynactin/dynein may also play an indirect role in modulating Rod transport by generating the inter-kinetochore tension required to trigger initiation of Rod streaming (Siller, 2005).

In summary, the data is consistent with and extends a model recently proposed for dynein function in checkpoint protein transport in Drosophila and mammalian cells. According to this model a Lis1/dynactin/dyneinRod/Zw10 complex, pre-assembled on unattached kinetochores, is critical for timely anaphase onset by promoting poleward streaming of checkpoint proteins away from kinetochores after correct kinetochore-MT attachment has occurred. The data demonstrate that in Drosophila, the Lis1 protein is an obligate component in this process. Although the Lis1-binding proteins NudE/Nudel have been implicated in facilitating dynein-dependent checkpoint protein transport, it remains to be directly tested whether Lis1 has a similar function in mammalian cells (Siller, 2005).

What is the link between Rod/Zw10 and Mad2 in mitotic checkpoint function? Two recent studies demonstrate that the Rod/Zw10 complex is required for efficient recruitment of Mad2 to unattached kinetochores in mammalian cells and Drosophila neuroblasts, and that Mad2 and Rod colocalize during poleward transport along kMTs in Drosophila neuroblasts. Although a physical link between the Rod/Zw10 complex and Mad2 has not been discovered, an attractive model is that Rod/Zw10 links Mad2 to the Lis1/dynactin/dynein complex during poleward checkpoint protein transport (Siller, 2005).

Epistasis of Lis1/dynactin localization at kinetochores Lis1/dynactin localization is regulated differently in worm and mammalian cells. In mammalian cells, dynactin is required for Lis1 kinetochore association, but Lis1 is not required for dynactin localization. Whereas in C. elegans, Lis1 localizes to kinetochores independently of dynactin. Surprisingly, a third mechanism is found in Drosophila neuroblasts, where Lis1 and dynactin (Gl) are co-dependent for their localization to kinetochores. In neuroblasts, Lis1 may have a 'structural' role in recruiting dynein/dynactin to the kinetochore, in addition to stimulating dynein/dynactin activity. Thus, despite the conservation of the physical interaction between Lis1/dynein/dynactin, subcellular localization of these proteins can be regulated differently in various organisms (Siller, 2005).

The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization

Drosophila melanogaster cellularization is a dramatic form of cytokinesis in which a membrane furrow simultaneously encapsulates thousands of cortical nuclei of the syncytial embryo to generate a polarized cell layer. Formation of this cleavage furrow depends on Golgi-based secretion and microtubules. During cellularization, specific Golgi move along microtubules, first to sites of furrow formation and later to accumulate within the apical cytoplasm of the newly forming cells. Golgi movements and furrow formation depend on cytoplasmic dynein. Furthermore, Lava lamp (Lva), a golgin protein that is required for cellularization, specifically associates with dynein, dynactin, cytoplasmic linker protein-190 (CLIP-190) and Golgi spectrin, and is required for the dynein-dependent targeting of the secretory machinery. The Lva domains that bind these microtubule-dependent motility factors inhibit Golgi movement and cellularization in a live embryo injection assay. These results provide new evidence that golgins promote dynein-based motility of Golgi membranes (Papoulas, 2005).

Following thirteen mitotic nuclear divisions, Drosophila syncytial embryos undergo a form of cytokinesis called cellularization. The process occurs during the interphase of nuclear cycle 14 over a 1-h period and relies on both microtubules and Golgi-based secretion. During cellularization, microtubules emanate from apically positioned centrosomes to form 'inverted baskets' around each nucleus at the embryo's’surface. The furrows form between adjacent nuclei, generating a honey comb pattern, and ultimately expand laterally at their base and fuse to seal each nucleus off from the inner yolk, creating a multicellular embryo. Cellularization requires an increase in cell surface area of approximately 20-fold, and because the disruption of Golgi function inhibits furrowing, a significant amount of this membrane is believed to come from de novo secretion. A portion of the Golgi undergoes two waves of apically directed movements during furrow formation that are coordinated with the dynamic changes in microtubule organization (Papoulas, 2005).

Golgi body movements can be visualized by injecting live embryos with dilute Cy5-tagged anti-Lava-lamp antibody, which fluorescently labels a small number of Golgi near the site of injection and does not affect Golgi movement or cellularization. Injection of colchicine to depolymerize microtubules blocks the Golgi movements, whereas microfilament depolymerization with cytochalasin D does not, consistent with the idea that microtubule-based Golgi movements support active membrane secretion (Papoulas, 2005).

The mechanism that drives the microtubule-dependent Golgi movements is unknown; however, the trajectory of the Golgi movements suggests that cytoplasmic dynein is involved. Cytoplasmic dynein is a multisubunit minus-end-directed microtubule motor that is known to transport a variety of cargos in animal cells. Several studies have suggested that dynein can associate with mammalian Golgi through Golgi-associated spectrin and dynactin, a protein complex that is required for processive movement of dynein cargo. α-spectrin is present on Drosophila Golgi, raising the possibility that it could recruit dynein/dynactin (Papoulas, 2005).

To determine whether cytoplasmic dynein is responsible for the apically directed Golgi movements, Dynein heavy chain (Dhc) activity was inhibited and the effects on Golgi movement were monitored. The use of transgenic flies that express green fluorescent protein (GFP)-tagged Myosin II (MyoII-GFP) permitted the simultaneous visualization of the furrow tip. Injection of function-blocking anti-Dhc antibodies into live MyoII-GFP embryos disrupted Golgi movements, reducing the number of Golgi that move processively. In addition, disruption of Dhc function blocks the progression and organization of the furrow front. Injection of a control [anti-glutathione-Stransferase (GST)] antibody had no effect. Next, embryos were collected from wild-type or Dhc6 mutant females, fixed, and prepared for indirect immunofluorescence using antibodies against tubulin and Lva. Embryos derived from mutant females that develop to nuclear cycle 14 fail to undergo normal furrow formation. These mutant embryos also accumulate fewer Golgi bodies in the apical cytoplasm compared with wild-type embryos at the same stage, despite the fact that microtubules seem abundant and oriented appropriately to support apically directed Golgi movement (Papoulas, 2005).

To explore the possibility that dynein directly mediates the Golgi movements, it was determined whether Dhc localizes to Golgi bodies. Fixed cellularizing embryos were prepared for immunofluorescence using antibodies against Lva and Dhc. Dhc is abundant throughout the cortical cytoplasm, but areas of Dhc enrichment colocalize with Golgi-associated Lva staining. Corroborating the immunofluorescence results, a portion of membrane-associated dynein and a significant fraction of α-spectrin in membrane extracts are present in Golgi-enriched membrane fractions with Lva on density gradients. Some membrane-associated dynein also sediments with an unknown membrane population that is not associated with Lva (Papoulas, 2005).

The role of spectrin and the dynactin complex in the recruitment of dynein to mammalian Golgi and the previous observation that the golgin Lva and Golgi spectrin associate in Drosophila, prompted an investigation of whether Lva might have a direct role in recruiting and/or regulating dynein function. To determine whether Lva associates with dynein, immunoprecipitations were performed using protein extracts prepared from embryos. Anti-Lva antibodies coimmunoprecipitate α-spectrin, as expected, and also coimmunoprecipitate Dhc, whereas control immunoprecipitations do not. These data demonstrate that a portion of the endogenous Lva and dynein associate in soluble embryo extracts. In conjunction with the colocalization and membrane cofractionation results, these data suggest that Lva and dynein may interact on the surface of Golgi in vivo (Papoulas, 2005).

So far, nearly all dynein-based motility of membrane vesicles requires the dynactin complex, and may also be facilitated by microtubule plus-end tracking proteins (+TIPs). +TIPs bind growing microtubule plus-ends and are believed to regulate microtubule dynamics and the docking of membranes to microtubules. Consistent with this model, the +TIPs p150^Glued and CLIP-190 were detected in the Golgi-enriched membrane fractions. p150^Glued, a subunit of the dynactin complex, extensively cosediments with Lva; and although anti-p150^Glued antibodies coimmunoprecipitate Lva and Dhc, anti-Lva antibodies do not detectably coimmunoprecipitate p150^Glued under the conditions used. These observations are consistent with the possibility that antibody binding to Lva disrupts adjacent binding of dynactin and previous data that show that anti-Lva antibody injections disrupt Golgi movements in live embryos. CLIP-190 is the Drosophila orthologue of mammalian CLIP-170, which has been implicated in linking vesicles to microtubules for dynein-dependent transport. CLIP-190 is known to cofractionate and colocalize with Lva, and as expected, CLIP-190 and Lva coimmunoprecipitate. Taken together, the association of Lva with dynein, p150^Glued, CLIP-190 and spectrin strongly suggests a role for Lva in the microtubule-dependent movement of Golgi during cellularization (Papoulas, 2005).

The ability of different portions of Lva to interact with these microtubule motility factors and with Golgi membrane was tested. Seven contiguous fragments spanning the full length of Lva were expressed as GST-Lva fusion proteins and used to make affinity columns. Chromatography was performed with native extracts that were derived from cellularizing embryos. Dynein exclusively binds the globular carboxyl terminus of Lva (Lva5), whereas both p150^Glued and CLIP-190 bind the coiled-coil central portion of Lva (Lva3) and the globular C terminus. Furthermore, α-spectrin binds to Lva3, but not to the dynein-binding C terminus. Therefore, spectrin and dynactin by themselves are insufficient to recruit dynein under the in vitro conditions. Moreover, dynein binds the C terminus of Lva in the absence of spectrin. Interestingly, Drosophila BicD fails to bind any of the seven Lva segments or colocalize with Golgi-associated Lva by immunofluorescence, despite the established role of its orthologues in dynein recruitment to mammalian Golgi membrane. Membrane-binding assays were used to map the region(s) of Lva that are required for Golgi association. Golgi-enriched membrane preparations were incubated with each of the GST-Lva fusion proteins and then collected by centrifugation and assayed for the presence of Lva fusion proteins by anti-GST immunoblotting. Only GST-Lva2A and 2B were found to associate with Golgienriched membranes with high affinity. No fusion proteins pelleted in the absence of membrane (Papoulas, 2005).

To test the functional significance of the Lva interactions with microtubule motility factors Lva fusion proteins were injected into live D. melanogaster embryos and the effects on Golgi movement and furrow formation were monitored. Injection of GST or Lva1, which do not bind the microtubule motility factors, does not inhibit Golgi movement or furrowing. In contrast, injection of Lva3 or Lva5 severely impairs the Golgi movements and furrowing. The number of Golgi bodies that move is significantly reduced by Lva5 and virtually eliminated by Lva3. The Golgi movements that remain in each case are significantly delayed, occurring exclusively during the time period when the second wave of apical movement is normally observed. The impaired furrowing caused by injection of Lva3 and Lva5 occurs primarily during the late (fast) phase, suggesting that the early Golgi movements are a prerequisite for rapid membrane growth during this later phase. Injection of Brefeldin A (BFA), a potent inhibitor of membrane transport, has a similar effect on the fast phase of membrane growth3. Staining with the lectin concanavalin A reveals normal plasma membrane ruffles at the apical margin of furrows and some subcortical membrane during the slow phase in GST-injected embryos. This plasma membrane topology has been previously described in greater detail and coincides with proposed sites of exocytosis. However, this plasma membrane structure is severely disrupted in Lva3-injected embryos and discontinuity in plasma membrane furrows is evident. These results are consistent with the idea that Golgi targeting to the cell surface is required for new plasma membrane secretion to form cleavage furrows (Papoulas, 2005).

These biochemical experiments suggest that the dominant-negative effect of Lva3 on Golgi body movement might result from displacing endogenous dynactin and CLIP-190 from the Golgi surface. To test this the subcellular localizations of Dhc, CLIP-190 and Lva were studied in fixed embryos after injection with GST or Lva3. The uniform expression of p150^Glued in embryos prevented identification of any potential effect on dynactin localization, and Dhc localization was indistinguishable between GST- and Lva3-injected embryos, consistent with the absence of dynein binding to Lva3 in vitro. By contrast, the normal CLIP-190 Golgi-association is disrupted in Lva3-injected embryos, particularly at the furrow front during the slow phase of cellularization. GST-injected embryos are unaffected, and Lva remains Golgi-associated in Lva3-injected embryos; although, Golgi are more dispersed (Papoulas, 2005).

Thus, biochemical and functional analysis of Lva suggests that it has a unique function among golgin proteins. The data are consistent with an adaptor model in which Lva stimulates dynein-dependent Golgi movement by binding cytoplasmic dynein in association with promoters of dynein-dependent motility. This model is based in part on the observation that dynein-dependent Golgi movement and furrow formation are significantly inhibited by injections of Lva3, which has no detectable affinity for dynein. This dominant-negative effect can be explained as a consequence of displacing CLIP-190 from Golgi bodies, but is likely to also displace dynactin, resulting in diminished dynein function. The microtubule motility factors bound to the central domain of Lva could facilitate dynein function indirectly by capturing microtubule plus ends, as has been proposed for dynactin7. Alternatively, Lva's predicted hinge region could facilitate interactions between microtubule motility factors that are bound to the central region of Lva and the dynein motor that is bound to the C terminus, and/or modulate dynein catalytic activity or microtubule binding. These possibilities are not mutually exclusive, and whether Lva contributes to dynein recruitment in vivo remains an open question, as does the possibility that two distinct Lva/dynein complexes exist, one containing dynactin and the other CLIP-190. Future studies will be required to distinguish between these possibilities and to assess the role of Lva in other developmental contexts (Papoulas, 2005).

Dynactin/Dynein is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons

How a nucleus is positioned within a highly polarized postmitotic animal cell is not well understood. The Dynactin complex (a regulator of the microtubule motor protein Dynein) has been shown to be required to maintain the position of the nucleus within post-mitotic Drosophila photoreceptor neurons. Multiple independent disruptions of Dynactin function cause a relocation of the photoreceptor nucleus toward the brain, and inhibiting Dynactin causes the photoreceptor to acquire a bipolar appearance with long leading and trailing processes. It has been found that while the minus-end directed motor Dynein cooperates with Dynactin in positioning the photoreceptor nucleus, the plus-end directed microtubule motor Kinesin acts antagonistically to Dynactin. These data suggest that the maintenance of photoreceptor nuclear position depends on a balance of plus-end and minus-end directed microtubule motor function (Whited, 2004).

The Dynactin complex is an assembly of 11 different subunits that functions as an activator of Dynein, serving as an adaptor for cargo and enhancing motor processivity. The Dynactin subunit Glued couples Dynactin to Dynein by binding to the Dynein intermediate chain (Dic, encoded by short wing). Overexpression of a truncated form of Glued that binds to Dic but cannot associate with the rest of the Dynactin complex acts as a powerful inhibitor of Dynein and Dynactin function. Overexpression of the Dynactin subunit Dynamitin disrupts Dynactin complex assembly and also inhibits Dynactin function. Biochemical studies have shown that the Dynactin complex also contains Capping Protein, a heterodimer composed of the Capping Protein alpha (Cpa) and Capping Protein beta (Cpb) subunits. Although best known for capping the barbed ends of filaments of actin, Capping Protein also associates with filaments of the actin-related Arp1 protein, which is a central element of the Dynactin complex (Whited, 2004 and references therein).

Patterning of the adult compound eye of Drosophila initiates during the third instar phase of larval life, and mutations in the Dynactin subunit Glued strongly disrupt eye development. Normally the nuclei of differentiating photoreceptors occupy apical regions of the eye disc. In animals heterozygous for the dominant-negative Glued allele Glued¹, many photoreceptor nuclei have been shown to accumulate within basal regions of the eye disc. The effect of Glued¹ on photoreceptor development was characterized using an antibody recognizing photoreceptor cell surfaces. In wild type, the region of the differentiating photoreceptor neuron containing the nucleus remained in the retina, while the photoreceptor axon extended through the optic stalk into the brain. However, in Glued¹ animals, while photoreceptors still extended axons into the brain, the region of the photoreceptor containing the nucleus often appeared to leave the retina and travel through the optic stalk into the brain. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in Glued¹ mutants (Whited, 2004).

To further establish that Glued¹ defects reflected disruptions in Dynactin function, two other approaches were used to disrupt the Dynactin complex. Drosophila Dynamitin, which also inhibits Dynactin function in flies, was overexpressed in photoreceptor neurons. Loss-of-function mutations in the Dynactin subunit Cpb were examined by generating animals whose visual systems contained homozygous mutant clones of the cpb strong loss-of-function mutation cpb^M143. In these cpb^M143 mosaic animals, the nuclear regions of many photoreceptors were observed in the optic stalk and brain (Whited, 2004).

To confirm that the cpb^M143 mutant photoreceptor defect was due to a loss of cpb function, an additional strong loss-of-function cpb allele, cpb^F44, was isolated from an EMS mutagenesis and a chromosomal deficiency uncovering the cpb locus, Df(2L)E.2, was obtained. When animals contained homozygous mutant clones of cpb^F44 cells or homozygous mutant clones of Df(2L)E.2, a similar movement of photoreceptor nuclear regions toward the brain was observed. cpb/Df(2L)E.2 animals did not survive to third instar, preventing the classic genetic demonstration that these cpb alleles behaved as strong loss-of-function mutations. Fortunately, it was found that the [pYES-ß] genomic transgene, which contains the CPB coding region, was able to rescue the lethality of cpb/Df(2L)E.2 animals, but did not rescue the previously described cpb bristle defect. This suggested that [pYES-ß] was a partially functional rescue construct that could be used to examine the visual systems of otherwise cpb/Df(2L)E.2 animals. It was found that [pYES-ß];cpb^M143/Df(2L)E.2 animals display a photoreceptor defect similar to that of other cpb mutants, consistent with nuclear mispositioning resulting from the loss of cpb function. It was further confirmed that the defect was due to the loss of cpb function by successfully rescuing the cpb^M143/Df(2L)E.2 photoreceptor defects (as well as the cpb bristle defects) by expression of a wild-type Cpb cDNA under the control of a heterologous promoter. Staining of photoreceptor nuclei directly demonstrated the movement of photoreceptor nuclei out of the eye disc and into the optic stalk in cpb mutants (Whited, 2004).

The bifunctional nature of Cpb, which associates with filaments of actin as well as filaments of Arp1, means that loss of Cpb also increases filamentous actin levels (Hopmann, 2003). Nonetheless, previous studies have shown that increases in filamentous actin alone, such as those observed in hypomorphic cpb alleles or in actup mutants, do not cause photoreceptor nuclear mispositioning. Together with the Glued¹ and Dynamitin data, the cpb observations yield a consistent picture that alterations in Dynactin subunits cause mispositioning of photoreceptor cell bodies and nuclei, and indicate that Dynactin, and not just the Glued subunit, has an important role in photoreceptor development (Whited, 2004).

The mispositioning of photoreceptor nuclei in Dynactin mutants raised the question of whether these disruptions reflect altered positioning of the nucleus within the photoreceptor or simply migration of the entire photoreceptor. To address this question, single photoreceptors were labeled in wild type and in Glued¹ mutants. Wild-type photoreceptors exhibit a highly polarized morphology in which the region of the photoreceptor containing the nucleus lies in the apical region of the eye disc and an axon extends basally into the brain. Glued¹ mutant photoreceptors whose nuclei have entered the optic stalk had highly altered morphologies, with both leading and trailing processes extending from the regions of the cell where the misplaced nucleus was located. Leading and trailing processes of misplaced Glued¹ photoreceptors were quantified, considering only those with no other labeled cells or processes nearby. Of these 13 neurons, 12 had clearly detectable leading and trailing processes. The leading process (axon) extended into the target region and the trailing process extended back into the eye disc. These data demonstrate that inhibition of Dynactin function dramatically alters the position of the nucleus within the photoreceptor (Whited, 2004).

The Dynactin complex also controls the pattern of mitoses within the Drosophila retina. To determine whether nuclear mispositioning is a secondary consequence of the earlier mitotic requirement for Dynactin, the effects of specifically inhibiting the Dynactin complex in postmitotic photoreceptors was examined. Conditional inhibition of Dynactin function can be achieved through inducible expression of a truncated, dominant-negative form Glued (Glued^DN) that resembles the protein product of Glued¹. Glued^DN was expressed under the control of the postmitotic photoreceptor-specific Glass 38-1 promoter, which initiates expression in the photoreceptors only after their axons have entered the brain. Expression of Glued^DN under the control of Glass 38-1 caused photoreceptor nuclei to move into the optic stalk. Overexpression of Dynamitin under the control of Glass 38-1 caused similar photoreceptor nuclear positioning defects. These data demonstrate that Dynactin is required postmitotically in photoreceptors to maintain nuclear position and that the disruptions in nuclear positioning observed are not simply a secondary consequence of mitotic defects (Whited, 2004).

The displacement of photoreceptor nuclei from apical regions of the eye disc toward more basal regions could reflect an overall disruption in apical/basal polarity of the eye disc. The apical/basal polarity of developing photoreceptors was assessed by examining the distribution of the Drosophila ß-catenin Armadillo and the PDZ-domain-containing protein PATJ. Armadillo localizes to the zonula adherens separating the apical and basolateral membrane domains of developing photoreceptors, while PATJ localizes to the apical membrane domain. In wild-type eye discs, Armadillo is concentrated just beneath the apical tips of the developing photoreceptors. In Glued¹ animals Armadillo was still present near apical regions of the eye disc, even in areas completely devoid of apical photoreceptor nuclei. Thus, this marker of apical/basal polarity was retained even when photoreceptor nuclei moved basally. Similar results were obtained when Glued¹ mutants were visualized in cross-section using both Armadillo and PATJ. Apical localization of PATJ and Armadillo were observed in Glued¹ and the relative apical/basal ordering of these markers was maintained. These data suggest that the alterations in photoreceptor morphology are not caused by a loss of apical/basal polarity within the developing photoreceptors (Whited, 2004).

Dynactin has important functions in the organization of the microtubule cytoskeleton in many systems. The microtubule cytoskeleton of developing photoreceptors is highly polarized, with microtubule minus ends concentrated apical to the nucleus as detected using antisera recognizing gamma-tubulin. A similar apical focus is observed when using the fusion protein Nod:LacZ, which often co-localizes with microtubule minus ends. The relatively ubiquitous expression of gamma-tubulin in the retina complicated the analysis of gamma-tubulin localization when retinal patterning was disrupted. Therefore, the effect of Glued on factors associated with the microtubule cytoskeleton was examined by expressing Nod:LacZ specifically in postmitotic photoreceptors. In animals expressing Glued^DN in postmitotic photoreceptors as well as in Glued¹ mutants, Nod:LacZ was no longer exclusively concentrated in apical regions of photoreceptors, but rather spread into the photoreceptor axons. Thus, while the overall apical/basal polarity of the photoreceptors was not disrupted in Glued mutants, the spatial organization of the microtubule cytoskeleton-associated factor Nod:LacZ was affected (Whited, 2004).

Dynactin activates the microtubule motor Dynein, and strong loss-of-function mutations in dynein intermediate chain (dic) are dominant enhancers of the rough eye phenotype of Glued¹ mutants. Since Dynein and Dynactin may play multiple roles together during eye development, the effect of a reduction in dic gene dosage upon photoreceptor nuclear positioning was examined in Glued¹ animals. A twofold reduction in dic gene dosage caused a further decrease in the number of photoreceptor nuclei in apical regions of Glued¹ mutant eye discs. This did not reflect a simple reduction in the number of photoreceptors generated; large numbers of photoreceptor nuclei were crowded at the base of the eye disc and entered the optic stalk in both animals. Thus, a larger fraction of photoreceptor nuclei left apical positions when the level of dic gene activity was reduced, consistent with Dynein and Dynactin acting together in this process (Whited, 2004).

To identify additional factors that interact with Dynactin to control nuclear positioning, a genetic screen was performed to identify genes that dominantly enhanced or suppressed the Glued¹ external eye phenotype. From a collection of approximately 1800 stocks containing transposon-induced lethal mutations, several stocks were identified that had no dominant effect on eye development in a wild-type background, but were dominant enhancers or suppressors of Glued¹. Two dominant suppressors of Glued¹, khc^k13219 and khc^k13314, were alleles of kinesin heavy chain (khc), which encodes a subunit of the plus-end directed microtubule motor kinesin. The interaction with Glued¹ was further confirmed using the null allele khc⁸. Examination of developing eye discs demonstrated that a twofold reduction of khc gene dosage greatly increased the number of photoreceptor nuclei present in apical regions of Glued¹ mutant eye discs. This suggested that khc acts antagonistically to Glued in photoreceptor nuclear positioning (Whited, 2004).

To determine whether khc mutations interacted with Glued¹ in postmitotic photoreceptors, khc gene dosage was reduced in animals expressing dominant-negative Glued under the control of the postmitotic Glass_38-1 promoter. Wild-type animals (n >50 hemispheres) or animals containing the dominant-negative Glued transgene without the Glass 38-1 promoter never contained photoreceptor nuclei within their optic stalks. Glass_38-1:Glued^DN animals contained an average of 11±1 photoreceptor nuclei within the optic stalk. However, Glass_38-1:Glued^DN animals heterozygous for either khc^k13314 or khc⁸ showed a significant reduction in the number of photoreceptor nuclei in the optic stalk. Thus, a twofold reduction in khc gene dosage suppressed the effects of postmitotic expression of dominant-negative Glued, consistent with Glued and khc acting antagonistically within differentiated photoreceptors to regulate nuclear positioning (Whited, 2004).

The interaction between Glued and khc in other photoreceptors was studied by examining the Bolwig organ, a cluster of 12 photosensitive neurons that differentiate during embryonic development and extend axons into the brain. By second and third instar larval stages, Bolwig photoreceptor nuclei are located near the anterior tip of the larva and their axons extend over the eye/antennal disc into the brain, a distance of >200 µm. In wild-type second instar animals, photoreceptor neuron differentiation has not yet begun in the eye disc and no neuronal nuclei are present there. However, when Glued^DN was expressed in postmitotic Bolwig photoreceptors, their nuclei appeared on the surface of the eye/antennal disc. Thus, as in the photoreceptors of the adult eye, expression of Glued^DN in Bolwig photoreceptors caused their nuclei to be positioned closer to their axon termini; in many cases, the Bolwig nuclei were over 150 µm closer than normal to their axon terminals in the brain (Whited, 2004).

The interaction between Glued and khc in Bolwig photoreceptors was assessed by counting the number of Bolwig nuclei on the surface of the eye/antennal disc. While wild-type and UAS:Glued^DN animals had no neuronal nuclei in this region, Glass_38-1:Glued^DN animals contained 7±1. A reduction of khc gene dosage in Glass_38-1:Glued^DN; khc^k13314/+ and Glass_38-1:Glued^DN; khc⁸/+ animals significantly reduced this to 4±1 and 3±1, respectively. These data further support the functional antagonism of Glued and khc in photoreceptor nuclear positioning (Whited, 2004).

The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Rhia arusy clarifies the interaction between EB1 and dynactin and shows that EB1 binds directly to the N-terminus of the p150^Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150^Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150^Glued on microtubule polymerization, and they show that p150^Glued has a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics (Ligon, 2003).

Dynactin is necessary for synapse stabilization

Evidence is presented that synapse retraction occurs during normal synaptic growth at the Drosophila neuromuscular junction (NMJ). An RNAi-based screen to identify the molecular mechanisms that regulate synapse retraction identified Arp-1/centractin, a subunit of the dynactin complex. Arp-1 dsRNA enhances synapse retraction, and this effect is phenocopied by a mutation in P150/Glued, also a dynactin component. The Glued protein is enriched within the presynaptic nerve terminal, and presynaptic expression of a dominant-negative Glued transgene enhances retraction. Retraction is associated with a local disruption of the synaptic microtubule cytoskeleton. Electrophysiological, ultrastructural, and immunohistochemical data support a model in which presynaptic retraction precedes disassembly of the postsynaptic apparatus. These data suggests that dynactin functions locally within the presynaptic arbor to promote synapse stability (Eaton, 2002).

Evidence is presented that regulated synaptic growth at the Drosophila NMJ includes synaptic retraction events in addition to the well-characterized addition of new synaptic boutons. Synaptic retraction events (footprints) are defined as the withdrawal of presynaptic antigens (synapsin, HRP, Futsch) from clearly defined regions of postsynaptic specialization defined by Discs-large immunoreactivity. Multiple lines of evidence, using a variety of analytic tools, including light level and ultrastructural analysis, are presented that a footprint represents a site where the nerve terminal once resided and has since retracted. Previous reports have likely failed to identify synaptic retraction as an important element during synapse development in this system because postsynaptic markers were employed to study synapse development. Synapse retraction events are more frequent at early developmental stages which correlate with the more rapid phase of synapse growth. These data suggest that regulated synaptic growth is achieved by a balance of synaptic growth and retraction at the Drosophila NMJ. Such a balance of growth and retraction may represent a general principle of synaptic growth control in this and other systems (Eaton, 2002).

Employing a functional genomic strategy, the dynactin protein complex has been identified as an essential component of the machinery that achieves synapse stabilization at the Drosophila NMJ. Disruption of the dynactin complex using any of three different perturbations, including Arp-1 RNAi, the Glued1 mutation, or presynaptic overexpression of the DN Glued transgene all result in an increase in the frequency and extent of synaptic retraction events at the NMJ. Presynaptic, but not postsynaptic, overexpression of the DN Glued transgene enhances synapse retraction, phenocopying genetic and RNAi perturbation of the dynactin complex. Consistent with this observation, P150/Glued protein is present at the NMJ and enriched in the presynaptic nerve terminal (Eaton, 2002).

Several lines of evidence suggest that dynactin functions locally within the presynaptic nerve terminal to control synapse stability. Retraction events do not result in the complete elimination of a synapse, but are generally restricted to a specific branch or portion of a branch within the synaptic arbor of a single motoneuron. In addition, MN 6/7 innervates multiple muscle targets, and retraction events are often specific to only one or a few of the muscle targets of this neuron, demonstrating that retraction events can be branch specific on different muscle targets (Eaton, 2002).

An obvious concern is that inhibition of dynactin function sufficiently impairs the health of the motoneuron to cause a secondary retraction of the synapse. Ultimately, an assessment of 'health' can only be achieved by assaying a number of independent variables such as electrophysiology, ultrastructure, morphology, and cell death. Synapse retraction events are not correlated with increased cell death in the motoneurons nor are they correlated with gross changes in the motoneuron microtubule cytoskeleton. Synapse retraction events can be local, affecting only a portion of a motoneuron arbor (demonstrated by analysis at both the light and ultrastructural level). In addition, motoneuron transmitter release properties are normal at a portion of the synapses that overexpress the DN Glued transgene (the most severe manipulation used). This is consistent with the observation that only a portion (40%) of the synapses reveal a retraction event despite the fact that DN Glued is expressed pan neuronally. These data argue against DN Glued simply poisoning the cell. Finally, retraction events are observed in the wild-type animal and occur with higher frequency during early larval development. Taken together, these data suggest that retraction is associated with synapse development rather than impaired health (Eaton, 2002).

The data indicate that increased synapse retraction caused by impaired dynactin activity has a functional consequence for the synapse. Increased synaptic retraction results in fewer synaptic boutons and decreased synaptic efficacy. This is predicted if synapse retraction helps to shape the outcome of synapse development. Thus, the increased frequency of retraction events observed during early synapse development (rapid growth) may be an important aspect of synaptic growth control. It is hypothesized, therefore, that presynaptic dynactin-mediated synapse stabilization may help set the balance between growth and retraction during normal synaptic development (Eaton, 2002).

Synaptic retraction caused by disruption of the dynactin complex has been characterized at the light level, ultrastructurally and electrophysiologically. At each level of analysis, the data support the conclusion that the presynaptic nerve terminal withdraws, followed by the disassembly of the postsynaptic apparatus. At the light level, retraction of presynaptic markers precedes the elimination of postsynaptic Discs-large staining and GluR clusters. In addition, no change was observed in quantal size when DN Glued is driven presynaptically, despite often severely impaired presynaptic release. By comparison, acetylcholine receptors are removed postsynaptically prior to presynaptic elimination at the vertebrate NMJ, and a decrease in quantal size is observed. Finally, it was observed ultrastructurally that the presynaptic membrane appears to separate from the postsynaptic density with high frequency at active zones when dynactin is disrupted presynaptically. This is again consistent with presynaptic retraction preceding disassembly of the postsynaptic apparatus (Eaton, 2002).

The dynactin protein complex binds microtubules and has been shown to bind a number of proteins that localize to the plus end of microtubules including Lis1, Eb1, and Clip170. In addition to their localization, these proteins have also been shown to effect microtubule stability and dynamics. In budding yeast, live imaging studies of microtubules have demonstrated that mutations in dynactin and EB1 lead to altered microtubule structure and defects in cortical association. Based on these observations, one hypothesis is that dynactin and associated proteins participate in the process of microtubule capture at the cell cortex. Alternatively, dynactin could be involved in more complex regulation of microtubule dynamics via the localization or trafficking of microtubule regulators (Eaton, 2002).

Dynactin regulation of the microtubule cytoskeleton is thought to occur at sites of intercellular adhesion and signaling. In epithelial cells, microtubules and cytoplasmic dynein have been observed to contact adherens junctions. In addition, overexpression of the p62 component of the dynactin complex localizes strongly to sites of focal adhesion. Furthermore, dynactin has been shown to bind proteins such as β-catenin and spectrin, which are known to be involved in cell adhesion (Eaton, 2002).

In Drosophila, dynactin has been studied during axon guidance. In dynactin mutations, axons reach their target regions but fail to branch normally. It was not determined whether synapses failed to form in these studies or whether synapse stability was disrupted. These data demonstrate that dynactin is not necessary for growth cone motility. The interpretation of these data has been a failure to transport important trophic molecules in a retrograde manner to the sensory neuron cell body via the dynein retrograde motor. However, these data are equally consistent with a failure of sensory neuron synapse stability within the central nervous system (Eaton, 2002).

The data at the Drosophila NMJ are consistent with an essential role for the dynactin complex in maintaining the appropriate microtubule architecture and, as a consequence, synapse stability. The possibility cannot be ruled out that an essential retrograde signal fails to reach the motoneuron soma. However, since retraction events are observed to affect only portions of a single synapse and since no change is seen in motoneuron cell death, it is suspected that failure to traffic an essential retrograde signal is not the primary cause of synapse retraction at the Drosophila NMJ (Eaton, 2002).

An important aspect of the current data is a possible link between synapse retraction and neural diseases with which dynactin function has been associated. Mutations in the human lis1 gene cause type 1 lissencephaly, a debilitating developmental disease of the nervous system characterized by severe mental retardation and reduced cerebral folds. Interestingly, in addition to defects in neuroblast proliferation and axonal transport, Drosophila Lis1 mutants have abnormal dendritic morphology. In addition, dynactin binds Huntingtin Associated Protein 1 (Hap-1) and is thought to play a role in the trafficking of this protein. The current data are most consistent with a local role for dynactin within the nerve terminal controlling synapse stabilization. With respect to vertebrate disease states, the altered control of synapse stability has not been fully investigated. As such, this aspect of dynactin function characterized is this study may be an important component of disease progression if affected cells lose essential target-derived trophic support as a result of impaired synapse stability (Eaton, 2002).

Dynein is regulated by the stability of its microtubule track

How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, spindle movement was found to be regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. Dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued. These results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. It is proposed that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning (Estrem, 2017).

Dynein is necessary for spindle movement in many cellular contexts, but the mechanisms that regulate dynein to control the accuracy of spindle positioning are poorly defined. This study provides three new insights into how dynein is regulated in budding yeast to accurately position the spindle at the nascent site of cytokinesis. First, microtubule stability is shown to be a limiting factor of the initiation and duration of dynein-dependent spindle movement. Second, evidence is provided that dynein combines both lateral sliding and plus-end destabilization activities to position the spindle, and both require the motor activity of dynein heavy chain. Third, dynactin plays a key role in stabilizing microtubules. It is proposed that the dynactin complex regulates the balance of dynein's activities by stabilizing microtubules, thereby promoting microtubule-cortex interactions and transitioning those interactions into force production for spindle movement (Estrem, 2017).

A previous study showed that yeast dynein heavy chain attached to a fabricated barrier can capture and destabilize microtubule plus ends in vitro. Comparing this study to the current results in yeast points to interesting similarities as well as differences. In both scenarios, microtubules that interact with dynein at the cortex exhibit dramatic increases in the frequency of catastrophes. Furthermore, in both scenarios, dynein's catalytic activity is necessary to destabilize microtubules. How dynein promotes catastrophe is an outstanding question. A previous study attributed the increased catastrophe frequency to dynein holding the plus end against the cortical barrier and preventing access to free tubulin subunits. Although a similar occlusion mechanism may contribute to catastrophe in yeast, this model is not sufficient to explain the results. First, this analysis comparing plus-end interactions at the cortex in num1Δ and dyn1Δ mutants indicates that dynein does not need to attach to the cortex to destabilize microtubule ends. Both mutants exhibit more frequent plus-end interactions at the cortex; however, these interactions are shorter lived in the num1Δ mutant than in the dyn1Δ mutant. This suggests that dynein at the plus end in the num1Δ mutants may still destabilize the microtubules, albeit to a lesser degree than normal. Second, sliding microtubules exhibit more frequent catastrophes, even though the plus ends are presumably parallel to the cell cortex as they move along it. Therefore, free tubulin subunits would be expected to have better access to the plus end of a sliding microtubule, and a catastrophe by a subunit occlusion model seems unlikely. An alternative model is favored, where dynein motor activity promotes catastrophe by directly destabilizing the microtubule lattice (Estrem, 2017).

Although dynein destabilizes microtubules in yeast, it is unlikely that this contributes force for spindle movement. This study finds that dynein-dependent spindle movement is initiated by polymerizing astral microtubules. In the majority of cases, these microtubules continue to polymerize as they slide along the cortex and move the spindle and remain in a polymerizing state for a mean of 12 s before undergoing catastrophe and switching to depolymerization. This indicates that polymerizing microtubules support spindle movement. In addition, this study found that increasing microtubule stability with mutants or drugs that inhibit depolymerization increases both the frequency and duration of spindle movement. These results are reminiscent of previous findings in C. elegans, where loss of the cortical protein EFA-6 increases the stability of astral microtubules and leads to increased cortical pulling forces by dynein, and studies of nuclear movements in Aspergillus mutants that alter microtubule dynamics. A simple interpretation of these results is that microtubule depolymerization is not required to move the spindle. This argues that microtubule depolymerization does not provide the energy needed for spindle movement, as has been proposed for kinetochore movement within the spindle. Instead, the results argue that stable microtubules promote spindle movement by enhancing lateral microtubule sliding by dynein. It is speculated that stable microtubules could enhance sliding either by providing more binding sites for dynein to move along the lateral sides of the microtubule and/or by enabling the microtubule to withstand stresses at the cell cortex, e.g., bending and force from dynein motility (Estrem, 2017).

Dynactin is required for virtually all known cellular functions of cytoplasmic dynein and is regarded as an obligate positive regulator. Typically, null mutations in dynactin subunits elicit phenotypes that are equivalent to null mutations in dynein. However, this study found that dynein and dynactin have opposite effects on microtubule stability. In contrast to heavy chain mutants, null mutations in the Nip100/p150glued subunit of dynactin diminish the frequency of microtubule-cortex interactions and lead to abnormally short astral microtubules. Importantly, double mutants combining null alleles of dynein heavy chain and NIP100 also exhibit shorter astral microtubules. Therefore, NIP100 is epistatic to dynein heavy chain with regard to astral microtubule stability, and functional Nip100 must be required to generate the longer astral microtubules found in the single dynein heavy chain null. These results support a model in which dynactin has a proximal role in stabilizing astral microtubules (Estrem, 2017).

The results indicate that astral microtubules are stabilized by the shoulder subcomplex of dynactin. The shoulder was first identified in electron microscopy analysis of purified vertebrate dynactin, where it lays across a short filament of the actin-related protein Arp1 (Schafer, 1994; Chowdhury, 2015; Urnavicius, 2015). The shoulder contains the subunits Nip100/p150glued, Jnm1/p50, and Ldb18/p24. The Nip100/p150glued subunit contains an N-terminal CAP-Gly domain, which binds to microtubules and suppresses catastrophes in vitro. This study found that mutants lacking the CAP-Gly domain exhibit shorter microtubules, similar to the nip100Δ null. Furthermore, mutants lacking the Jnm1/p50 subunit exhibit the same phenotype. Interestingly, mutants lacking the Arp1 filament exhibit longer microtubules, reminiscent of dynein-null mutants. This indicates that the shoulder stabilizes microtubules through a mechanism that involves the CAP-Gly domain of Nip100/p150glued but does not require the Arp1 filament. Although the shoulder is known to form a stable complex on its own, this study is the first evidence of an independent cellular function for the shoulder (Estrem, 2017).

It is proposed that a balance of dynactin and dynein activities controls the initiation and magnitude of spindle movement. Dynein and dynactin are proposed to be first recruited to the plus ends of astral microtubules, with an approximate stoichiometry of two dynein motors for every dynactin complex. Dynactin stabilizes the plus end, promoting microtubule polymerization that eventually delivers dynein-dynactin to the cortex. At the cortex, 1:1 complexes of dynein/dynactin offload by binding to Num1 and begin to move along the microtubule toward the minus end. In this way, dynein motility depletes dynactin's stabilizing activity from the plus end. Because the plus end begins with a two-times excess of dynein to dynactin, offloading equal amounts of dynein and dynactin causes an increase in the relative amount of dynein to dynactin that remains at the plus end. This shifts the balance toward the destabilizing activity of dynein and leads to catastrophe. Then, the microtubule depolymerizes at a rate of 2.3 μm/min, which is 1.4× faster than dynein motility. This faster rate may allow depolymerization to catch up with dynein-dynactin along the microtubule lattice and terminate spindle movement by destroying its microtubule substrate. In summary, the model proposes that the activation of dynein motility creates a negative feedback that leads to destruction of its microtubule track by titrating dynactin away from the plus end (Estrem, 2017).

Regulation of dynactin through the differential expression of p150Glued isoforms

Cytoplasmic dynein and dynactin interact to drive microtubule-based transport in the cell. The p150Glued subunit of dynactin binds to dynein, and directly to microtubules. This study has identified alternatively spliced isoforms of p150Glued that are expressed in a tissue-specific manner and which differ significantly in their affinity for microtubules. Live cell assays indicate that these alternatively spliced isoforms also differ significantly in their microtubule plus end-tracking activity, suggesting a mechanism by which the cell may regulate the dynamic localization of dynactin. To test the function of the microtubule-binding domain of p150Glued, RNAi was used to deplete the endogenous polypeptide from HeLa cells, followed by rescue with constructs encoding either the full-length polypeptide or an isoform lacking the microtubule-binding domain. Both constructs fully rescued defects in Golgi morphology induced by depletion of p150Glued, indicating that an independent microtubule-binding site in dynactin may not be required for dynactin-mediated trafficking in some mammalian cell types. In neurons, however, a mutation within the microtubule-binding domain of p150Glued results in motor neuron disease; this study investigated the effects of four other mutations in highly conserved domains of the polypeptide (M571T, R785W, R1101K, and T1249I) associated in genetic studies with Amyotrophic Lateral Sclerosis. Both biochemical and cellular assays reveal that these amino acid substitutions do not result in functional differences, suggesting that these sequence changes are either allelic variants or contributory risk factors rather than causative for motor neuron disease. Together, these studies provide further insight into the regulation of dynein-dynactin function in the cell (Dixit, 2008).

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. This study used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. The results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. The CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, the experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. The data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150^Glued subunit. CLIP-170 mutants alter p150^Glued localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin (Goodson, 2003).

The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1)

p150Glued was first identified as a polypeptide that copurifies with cytoplasmic dynein, the minus-end-directed microtubule-based motor protein, and has more recently been shown to be present as a member of the oligomeric dynactin complex, which includes the actin-related protein centractin (Arp-1). Dynactin is thought to mediate dynein-driven vesicle motility, as well as nuclear transport, in lower eukaryotes. The mechanism by which dynactin may function in these cellular processes is unknown. To examine the role of the dynactin complex in vivo, this study overexpressed the rat cDNA encoding p150Glued in Rat-2 fibroblasts. Overexpression of full-length, as well as C-terminal deletion, constructs resulted in the decoration of microtubules with the p150Glued polypeptides. This cellular evidence for microtubule association was corroborated by in vitro microtubule-binding assays. Amino acids 39-150 of p150Glued were determined to be sufficient for microtubule association. The possibility of a direct interaction between p150Glued and centractin was tested. In vitro translated centractin was specifically retained by a p150Glued affinity column, and this interaction was blocked by a synthetic peptide which corresponds to a highly conserved motif from the C terminus of p150Glued. These results demonstrate that p150Glued, a protein implicated in cytoplasmic dynein-based microtubule motility, is capable of direct binding to both microtubules and centractin (Waterman-Storer, 1995).

Search PubMed for articles about Drosophila Dynactin

Amrute-Nayak, M. and Bullock, S. L. (2012). Single-molecule assays reveal that RNA localization signals regulate dynein-dynactin copy number on individual transcript cargoes. Nat Cell Biol 14(4): 416-423. PubMed ID: 22366687

Ayloo S., Lazarus J.E., Dodda A., Tokito M., Ostap E.M., Holzbaur E.L. (2014). Dynactin functions as both a dynamic tether and brake during dynein-driven motility. Nat. Commun. 5:4807

Bulgari, D., Zhou, C., Hewes, R. S., Deitcher, D. L. and Levitan, E. S. (2014). Vesicle capture, not delivery, scales up neuropeptide storage in neuroendocrine terminals. Proc Natl Acad Sci U S A 111(9): 3597-3601. PubMed ID: 24550480

Cavolo, S. L., Zhou, C., Ketcham, S. A., Suzuki, M. M., Ukalovic, K., Silverman, M. A., Schroer, T. A. and Levitan, E. S. (2015). Mycalolide B dissociates dynactin and abolishes retrograde axonal transport of dense-core vesicles. Mol Biol Cell 26(14): 2664-2672. PubMed ID: 26023088

Cavolo, S. L., Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2016). Activity induces Fmr1-sensitive synaptic capture of anterograde circulating neuropeptide vesicles. J Neurosci 36(46): 11781-11787. PubMed ID: 27852784

Chang, L., Kreko, T., Davison, H., Cusmano, T., Wu, Y., Rothenfluh, A. and Eaton, B. A. (2013). Normal dynactin complex function during synapse growth in Drosophila requires membrane binding by Arfaptin. Mol Biol Cell 24(11): 1749-1764, S1741-1745. PubMed ID: 23596322

Chowdhury, S., Ketcham, S. A., Schroer, T. A. and Lander, G. C. (2015). Structural organization of the dynein-dynactin complex bound to microtubules. Nat Struct Mol Biol 22(4): 345-347. PubMed ID: 25751425

Dix, C. I., Soundararajan, H. C., Dzhindzhev, N. S., Begum, F., Suter, B., Ohkura, H., Stephens, E. and Bullock, S. L. (2013). Lissencephaly-1 promotes the recruitment of dynein and dynactin to transported mRNAs. J Cell Biol 202(3): 479-494. PubMed ID: 23918939

Dixit, R., Levy, J. R., Tokito, M., Ligon, L. A. and Holzbaur, E. L. (2008). Regulation of dynactin through the differential expression of p150Glued isoforms. J Biol Chem 283(48): 33611-33619. PubMed ID: 18812314

Eaton, B. A., Fetter, R. D. and Davis, G. W. (2002). Dynactin is necessary for synapse stabilization. Neuron 34(5): 729-741. PubMed ID: 12062020

Estrem, C., Fees, C. P. and Moore, J. K. (2017). Dynein is regulated by the stability of its microtubule track. J Cell Biol 216(7): 2047-2058. PubMed ID: 28572117

Farrer, M. J., Hulihan, M. M., Kachergus, J. M., Dachsel, J. C., Stoessl, A. J., Grantier, L. L., Calne, S., Calne, D. B., Lechevalier, B., Chapon, F., Tsuboi, Y., Yamada, T., Gutmann, L., Elibol, B., Bhatia, K. P., Wider, C., Vilarino-Guell, C., Ross, O. A., Brown, L. A., Castanedes-Casey, M., Dickson, D. W. and Wszolek, Z. K. (2009). DCTN1 mutations in Perry syndrome. Nat Genet 41(2): 163-165. PubMed ID: 19136952

Folker, E. S., Schulman, V. K. and Baylies, M. K. (2012). Muscle length and myonuclear position are independently regulated by distinct Dynein pathways. Development 139(20): 3827-3837. PubMed ID: 22951643

Goodson, H. V., Skube, S. B., Stalder, R., Valetti, C., Kreis, T. E., Morrison, E. E. and Schroer, T. A. (2003). CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms. Cell Motil Cytoskeleton 55(3): 156-173. PubMed ID: 12789661

Hayashi, I., Wilde, A., Mal, T. K. and Ikura, M. (2005). Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex. Mol Cell 19(4): 449-460. PubMed ID: 16109370

Heber, S., McClintock, M. A., Simon, B., Mehtab, E., Lapouge, K., Hennig, J., Bullock, S. L., Ephrussi, A. (2024). Tropomyosin 1-I/C coordinates kinesin-1 and dynein motors during oskar mRNA transport. Nat Struct Mol Biol, 31(3):476-488 PubMed ID: 16949363

Hosaka, Y., Inoshita, T., Shiba-Fukushima, K., Cui, C., Arano, T., Imai, Y. and Hattori, N. (2017). Reduced TDP-43 expression improves neuronal activities in a Drosophila model of Perry syndrome. EBioMedicine [Epub ahead of print]. PubMed ID: 28625517

Kim, H., Ling, S. C., Rogers, G. C., Kural, C., Selvin, P. R., Rogers, S. L. and Gelfand, V. I. (2007). Microtubule binding by dynactin is required for microtubule organization but not cargo transport. J Cell Biol 176(5): 641-651. PubMed ID: 17325206

Lazarus, J. E., Moughamian, A. J., Tokito, M. K. and Holzbaur, E. L. (2013). Dynactin subunit p150^Glued is a neuron-specific anti-catastrophe factor. PLoS Biol 11(7): e1001611. PubMed ID: 23874158

Ligon, L. A., Shelly, S. S., Tokito, M. and Holzbaur, E. L. (2003). The microtubule plus-end proteins EB1 and dynactin have differential effects on microtubule polymerization. Mol Biol Cell 14(4): 1405-1417. PubMed ID: 12686597

Lloyd, T. E., Machamer, J., O'Hara, K., Kim, J. H., Collins, S. E., Wong, M. Y., Sahin, B., Imlach, W., Yang, Y., Levitan, E. S., McCabe, B. D. and Kolodkin, A. L. (2012). The p150^Glued CAP-Gly domain regulates initiation of retrograde transport at synaptic termini. Neuron 74(2): 344-360. PubMed ID: 22542187

Ma, L., Johns, L. A. and Allen, M. J. (2009). A modifier screen in the Drosophila eye reveals that aPKC interacts with Glued during central synapse formation. BMC Genet 10: 77. PubMed ID: 19948010

Morgan, J. L., Song, Y. and Barbar, E. (2011). Structural dynamics and multiregion interactions in dynein-dynactin recognition. J Biol Chem 286(45): 39349-39359. PubMed ID: 21931160

Moughamian, A. J. and Holzbaur, E. L. (2012). Dynactin is required for transport initiation from the distal axon. Neuron 74(2): 331-343. PubMed ID: 22542186

Papoulas, O., Hays, T. S. and Sisson, J. C. (2005). The golgin Lava lamp mediates dynein-based Golgi movements during Drosophila cellularization. Nat Cell Biol 7(6): 612-618. PubMed ID: 15908943

Rome, P., Montembault, E., Franck, N., Pascal, A., Glover, D. M. and Giet, R. (2010). Aurora A contributes to p150^glued phosphorylation and function during mitosis. J Cell Biol 189(4): 651-659. PubMed ID: 20479466

Sakuma, C., Kawauchi, T., Haraguchi, S., Shikanai, M., Yamaguchi, Y., Gelfand, V. I., Luo, L., Miura, M. and Chihara, T. (2014). Drosophila Strip serves as a platform for early endosome organization during axon elongation. Nat Commun 5: 5180. PubMed ID: 25312435

Schafer, D. A., Gill, S. R., Cooper, J. A., Heuser, J. E. and Schroer, T. A. (1994). Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin. J Cell Biol 126(2): 403-412. PubMed ID: 7518465

Siller, K. H., Serr, M., Steward, R., Hays, T. S. and Doe, C. Q. (2005). Live imaging of Drosophila brain neuroblasts reveals a role for Lis1/Dynactin in spindle assembly and mitotic checkpoint control. Mol. Biol. Cell 16(11): 5127-40. 16107559

Siller, K. H. and Doe, C. Q. (2008). Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts. Dev Biol 319(1): 1-9. PubMed ID: 18485341

Tacik, P., Fiesel, F. C., Fujioka, S., Ross, O. A., Pretelt, F., Castaneda Cardona, C., Kidd, A., Hlavac, M., Raizis, A., Okun, M. S., Traynor, S., Strongosky, A. J., Springer, W. and Wszolek, Z. K. (2014). Three families with Perry syndrome from distinct parts of the world. Parkinsonism Relat Disord 20(8): 884-888. PubMed ID: 24881494

Urnavicius, L., Zhang, K., Diamant, A. G., Motz, C., Schlager, M. A., Yu, M., Patel, N. A., Robinson, C. V. and Carter, A. P. (2015). The structure of the dynactin complex and its interaction with dynein. Science 347(6229): 1441-1446. PubMed ID: 25814576

Waterman-Storer, C. M., Karki, S. and Holzbaur, E. L. (1995). The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). Proc Natl Acad Sci U S A 92(5): 1634-1638. PubMed ID: 7878030

Whited, J. L., Cassell, A., Brouillette, M. and Garrity, P. A. (2004). Dynactin is required to maintain nuclear position within postmitotic Drosophila photoreceptor neurons. Development 131(19): 4677-4686. PubMed ID: 15329347

Wong, M. Y., Zhou, C., Shakiryanova, D., Lloyd, T. E., Deitcher, D. L. and Levitan, E. S. (2012). Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture. Cell 148(5): 1029-1038. PubMed ID: 22385966

Yang, J. S., Bai, J. M. and Lee, T. (2008). Dynein-dynactin complex is essential for dendritic restriction of TM1-containing Drosophila Dscam. PLoS One (10): e3504. PubMed Citation: 18946501

date revised: 23 October 2023

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