BIOLOGICAL OVERVIEW

In normal flies each of the three thoracic and eight abdominal segments has a characteristic belt of hairs (denticles); the hairs in the abdominal segments generally are thicker, and hence their denticle belts are more readily apparent. Mutation of BicD results in a bicaudal phenotype: in the bicaudal embryo, the head, thorax, and anterior-most three to five abdominal segments are replaced by a mirror image of the posterior abdominal segments and terminalia, including the posterior spiracles (Suter, 1989, Wharton, 1989 and Mohler, 1986).

Earlier literature on Bicaudal-D provides an impressive number of explanations for the origin of bicaudality. One explanation suggests that Bic-D has a direct role in the localization of the posterior determinant nanos (Wharton, 1989). Equally likely was the belief that Bic-D regulates the anterior localization of Bicoid mRNA (Suter, 1989). A third idea was that Bic-D is required for the determination of the one cystocyte to become the oocyte (the egg has a total of 16, 15 of which become nurse cells). This determination is not made in BicD mutants, thereby preventing the posterior migration of the oocyte (Ran, 1994). However, over time it has become clear that establishment of oocyte identity is a multistage process involving multiple factors and events and that BicD regulates multiple aspects of this process. BicD is indeed involved in establishing the developmental fate of the the oocyte as distinct from that of nurse cells, and a failure of this process interfers with the localization of anterior and posterior determinants of oocyte polarity. A major aspect of the determination of developmental fate of the oocyte is, in fact, localization of various determinants in the presumptive oocyte, and in this process, Bic-D seems to play an important role. These localization events take place during mid-oogenesis, after the establishment of oocyte fate. BicD plays a role in early fate determination of the oocyte and in subsequent localization of the mRNA determinants of oocyte polarity (Mach, 1997 and Swan, 1996).

Three proteins have been implicated in oocyte determination: Stonewall, a presumptive transcription factor, and BicD and Egalitarian, both with novel sequences. The microtubule cytoskeleton is an additional element in oocyte determination. Treating wild-type flies with microtubule-depolymerizing drugs such as colchicine also causes a 16-nurse-cell phenotype, implicating microtubule structure as an important component of the oocyte determination mechanism. A microtubule organizing center (MTOC) forms in the presumptive oocyte just after the formation of the 16-cell cluster; in Bic-D mutants this MOTC does not form (Theurkauf, 1993). Because BicD protein localizes to a single cell in Bic-D mutants, BicD can localize without the formation of the MOTC (Mach, 1997).

The beginnings of determination of oocyte fate precede the function of Bic-D. In fact, the oocyte fate is determined earlier than the 16 cell stage. A bias toward the oocyte fate already may be set during the first division of the oocyte precursor, or cystoblast, when one daughter inherits the spectrosome, a spectrin-rich organelle that seems to play a role in orienting the plane of division of the cystoblast progeny. Of particular interest is the association of spectrosomes with the pole of the mitotic spindle. During the first cystoblast division, spectrosome material is associated with only one pole of the mitotic spindle, revealing that this division is asymmetric. During the subsequent three divisions, the growing spectrosome always associates with the pole of each mitotic spindle that remains in the mother cell, and only extends through the newly formed ring canals after each division is completed (Lin, 1995).

What then is the role of Bic-D in establishing oocyte fate? The recent characterization of the egalitarian gene has provided new insight into this question. Like BicD, Egalitarian is a novel protein that colocalizes with BicD protein at all stages of oogenesis. Egl and BicD proteins localize to the oocyte in three stages that correlate with the stepwise polarization of the oocyte. From stage 2 until stage 7 of oogenesis, EGL mRNA is concentrated at the posterior cortex of the oocyte. The distribution of Egl protein strikingly resembles the localization of the minus end of microtubules (see ß1 tubulin for more information). During stages 1-6 of oogenesis, a microtubule network extends from an MTOC located at the posterior cortex of the early oocyte into the nurse cells. During stage 8 of oogenesis the microtubule network repolarizes and the microtubules orient from the anterior cortex (Theurkauf, 1992). At this time EGL transcript localizes in an anterior ring at the nurse cell-oocyte boundary. This distribution is similar to that of other RNAs, such as K10, oo18 RNA-binding protein (orb), BicD, and oskar, all of which accumulate early in the oocyte and sometime later form a transient anterior ring. By stage 10 of oogenesis, EGL mRNA is distributed evenly throughout the oocyte but persists in the oocyte into early embryogenesis (Mach, 1997).

EGL protein is detectable in the germarium and initially is distributed evenly within the newly formed 16-cell cyst. Once the cyst flattens in germarial region 2B, the protein is often concentrated in the two cells that have four canals. By stage 1 of oogenesis, Egl protein localizes to a single cell, the future oocyte. From stage 2 to stage 7 of oogenesis, Egl protein is enriched at the posterior cortex of the oocyte. At stage 8 of oogenesis, Egl protein shifts to the anterior cortex in a ring around the margin of the oocyte, where the oocyte, nurse cells, and follicle cells meet. Disrupting the microtubule network with colchicine abolishes EGL localization in the oocyte. Thus, EGL localization requires microtubule organization. Morover, it has been shown (Theurkauf, 1993) that in egl and BicD mutants the microtubule network, although established initially, is not maintained. It is therefore likely that the effect of microtubules on oocyte determination is mediated by EGL and BicD, and that these two proteins in turn reinforce or maintain the microtubule network after an initial polarity has been established. There is no hierarchical relationship between EGL and BicD localization; instead, localization of each requires the other (Mach, 1997).

Experiments with antibodies to EGL and BicD make it clear that there is a physical interaction between the two proteins. Since mutation of either protein results in the loss of oocyte identity with the consequent adoption of nurse cell fate by all 16 cystocytes, both proteins play a role in oocyte determination. They are also required to establish oocyte polarity. Females carrying a dominant BicD mutation produce embryos that develop with reduced head structures attributable to the partial mislocalization of OSK mRNA to the anterior of the oocyte (Ephrussi, 1991). Antibody staining reveals that compared with wild type, BicD protein accumulation in the early oocyte is more pronounced in BicD-Dominant mutants and that the protein remains at the anterior pole of mutant oocytes during later stages of oogenesis and early embryogenesis (Wharton, 1989).

To determine whether egl and Bic-D directly affects the extent to which OSK mRNA mislocalizes in embryos, the distribution of OSK mRNA was examined in BicD-Dominant mutants. Reducing the amount of egl wild-type product decreases ectopic localization of osk to the anterior and increasing the amount of egl wild-type product enhances the mislocalization of OSK to the anterior. Because the effect of BicD-Dominant mutants depends on egl wild type function, it is concluded that egl and BicD act in the same pathway and that the two function in concert to control OSK mRNA localization. It is also thought that Egl and BicD have a role in dorsoventral polarity, as mutation of the two genes reduce the level of GURKEN mRNA. Localization of GUR is also known to require an intact microtubule cytoskeleton (Mach, 1997).

Each step - first determination of oocyte fate, then specification of the anterior-posterior axis, and finally specification of the dorsoventral axis - requires both RNA transport along a polarized microtubule network and the function of the EGL-BicD complex. The distribution of the two proteins resembles that of the minus ends of microtubules, and mutations in either disrupt microtubule stability. Although it is still possible that the EGL-BicD complex affects RNA localization solely by stabilizing microtubule structure, it is likely that the complex also acts as a link between microtubules and the RNA localization machinery. If the two proteins act directly to localize RNAs, these proteins may either bind RNA or associate with an RNA-binding protein such as Orb, whose distribution is strikingly similarly to that of Egl and BicD, forming a pattern dependent on Egl and BicD function (Mach, 1997, Lantz, 1994 and Christerson, 1994)

Interaction between Polo and BicD proteins links oocyte determination and meiosis control in Drosophila

Meiosis is a specialized cell cycle limited to the gametes in Metazoa. In Drosophila, oocyte determination and meiosis control are interdependent processes, and BicD appears to play a key role in both. However, the exact mechanism of how BicD-dependent polarized transport could influence meiosis and vice versa remains an open question. This article reports that the cell cycle regulatory kinase Polo binds to BicD protein during oogenesis. Polo is expressed in all cells during cyst formation before specifically localizing to the oocyte. This is the earliest known example of asymmetric localization of a cell-cycle regulator in this process. This localization is dependent on BicD and the Dynein complex. Loss- and gain-of-function experiments showed that Polo has two independent functions. On the one hand, it acts as a trigger for meiosis. On the other, it is independently required, in a cell-autonomous manner, for the activation of BicD-dependent transport. Moreover, Polo overexpression can rescue a hypomorphic mutation of BicD by restoring its localization and its function, suggesting that the requirement for Polo in polarized transport acts through regulation of BicD. Taken together, these data indicate the existence of a positive feedback loop between BicD and Polo, and it is proposed that this loop represents a functional link between oocyte specification and the control of meiosis (Mirouse, 2006).

This paper describes the localization of the Polo protein and its genetic control in the Drosophila germline during early oogenesis. Polo has a peculiar subcellular localization in cytoplasmic dots that do not correspond to any well-known structures of germline cysts or to microtubule minus-ends where BicD accumulates. Polo has previously been described as colocalizing with several subcellular structures depending on cell cycle phase, but none of these corresponds to the localization observed in this study. Similar cytoplasmic dots were observed in the primordial germline cells of the Drosophila embryo as soon as they were formed, suggesting that this unusual localization could be a specific feature of the germline (Mirouse, 2006).

From region 2a onward, Polo dots are present mostly in the cells containing SCs. This is the first report of a cell-cycle regulator whose localization is spatially and temporally correlated with meiotic progression during early oogenesis. Moreover this correlation is still conserved in mutants that affect polarized transport and the restriction and maintenance of meiosis. This indicates that Polo localization is dependent on polarized transport. One possibility is that Polo itself is directly transported to the oocyte. This hypothesis is reinforced by the physical interaction between BicD and Polo proteins, according to the proposed function of BicD as adapter for Dynein cargos. However, the BicD-dependent localization of Polo is not sufficient to explain its expression profile. Polo is strongly expressed in region 1 of the germarium, and the overall amount of the protein in the cyst progressively decreases, becoming undetectable after stage 2. This degradation seems to be compensated in meiotic cells and then in the oocyte by the polarized transport. The progressive degradation of Polo is also observed in egl and BicD null mutants. Degradation in association with a complete absence of Polo transport may explain why all the cells of a cyst enter into meiosis in these mutants (all the cells contain the same amount of Polo), and then exit meiosis simultaneously (none of the cells preferentially accumulates enough Polo). Alternatively, rather than by direct transport of Polo to the oocyte, its asymmetric distribution in the cyst could be due to a differential control of its stability between nurse cells and oocyte under the control of the BicD-dependent polarized transport (Mirouse, 2006).

BicD and egl null mutants showed a very similar phenotype, in which all 16 cells of a cyst first enter into meiosis but subsequently lose the synaptonemal complexes (SCs). This phenotype cannot be compared with null mutants of the dhc, since Dynein is required at earlier steps of cyst formation. The human homolog of BicD interacts directly with Dynamitin, and this interaction is thought to mediate the interaction of BicD with the Dynein complex. In contrast to BicD, Dynamitin is not involved in the initial restriction of meiosis, showing that the interaction of BicD with Dynamitin, and thus probably Dynein, is not required for the initial restriction of meiosis. In a similar way, LC8 (cut up) null mutants or egl mutants that specifically block the interaction between Egl and LC8 do not interfere with the initiation of meiosis in only four cells. Transport of the BicD protein between the cyst cells is apparently not required for this first step, sinc the BicDPA66 allele or drug-induced microtubule depolymerization does not affect this initial restriction, although BicD is diffuse throughout the entire cyst. Finally, a null mutant for the plakin shot, which has been proposed to be an essential upstream component of the Dynein function in centrosome migration, exhibits variable meiotic phenotypes but allows a normal initial restriction of meiosis to four cells. These data are consistent with a function of BicD and Egl independent of Dynein in the initial restriction of meiosis (Mirouse, 2006).

Polo is involved in many crucial steps of the cell cycle, including the G2/M transition of mitosis and meiosis processes. This study shows that hypomorphic polo alleles lead to a delay in meiotic entry and that Polo overexpression can trigger meiosis in more than four cells per cyst in region 2a. These phenotypes could be related to the function of Polo in the G2/M transition. In vertebrates, Polo is an activator of the String/CDC25 phosphatase, and it has also been proposed that Polo can repress the kinases Myt1 and Wee1. String is the main activator of the cyclinB/CDC2 complex, the activity of which triggers the G2/M transition, whereas Myt1 and Wee are repressors of this complex. However, the role of the cyclin B and CDC25 in meiosis in Drosophila oogenesis is not yet well understood because, for example, CDC25 seems to act as a negative regulator of meiotic oocyte cell fate. Further investigations will be needed to determine how Polo triggers meiotic entry during early oogenesis (Mirouse, 2006).

This study has shown that in mutants with partial loss of polo function, SCs start to disassemble in region 3 but are well formed again in stage 2/3 before disappearing in the following stages. One possible hypothesis to explain how meiosis is finally properly maintained in polo hypomorphic mutants is that the repression of cyclin E by Dacapo during stage 2/3 represses endoreplication, and thus allows meiotic progression. This is consistent with the finding that the specific localization of Dacapo to the oocyte and its requirement for meiosis maintenance begins only in region 3. Moreover, null mutations of dacapo do not lead to a fully penetrant 16-nurse-cell phenotype, confirming the existence of a partially redundant control. Therefore, it is proposed that the balance in favor of meiosis is initially due to the localized activation of meiosis by Polo, and later to the localized inhibition of endoreplication by Dacapo, and that both mechanisms partially overlap (Mirouse, 2006).

It was also observed that Polo is required for the normal restriction of meiosis. Moreover, the defects in the restriction of meiosis caused by both loss and gain of polo function are correlated with defects in oocyte determination. Meiosis restriction and oocyte specification both depend on the Dynein complex and the BicD polarized transport system. Thus, it is assumed that these Polo phenotypes indicate that Polo is involved in polarized transport. This role may be indirect and thus reveals the influence of meiosis and cell-cycle control on oocyte differentiation. Such influence has been observed in situations where there is activation of the meiotic checkpoint due to a failure in DNA double-stand break repair. However, at least two results argue for a direct role of Polo in polarized transport, independently of its meiotic function. First, in mosaic germline cysts, nonmeiotic cells mutant for polo retain BicD protein. Thus, this phenotype cannot be due to the activation of the meiotic checkpoint. This strongly suggests that Polo is required in each cell of the cyst to initiate BicD-dependent transport to the presumptive oocyte. Second, the overexpression of Polo is able to restore the localization and therefore the function of BicDPA66 protein. Interestingly, this mutant allele is due to a single amino acid substitution (A40V) that leads to a hypophosphorylation of BicD, and genetic evidence indicates that this phosphorylation is crucial for BicD function. Polo overexpression might restore a functional level of BicDPA66 phosphorylation. Therefore, even if no significant changes were observed in the gel mobility of BicD in polo hypomorph mutants, it is tempting to propose that the function of Polo in the polarized transport could be to activate, directly or indirectly, BicD by phosphorylation (Mirouse, 2006).

Taken together, these results lead to a model that can explain a reciprocal requirement between the control of meiosis and oocyte specification. This model is based on four major points: (1) BicD is required for the Dynein-dependent polarized transport of oocyte determinants; (2) BicD is also required for the progressive localization of Polo to the oocyte; (3) Polo appears to trigger meiosis in the germarium; (4) Polo is required to activate the BicD and Dynein-dependent polarized transport. These findings suggest the existence of a positive feedback loop between Polo and BicD proteins, and therefore between oocyte specification and meiosis (Mirouse, 2006).

Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. This study reports that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. These results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle (Li, 2010).

The genetic requirement of BicD at the organismal level had previously only been investigated in detail during maternal stages in Drosophila. This study describes the unexpected finding that zygotic BicD function is obligatory only in the nervous system, despite its widespread expression during larval stages. This function seems to be independent of BicD's well-known role in Egl-dependent mRNA transport (Li, 2010).

Chc was identified as the major BicD-associated protein in head extracts, and a direct interaction was mapped between the C-terminal third of BicD and the Chc ankle domain. This region of BicD provides a link between cellular cargoes and the dynein motor. Consistent with clathrin acting as a cargo for BicD/dynein, the directed transport of Chc::eGFP in association with microtubules is strongly reduced in BicD mutant presynaptic boutons leading to aberrant accumulation of the protein internally and a partial reduction in clathrin levels at the plasma membrane. Changes in microtubule integrity were not observed in BicD mutant boutons. In addition, a statistically significant portion of Chc signals overlapped with BicD signals within boutons and highly motile structures containing both Chc and BicD were observed in egg chambers, which are suited to sensitive time-lapse imaging. Collectively, these data build a strong case for disruption of Chc motility in boutons being a consequence of a direct requirement for BicD in microtubule-based transport complexes (Li, 2010).

In the case of embryonic transport of mRNA and lipid droplets in Drosophila, BicD does not seem to be obligatory for linkage of cargoes to the bidirectional motor complex, but leads to efficient transport by augmenting the persistence of motor movement. The presence of some residual directed motion of Chc::eGFP in mutant boutons raises the possibility that BicD serves an analogous, stimulatory function in the transport of clathrin-associated cargoes (Li, 2010).

It should also be pointed out that although the readily discernible phenotypic requirement for Drosophila BicD is restricted to neuronal tissue, this does not rule out a role for BicD in modulating the kinetics of clathrin-mediated mechanisms in non-neuronal cells, especially in other species. Highly motile clathrin-labelled structures within cultured mammalian cells are translocated by dynein on microtubules , raising the possibility of a conserved involvement of the BicD-Chc interaction (Li, 2010).

Functional assays show a novel requirement for BicD and dynein in the maintenance of normal levels of neurotransmission during high-frequency electrical stimulation. The function of these factors is not, however, limiting during low-frequency stimulation. BicD and dynein therefore join a group of other clathrin-associated proteins in Drosophila, such as Dap160, AP180, EndophilinA and Synaptojanin, in being required to maintain normal levels of neurotransmission specifically during periods of intense stimulation (Li, 2010).

Experiments assaying FM1-43 dye uptake and release showed that BicD and dynein are required for efficient membrane uptake specifically during intense electrical stimulation. This defect is associated, at least in part, with a requirement for augmenting the rate of release of pre-internalised vesicles. However, because rates of membrane uptake are intimately linked to rates of membrane release, the experiments performed could not directly measure endocytic rates in isolation. Ultrastructural analysis did not show dramatic changes in the organisation or morphology of the membrane trafficking system in resting or stimulated BicD mutant synapses. Collectively, these data indicate that BicD has a kinetic function in stimulating the rate of recycling of pre-internalised vesicles, as opposed to an obligatory role in any one step. This requirement for BicD is almost certainly associated with its well-characterised role in stimulating cargo transport by microtubule-based motors, as inhibition of dynein heavy chain and dynactin resulted in very similar effects on vesicle recycling to those seen in BicD mutants (Li, 2010).

To date the actin cytoskeleton has been heavily implicated in synaptic membrane recycling. However, the absence of chronic problems in synaptic morphology and membrane organisation in BicD mutant boutons is consistent with microtubule-based transport also participating directly in the synaptic vesicle cycle. In support of this notion, microtubules are prominent in boutons and acute, efficient interference with their integrity inhibits neurotransmission (Li, 2010).

BicD is likely to have a role in the transport of a subset of cargoes by dynein. However, the previously known BicD interaction partners do not seem to contribute significantly to the synaptic vesicle recycling phenotype in the BicD mutants; Egl function appears to be strictly maternal and Rab6 is not detectable in presynaptic boutons and its distribution in axons of motor neurons is not sensitive to the absence of BicD (Li, 2010).

It is possible that other, potentially unidentified, BicD cargoes contributing to the synaptic vesicle recycling phenotype in the mutants -- in fact, it is quite plausible. However, several lines of evidence point towards an important involvement of the interaction of BicD with Chc: (1) Chc is by far the most abundant protein stably associated with BicD in head extracts; (2) presynaptic overexpression of EndoA, which is rate-limiting for clathrin-mediated endocytosis and/or uncoating, is sufficient to completely suppress the defects in synaptic vesicle recycling of BicD mutants; (3) the requirement for BicD and dynein in synaptic vesicle recycling mirrors the requirement for high rates of clathrin-mediated membrane retrieval; (4) synaptic vesicle diameter and synaptic bouton number are increased in BicD mutants, reminiscent of when components of the machinery for clathrin-mediated endocytosis are mutated; and (5) despite extensive efforts, no factor other than Chc was found that was mislocalised in BicD mutant synapses; the factors tested include markers of membrane compartments (Hrs, Rabs 5, 6, 7 and 11), active zones (nc82) and synaptic vesicles (Vglut, Csp and Synaptotagmin (Syt) (Li, 2010).

How might a BicD-Chc interaction contribute to efficient synaptic membrane recycling? It seems unlikely that BicD has a direct role in stimulating clathrin-mediated internalisation of the plasma membrane because BicD, unlike several components of the clathrin-mediated endocytic machinery, is not enriched within periactive zones. BicD accumulates predominantly beneath these zones and the overlap of Chc and BicD puncta can occur even more internally within the bouton. These observations also suggest that BicD does not have a critical role in uncoating of clathrin from synaptic vesicles, which occurs very shortly after scission from the plasma membrane. Consistent with this notion, an accumulation of densely coated vesicles, indicative of uncoating defects in NMJ synapses of other mutants, was not detectable in electron micrographs of either resting or stimulated BicD mutant synapses (Li, 2010).

One possibility is that BicD augments dynein-based transport of clathrin that has disassociated from internalised vesicles back to the plasma membrane. This could account for two observations in the BicD mutants: the partially reduced concentration of Chc at periactive zones and the lack of co-localisation of internally accumulated Chc with clathrin adaptor proteins and markers of membrane compartments tested (AP180, α-adaptin/AP2, Hrs and Rab5). Reduced levels of available clathrin can compromise the ability to sustain high rates of membrane uptake. Thus, rates of Chc recycling to the plasma membrane in BicD and Dhc mutants may not be limiting during low-frequency stimulation, but may be unable to maintain sufficient levels of plasma membrane clathrin during bouts of intense stimulation. Recent studies in Drosophila synapses show that the membrane internalised in the absence of clathrin function is greatly increased in size and is not competent for recycling (Heerssen, 2008; Kasprowicz, 2008). Thus, a partial decrease in clathrin availability at the plasma membrane could conceivably contribute to the subtle increase in vesicle diameter and the inefficient recycling of pre-internalised membrane in BicD mutants. Alternatively, the internally mislocalised Chc in BicD mutants may interfere with normal sorting or maturation of synaptic vesicles by acting at ectopic sites or by sequestering important, as-of-yet unidentified, co-factors. Sequestration of clathrin co-factors could also account for changes in the diameter of nascent clathrin-coated vesicles in the mutants (Li, 2010).

Another possibility, which is by no means mutually exclusive, is that the ability of BicD to stimulate Chc transport on microtubules could directly augment translocation of recycling membrane during the time it is associated with clathrin (note that recent EM tomography studies in non-neuronal cells suggest that an incomplete uncoating reaction leads to the retention of some clathrin on vesicles until the exocytic event. Once again, such a kinetic requirement for BicD may only be limiting when there is a demand for a rapid rate of membrane recycling. BicD-Chc could potentially have a role in a process analogous to the microtubule-based pre-endosomal sorting process operating shortly after internalisation in other cell types. Alternatively, BicD might recognise Chc associated with endosomal structures. Indeed, dynein has a key role in non-neuronal cells in the sorting and subsequent transport of specific subsets of endosomal vesicles. Long-term experiments in neuronal and non-neuronal cells will be needed to resolve to what extent BicD participates in motor complexes that directly transport different post-endocytic intermediates, and the involvement of the interaction of BicD with Chc in these events. Future studies will also test directly the contribution of the interaction with Chc to other BicD-dependent processes, including sculpting of NMJ morphology (Li, 2010).

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal-D

Bicaudal-D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. As BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize them to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles, and the tandem spindles during female meiosis II. Furthermore, BicD is required to correctly localize clathrin and the microtubule-stabilizing factors, D-TACC and Msps, to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, right after the establishment of the female pronucleus, D-TACC and C. elegans BicD, tacc, and Chc are also needed for pronuclear fusion, pointing to the possibility that the underlying mechanism might be more widely used (Vazquez-Pianzola, 2022).

Encoded by a single gene, the Drosophila Bicaudal D (BicD) protein is part of a family of evolutionarily conserved dynein adaptors responsible for the transport of different cargoes along microtubules (MTs). The founding member of this protein family, Drosophila BicD, was identified because of its essential role during oogenesis and embryo development, in which it transports mRNAs that control polarity and cell fate. This process is mediated by its binding to the RNA-binding protein Egalitarian (Egl). Since its initial discovery, BicD and its orthologs have been shown to control a diverse group of MT transport processes through binding to different cargoes or adaptor proteins (Vazquez-Pianzola, 2022).

BicD can alternatively bind to Clathrin heavy chain (Chc) and this interaction facilitates Chc transport of recycling vesicles at the neuromuscular junctions and regulates endocytosis and the assembly of the pole plasm during oogenesis. The best-known function of Chc is in receptor-mediated endocytosis, in which it forms part of clathrin, a trimeric scaffold protein (called a triskelion), composed of three Chc and three Clathrin light chains (Clc). Aside from this, clathrin was shown to localize to mitotic spindles in mammalian and Xenopus cells and to have non-canonical activity by stabilizing the spindle MTs during mitosis. This function depends on clathrin trimerization and its interaction with Aurora A-phosphorylated Transforming Acidic Coiled-Coil protein 3 (TACC3) and the protein product of the colonic hepatic Tumor Overexpressed Gene (ch-TOG). This heterotrimer forms intermicrotubule bridges between kinetochore fibers (K-fibers), stabilizing these fibers and promoting chromosome congression. Recently, TACC3 and a mammalian homolog of Chc (CHC17) were shown to control the formation of a new liquid-like spindle domain (LISD) that promotes the assembly of acentrosomal mammalian oocyte spindles (Vazquez-Pianzola, 2022).

In order to transport its cargos along MTs, BicD interacts with the dynein/dynactin motor complex, a minus-end-directed MT motor. This complex is involved in different cellular processes, including intracellular trafficking of proteins and RNAs, organelle positioning and microtubule organization, some of which also require BicD. The dynein/dynactin complex also plays essential roles during cell division, in which it is required for centrosome separation, chromosome movements, spindle organization and positioning and mitotic checkpoint silencing (Vazquez-Pianzola, 2022).

Given that Drosophila BicD forms complexes with Chc and Dynein, both of which, as described above, perform essential activities during mitosis, this study set out to investigate possible BicD functions during cell division. Reducing BicD levels by specific protein-targeted degradation in freshly laid eggs revealed that BicD is essential for pronuclear fusion. In addition, it is required for metaphase arrest of female meiotic products after meiosis II completion. This activity appears to be mediated by the role of BicD in localizing the spindle assembly checkpoint (SAC) components. Furthermore, BicD interacts with its cargo protein, Chc, and they both localize to the mitotic spindles and centrosomes and the female tandem meiotic II spindles. In addition, BicD localizes D-TACC, clathrin, and Mini spindles (Msps; ch-TOG homolog) to the meiosis II spindles. The failure to localize these proteins accurately might also contribute to the SAC function defects observed in embryos with reduced BicD levels. D-TACC and Caenorhabditis elegans bicd-1, tac-1 and chc-1 are also needed after fertilization for pronuclear fusion, revealing an evolutionary conserved and essential role of these proteins in early zygote formation and suggesting that their mechanism of action on MTs might be widely used across species (Vazquez-Pianzola, 2022).

This study found that BicD localizes to the female tandem spindles and the central aster during MII. After fertilization, BicD also localizes to the mitotic spindles and the centrosomes. BicD^null mutants rarely survive and are sterile, but this study generated embryos with reduced levels of BicD at the beginning of embryogenesis (BicDhb-deGradFP embryos) by setting up a strategy based on the deGradFP technique. Consistent with BicD localization at the female MII spindles, it was discovered that BicDhb-deGradFP embryos arrest development, displaying aberrant meiotic products and no pronuclear fusion. Especially if combined with the CRIPSP-Cas9 strategy first to produce functional GFP-tagged proteins of interest, the construct designed in this study could be helpful for studying the role of female-sterile and lethal mutations during very early embryonic development (Vazquez-Pianzola, 2022).

In unfertilized BicDhb-deGradFP eggs, the female meiotic products were not arrested in metaphase as normally happens. Instead, they underwent additional rounds of replication. They failed to recruit or maintain the recruitment of the SAC pathway components BubR1 and Mad2, which are normally present at the kinetochores in the wild-type female meiotic polar bodies. Interestingly, in Drosophila eggs mutated for Rod, mps1 and BubR1, well-conserved orthologs of the SAC pathway, the polar bodies also cannot remain in a SAC-dependent metaphase-like state and decondense their chromatin. Furthermore, in these mutants, the polar bodies cycle in and out of M-phase, replicating their chromosomes similarly to those in BicDhb-deGradFP eggs. Thus, it appears that BicD functions to localize the SAC components to induce and/or maintain metaphase arrest of the polar bodies. Several mechanisms could explain the failure to maintain SAC activation observed in BicDhb-deGradFP embryos. BicD might be needed to recruit the SAC components to kinetochores directly. By contrast, during mitosis, the Rod-Zw10-Zwilch (RZZ) complex binds to the outer kinetochore region and recruits Mad2, Spindly and the dynactin complex. Spindly and dynactin act cooperatively to recruit dynein, which then transports the SAC components along the MTs away from kinetochores as a mechanism to trigger checkpoint silencing and anaphase onset. Given that the BicD N-terminal domain binds dynein and dynactin and promotes their interaction, it is also possible that BicD helps to move the SAC components away from the kinetochores. If this does not happen, the SAC remains persistently activated. It was also found that BicD activity in BicDhb-deGradFP embryos is insufficient to localize clathrin, TACC and Msps efficiently along the MTs of the spindle. During mitosis, impairment of MT motors, such as dynein, and treatments that prevent the TACC/clathrin complex from binding to the mitotic spindles and affecting K-fiber stability, also persistently activate the SAC. Thus, reduced levels of BicD in BicDhb-deGradFP embryos could additionally trigger SAC hyperactivation through its role in stabilizing the K-fibers. Although these data strongly suggest that the lack of BicD contributes to SAC defects through its role in localizing clathrin, D-TACC and Msps, further work is needed to elucidate whether BicD also acts more directly by binding to, and localizing, the SAC components, or indirectly by affecting the function of dynein (Vazquez-Pianzola, 2022).

Whereas persistent SAC activation leads to metaphase arrest and delayed meiosis (D-meiosis), this delay is known to be rarely permanent, at least during mitosis. Most cells that cannot satisfy the SAC ultimately escape delayed mitosis (D-mitosis) and enter G1 as tetraploid cells by a currently poorly understood mechanism. It is possible that, in BicDhb-deGradFP, the SAC pathway is constantly activated, delaying meiosis. However, at one point, the nuclei might escape metaphase II arrest, cycling in and out of M-phase, thereby replicating their chromosomes and decondensing their chromatin. The fact that female meiotic products over-replicate in BicDhb-deGradFP eggs and show no or only pericentromeric PH3 staining supports the notion that these nuclei are on an in-out metaphase arrest phase. That meiotic products in about half of the BicDhb-deGradFP embryos failed to stain for the SAC components BubR1 and Mad2 supports this hypothesis (Vazquez-Pianzola, 2022).

Chc, its partner Clc and BicD are enriched at mitotic spindles and centrosomes. Furthermore, these proteins and the clathrin-interacting partners D-TACC and Msps localize to the tandem spindles and the central aster of the female MII apparatus. The interaction of Drosophila Chc with D-TACC is conserved, and Chc interacts through the same protein domain directly with D-TACC and BicD. Moreover, BicD is needed for localizing D-TACC, Msps and clathrin throughout the MII tandem spindles. The TACC3/Chc interaction was proposed to form a domain in tandem to bind spindle MTs. It is hypothesize that BicD could help recruit Chc to the MTs by association with dynein. Given that Chc usually acts as a trimer with Clc (triskelion), each Chc might interact with either BicD or D-TACC. Thus, a mixed complex could be formed, and BicD might help to move, recruit or stabilize Chc and D-TACC along the spindles via the interaction of BicD/Chc in the same trimer. The fact that expression of D-TACC enhances the Chc/BicD interaction and that overexpression of Chc and D-TACC (tacc) arrested early development in a background in which BicD is reduced to a level that does not produce visible phenotypes on its own, supports this model. These results suggest that, with respect to BicD, the levels of D-TACC and Chc should be tightly balanced for these proteins to perform their normal function during early development, as has been shown previously for other BicD transport processes (Vazquez-Pianzola, 2022).

BicD has a role in pronuclear fusion that is conserved during evolution given that C. elegans eggs depleted for bicd-1 also failed to undergo pronuclear fusion. Moreover, Drosophila D-TACC and C. elegans chc-1 and tac-1 are also needed for pronuclear fusion. These genes might be required indirectly through their role in meiosis because preliminary data suggest that MII is also compromised in bicd-1 and chc-1 dsRNA-fed worms. Alternatively, they might play a more direct role in pronuclear migration, which depends on dynein and MTs in bovine, primate and C. elegans embryos. This would then suggest that the underlying mechanism may be used to build correctly or stabilize different types of MT. Determining their precise mechanistic involvement in pronuclear fusion is an interesting question for further studies (Vazquez-Pianzola, 2022).

Subcellular spatial transcriptomics identifies three mechanistically different classes of localizing RNAs

Intracellular RNA localization is a widespread and dynamic phenomenon that compartmentalizes gene expression and contributes to the functional polarization of cells. Thus far, mechanisms of RNA localization identified in Drosophila have been based on a few RNAs in different tissues, and a comprehensive mechanistic analysis of RNA localization in a single tissue is lacking. By subcellular spatial transcriptomics this study has identified RNAs localized in the apical and basal domains of the columnar follicular epithelium (FE) and the mechanisms mediating their localization were analyzed. Whereas the dynein/BicD/Egl machinery controls apical RNA localization, basally-targeted RNAs require kinesin-1 to overcome a default dynein-mediated transport. Moreover, a non-canonical, translation- and dynein-dependent mechanism mediates apical localization of a subgroup of dynein-activating adaptor-encoding RNAs (BicD, Bsg25D, hook). Altogether, this study identifies at least three mechanisms underlying RNA localization in the FE, and suggests a possible link between RNA localization and dynein/dynactin/adaptor complex formation in vivo (Cassella, 2022).

Only few examples of localizing RNAs in the FE have been described to date, with little mechanistic insight. To explore the extent of RNA localization in a somatic tissue in vivo and gain insight into the mechanisms underlying the phenomenon, laser-capture microdissection of apical and basal subcellular fragments of columnar follicle cells was used coupled with RNA-seq to identify localizing RNAs in this tissue. This allowed investigation in detail the landscape of mechanisms that mediate both apical and basal RNA localization in the FE. This study found that basal RNA localization is mechanistically analogous to posterior RNA localization in the oocyte (represented by osk), reflecting MT plus end enrichment. Khc, aTm1 (atypical Tropomyosin-1 isoform I/C), and the Exon Junction Complex (EJC) appear to be core components of a general basal RNA localization machinery. These results are in line with previous findings on osk RNA indicating that Khc/aTm1 bind to the 3'UTR20 and the EJC activates kinesin-1 transport through association with the coding sequence (Cassella, 2022).

According to this analysis, deposition of the EJC is necessary but not sufficient to determine RNA localization, as it was found that the EJC is deposited on both apically- and basally-directed RNAs. Interestingly, another study found that the EJC specifically localizes to the basal body of the primary cilium in mono-ciliated cells, where it controls the centrosomal localization of NIN RNA towards MT minus ends. In contrast, the current data suggest that in columnar follicle cells, which are characterized by non-centrosomal MTOCs, the EJC may play a role in MT plus end-directed (basal) RNA transport by acting synergistically with kinesin-1 and aTm1. Strikingly, the localization of osk RNA to the posterior pole of the oocyte also relies on the presence of MTs generated from non-centrosomal MTOCs65.Therefore, the mammalian EJC might have acquired a specific role in the localization of NIN RNA at basal bodies of mono-ciliated cells, while the Drosophila EJC appears to contribute to the MT plus end-directed localization of several RNAs through a centrosome-independent mechanism both in the somatic follicular epithelium (basal RNAs) and in the germline (osk RNA) (Cassella, 2022).

Interestingly, when either component of the kinesin-1 transport complex was lacking, basal RNAs were mislocalized to the apical domain in a dynein-dependent process. Therefore, dynein-mediated apical localization represents a default mechanism that must be overcome by kinesin-1 to drive basal RNA localization. Two possible scenarios could explain dynein-mediated apical mislocalization upon kinesin inhibition. Dynein and kinesin-1 could be engaged in a tug-of-war, pulling the RNAs in opposing directions, a phenomenon observed in the transport of vesicles and lipid droplets67. Alternatively, the dynein complex could be kept in an inhibited state and activated upon disruption of kinesin-1 and its regulators. If the tug-of-war scenario were correct, a change would be expected in zip RNA localization in all RNAi conditions including egl RNAi alone, namely a shift to a more basal localization due to the enhanced Khc-dependent motility. However, since no a significant change was seen in zip localization when only Egl was knocked down, the tug-of-war hypothesis appears to be less likely than the inhibition hypothesis. In addition, this phenomenon recalls osk RNA mislocalization to the oocyte anterior upon disruption of kinesin-1, aTm1 or EJC components which was hypothesized to occur due to a failure to inactivate dynein-mediated RNA transport (Cassella, 2022).

Apical RNA localization, on the other hand, can be divided into two mechanistically distinct categories, both based on dynein-mediated transport. The first category includes those RNAs that are transported apically by the dynein/BicD/Egl machinery, a well characterized RNA transport complex that directs RNAs towards MT minus ends in a variety of tissues. The data suggest that the majority of apically localizing RNAs may belong to this class, as the localization of most of the randomly chosen apical RNAs was affected in both Dhc RNAi and egl RNAi conditions. This hypothesis is consistent with previous studies that identified several apical RNAs as BicD/Egl cargoes, in a variety of Drosophila tissues. The BicD/Egl machinery has been hypothesized to be part of a larger RNP complex that ensures a tight translational control of the transported RNA. The finding that basal RNAs are on average translated more than apical RNAs suggests that RNAs transported apically in the FE by the dynein/BicD/Egl transport complex might indeed be kept in a translationally silent state until they have reached their final destination (Cassella, 2022).

The second category of dynein-dependent apical RNAs does not involve Egalitarian activity for their localization. This includes a subgroup of dynein-activating adaptors, namely BicD, hook, and Bsg25D (BICD2, HOOK1-3, and NIN/NINL in mammals). Common features of their apical RNA localization include sensitivity to puromycin and partial co-localization with cortical dynein foci containing also Dhc RNA. Moreover, both Bsg25D74 and BicD RNA constructs containing the CDS alone are sufficient for the accumulation of their encoded protein at MT minus ends. Puromycin causes the disassembly of the translational machinery and the release of the N-terminal peptides emerging from ribosomes. As the N-terminal portion of these adaptors binds dynein or dynactin subunits, it is proposed that the apical localization of BicD, hook, and Bsg25D depends on the co-translational association between dynein components and nascent adaptors at cortical dynein foci. This process might also be conserved in mammals, since the localization of both BICD2 and NIN RNA was shown to be puromycin-sensitive. Previous studies have shown that the presence of either BICD2, HOOK3 or NIN/NINL promotes the formation of highly processive dynein/dynactin complexes. Therefore, it is possible that co-translational assembly of components of the dynein-adaptor complexes is necessary to overcome dynein auto-inhibition. BicD, hook, and Bsg25D may co-translationally associate with dynein soon after nuclear export of the RNA, promoting its apical transport in a manner similar to what has been proposed for PCNT RNA targeting at centrosomes. Alternatively, since dynein can also function as a MT-tethered static anchor in mid-oogenesis oocytes and follicle cells, the interaction between dynein and nascent adaptor proteins could occur after the RNA has reached the cell cortex by dynein-mediated transport. Indeed, puromycin treatment did not completely abolish the apical enrichment of adaptor-encoding RNAs, despite causing a marked decrease in their signal close to the apical cortex, where they decorate dynein cortical foci (Cassella, 2022).

In vitro studies have shown that full-length BicD/BICD2 adopts an autoinhibitory conformation resulting from CC1/2 folding onto the CTD-containing CC3. Although the leading hypothesis in the field is that cargo binding to the CTD is responsible for the alleviation of auto-inhibition by freeing up the N-terminal dynein-binding domain, it is possible that in vivo both nascent BicD interaction with dynein and cargo binding to the CTD might cooperate in preventing BicD intramolecular inhibition in the cellular environment. Strikingly, whereas the mechanism underlying oocyte localization of BicD RNA during mid-oogenesis resembles that observed in follicle cells, the nurse cell-to-oocyte transport of BicD RNA appears to be governed by a different, translation-independent mechanism that may not involve interaction with Dhc/Dhc RNA particles, consistent with a previous study indicating that BicD RNA is translationally inhibited by Me31B in the nurse cells. In contrast to early egg chambers in which the MT network emanates from a posteriorly-positioned microtubule organizing center in the oocyte, mid-stage oocytes and columnar follicle cells are both characterized by non-centrosomal MTs (ncMTs) tethered to the cell cortex. Therefore, the establishment of ncMTs could be at the basis of the mechanistic switch from translation-independent to co-translational BicD RNA localization in these compartments. A recent report has shown that NIN RNA (the mammalian ortholog of Bsg25D) localizes at ncMTs and its expression is essential for apico-basal MT formation and columnar epithelial shape. Therefore, it is possible that the co-translational transport of adaptor-encoding RNAs may be important for correct ncMT nucleation at the apical cortex of the follicular epithelium (Cassella, 2022).

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).

GENE STRUCTURE

Two transcripts, one of 3.8 kb and the other of 4.4 kb are detected in RNA prepared from adult females. A third transcript of 5.7 kb is found in late embryos, pupae and adult males. The two transcripts differ in the length of the 3' tail. There are multiple polyadenylation signals that determine transcript length (Suter, 1989)

Bases in 5' UTR - 934

Bases in 3' UTR - 912

PROTEIN STRUCTURE

Amino Acids - 782

Structural Domains

The sequence of Bic-D shows some similarity to the rod region of myosin heavy chains and to lamin, desmin, keratin, and other intermediate filament proteins, but there is no reason to believe that the protein is a myosin motor. The common feature of fibrous proteins such as the myosin heavy chain, is an extended alpha-helical coiled-coil structure that is built with a characteristic heptad repeat pattern, with hydrophobic residues at the first and fourth position (Suter, 1989 and Wharton, 1989).

A cDNA fragment homologous to the Drosophila Bicaudal-D gene (Bic-D) has been isolated using a hybridization selection procedure with cosmids derived from the short arm of human chromosome 12. A PCR-mediated cDNA cloning strategy was applied to obtain the coding sequence of the human homolog (BICD1) and to generate a partial mouse (Bicdh1) cDNA. The Drosophila Bicaudal-D gene encodes a coiled coil protein, characterized by five alpha-helix domains and a leucine zipper motif; the Drosophila protein forms part of the cytoskeleton and mediates the correct sorting of mRNAs for oocyte- and axis-determining factors during oogenesis. Analysis of the predicted amino acid sequence of the BICD1 cDNA clones indicates that the sequence similarity is essentially limited to the amphipatic helices and the leucine zipper, but the conserved order of these domains suggests a similar function of the protein in mammalians. A database search further indicates the existence of a second human homolog on chromosome arm 9q and a Caenorhabditis elegans homolog. Northern blot analysis indicates that both the human and the murine homologs are expressed in brain, heart, and skeletal muscle and during mouse embryonic development. The conserved structural characteristics of the BICD1 protein and its expression in muscle and especially brain suggest that BICD1 is a component of a cytoskeleton-based mRNA sorting mechanism conserved during evolution (Baens, 1997).

Genetic analysis in Drosophila suggests that Bicaudal-D functions in an essential microtubule-based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. A mammalian homolog of Bicaudal-D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co-localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein-tagged forms of BICD2 verify its intracellular distribution and co-localization with dynactin, and indicate that the C-terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N-terminal domain of BICD2 disrupts minus-end-directed organelle distribution and this portion of BICD2 co-precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2-dynactin-dynein co-localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein-dynactin interaction on the surface of membranous organelles, by associating with these complexes (Hoogenraad, 2001).

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. A yeast two-hybrid approach has been used to identify binding partners of Rab6a. In particular, the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologs of Drosophila Bicaudal-D, was identifed. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport (Matanis, 2002).

Human Nek8 is a new mammalian NIMA-related kinase, and its candidate substrate is Bicd2. Nek8 was isolated as a beta-casein kinase activity in rabbit lung and has an N-terminal catalytic domain homologous to the Nek family of protein kinases. Nek8 also contains a central domain with homology to RCC1, a guanine nucleotide exchange factor for the GTPase Ran, and a C-terminal coiled-coil domain. Like Nek2, Nek8 prefers beta-casein over other exogenous substrates, has shared biochemical requirements for kinase activity, and is capable of autophosphorylation and oligomerization. Nek8 activity is not cell cycle regulated, but like Nek3, levels are consistently higher in G(0)-arrested cells. During the purification of Nek8 a second protein co-chromatographed with Nek8 activity. This protein, Bicd2, is a human homolog of the Drosophila protein Bicaudal D, a coiled-coil protein. Bicd2 is phosphorylated by Nek8 in vitro, and the endogenous proteins associate in vivo. Bicd2 localizes to cytoskeletal structures, and its subcellular localization is dependent on microtubule morphology. Treatment of cells with nocodazole leads to dramatic reorganization of Bicd2, and correlates with Nek8 phosphorylation. This may be indicative of a role for Nek8 and Bicd2 associated with cell cycle independent microtubule dynamics (Holland, 2002).

Bicaudal D is an evolutionarily conserved protein that is involved in dynein-mediated motility both in Drosophila and in mammals. The N-terminal portion of human Bicaudal D2 (BICD2) is capable of inducing microtubule minus end-directed movement independently of the molecular context. This characteristic offers a new tool to exploit the relocalization of different cellular components by using appropriate targeting motifs. The BICD2 N-terminal domain has been used as a chimera with mitochondria and peroxisome-anchoring sequences to demonstrate the rapid dynein-mediated transport of selected organelles. Surprisingly, unlike other cytoplasmic dynein-mediated processes, this transport shows very low sensitivity to overexpression of the dynactin subunit dynamitin. The dynein-recruiting activity of the BICD2 N-terminal domain is reduced within the full-length molecule, indicating that the C-terminal part of the protein might regulate the interaction between BICD2 and the motor complex. These findings provide a novel model system for dissection of the molecular mechanism of dynein motility (Hoogenraad, 2003).

In mammals, two homologs of Bicaudal D, BICD1 and BICD2, are present (Baens, 1997; Hoogenraad, 2001). Studies in cultured mammalian cells have shown that BICD proteins bind to the small GTPase Rab6, as well as to dynein and dynactin complexes, and therefore participate in recruitment of dynein motor to Rab6-positive membranes of the Golgi apparatus and cytoplasmic vesicles (Hoogenraad, 2001; Matanis, 2002; Short, 2002). However, in addition to BICD proteins, Rab6 GTPase can also interact directly with the p150Glued component of the dynactin complex (Short, 2002). This raises the possibility that BICD acts as an accessory factor for the dynein motor, but is not sufficient by itself to recruit it to organelles (Hoogenraad, 2003 and references therein).

BICD proteins consist of several coiled-coil domains, and previous studies have demonstrated that while the C-terminal domain is responsible for interaction with membranes via Rab6, the N-terminal domain binds to cytoplasmic dynein (Hoogenraad, 2001; Matanis, 2002). In addition, the N- and C-terminal domains of BICD can interact with each other. Based on these findings, it is proposed that when BICD binds to the cargo (cytoplasmic vesicle) via its C-terminal domain, the N-terminal domain of BICD2 becomes available for interaction with dynein motor, which, in its turn, would transport the vesicle. If this model is correct, tethering of the BICD N-terminus to membranous organelles, which are normally devoid of BICD (such as mitochondria or peroxisomes), should be sufficient to induce their transport by cytoplasmic dynein. In this study, this idea is tested, and it is shown that the N-terminal part of BICD2 protein is indeed a potent recruitment factor for dynein, and that it can act in different molecular contexts (Hoogenraad, 2003).

Multiple approaches were used to investigate (1) the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and (2) conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. It is concluded that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG (Sun, 2007).

The Rab6 subfamily of small GTPases consists of three different isoforms: Rab6A, Rab6A' and Rab6B. Both Rab6A and Rab6A' are ubiquitously expressed whereas Rab6B is predominantly expressed in brain. Recent studies have shown that Rab6A' is the isoform regulating the retrograde transport from late endosomes via the Golgi to the ER and in the transition from anaphase to metaphase during mitosis. Since the role of Rab6B is still ill defined, its intracellular environment and dynamic behavior were characterized. A Y-2H search for novel Rab6 interacting proteins identified Bicaudal-D1, a large coiled-coil protein known to bind to the dynein/dynactin complex and previously shown to be a binding partner for Rab6A/Rab6A'. Co-immunoprecipitation studies and pull down assays confirmed that Bicaudal-D1 also interacts with Rab6B in its active form. Using confocal laser scanning microscopy it was established that Rab6B and Bicaudal-D1 co-localize at the Golgi and vesicles that align along microtubules. Furthermore, both proteins co-localized with dynein in neurites of SK-N-SH cells. Live cell imaging revealed bi-directional movement of EGFP-Rab6B structures in SK-N-SH neurites. It is concluded that the brain-specific Rab6B is linked via Bicaudal-D1 to the dynein/dynactin complex, suggesting a regulatory role for Rab6B in the retrograde transport of cargo in neuronal cells (Wanschers, 2007).

Chlamydia species are obligate intracellular bacteria that replicate within a membrane-bound vacuole, the inclusion, which is trafficked to the peri-Golgi region by processes that are dependent on early chlamydial gene expression. Although neither the host nor the chlamydial proteins that regulate the intracellular trafficking have been clearly defined, several enhanced green fluorescent protein (EGFP)-tagged Rab GTPases, including Rab6, are recruited to Chlamydia trachomatis inclusions. To further characterize the association of Rab6 with C. trachomatis inclusions, the intracellular localization of guanine nucleotide-binding mutants of Rab6 was examined, and it was demonstrated that only active GTP-bound and not inactive GDP-bound EGFP-Rab6 mutants were recruited to the inclusion, suggesting that EGFP-Rab6 interacts with the inclusion via a host Rab6 effector or a chlamydial protein that mimics a Rab6 effector. Using EGFP-tagged fusion proteins, it was also demonstrated that the Rab6 effector Bicaudal D1 (BICD1) localized to C. trachomatis inclusions in a biovar-specific manner. In addition, EGFP-Rab6 and its effector EGFP-BICD1 are recruited to the inclusion in a microtubule- and Golgi apparatus-independent but chlamydial gene expression-dependent mechanism. Finally, in contrast to the Rab6-dependent Golgi apparatus localization of endogenous BICD1, EGFP-BICD1 was recruited to the inclusion by a Rab6-independent mechanism. Collectively, these data demonstrate that neither Rab6 nor BICD1 is trafficked to the inclusion via a Golgi apparatus-localized intermediate, suggesting that each protein is trafficked to the C. trachomatis serovar L2 inclusion by a unique, but as-yet-undefined, mechanism (Moorhead, 2007).

Bicaudal D family adaptor proteins control the velocity of Dynein-based movements

Cargo transport along microtubules is driven by the collective function of microtubule plus- and minus-end-directed motors. How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. This study, carried out in primary hippocampal cultures, combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. The velocities of kinesin-based vesicle movements were found to be slower and more homogeneous than those of dynein-based movements. It was also found that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. This study suggests that adaptor proteins can modulate the velocity of dynein-based motility and thereby control the distribution of transport carriers (Schlager, 2014: PubMed).

A role for Bicaudal-D2 in radial cerebellar granule cell migration

Bicaudal-D (BICD) belongs to an evolutionary conserved family of dynein adaptor proteins. It was first described in Drosophila as an essential factor in fly oogenesis and embryogenesis. Missense mutations in a human BICD homologue, BICD2, have been linked to a dominant mild early onset form of spinal muscular atrophy. This study further examine the in vivo function of BICD2 in Bicd2 knockout mice. BICD2-deficient mice develop disrupted laminar organization of cerebral cortex and the cerebellum, pointing to impaired radial neuronal migration. Using astrocyte and granule cell specific inactivation of BICD2, it was shown that the cerebellar migration defect is entirely dependent upon BICD2 expression in Bergmann glia cells. Proteomics analysis reveals that Bicd2 mutant mice have an altered composition of extracellular matrix proteins produced by glia cells. These findings demonstrate an essential non-cell-autonomous role of BICD2 in neuronal cell migration, which might be connected to cargo trafficking pathways in glia cells (Jaarsma, 2014).

date revised: 22 December 2023 

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.