BIOLOGICAL OVERVIEW

Kinesin is expressed in virtually all cells of both vertebrates and invertebrates. The majority of kinesin appears to be free in cytosol, but various studies have shown that it can associate with endoplasmic recticulum (ER), vesicles, mitochondria, and other organelles. Function disruption tests indicate that it is critical for fast organelle transport in axons, although the set of cargoes it carries is not clearly defined (see for example Goldstein, 2000). Other studies of non-neuronal cells or cell-free systems suggest that kinesin is important for the positioning of lysosomes, mitochondria, and ER. It is also thought to be important for vesicle transport from the Golgi to the plasma membrane, one of the late steps in the secretion pathway, and in Golgi-to-ER membrane recycling, which is an indirect but essential part of the early secretion pathway. For example, based on the effects of anti-kinesin heavy chain antibodies or a KHC tail fragment microinjected into sea urchin embryos, kinesin has been proposed to be important for outward transport, to the cortex, of a class of vesicles used for rapid membrane repair. Furthermore, based on immunolocalization and on the effects of anti-KHC antibodies on brefeldin A-induced Golgi-to-ER membrane transport in vertebrate cultured cells, kinesin has been proposed to be the motor for Golgi-to-ER membrane recycling (R. P. Brendza, 2000a and references therein).

Kinesin heavy chain (Khc) is the plus end directed microtubule motor protein of Drosophila. Conventional kinesin (often referred to simply as kinesin) is an abundant microtubule motor protein that functions in a number of important intracellular transport processes. Kinesin is a heterotetramer, comprised of 2 kinesin heavy chains and 2 light chains. The heavy chains dimerize to form an elongated stalk with 2 amino-terminal globular domains ('motor domains') at one end and the light chains at the other end. The light chains and stalk are expected to bind the cytoplasmic cargoes that conventional kinesin transports. Each motor domain couples a cycle of ATP turnover to conformational changes and a cycle of microtubule binding and release that generates displacement toward the microtubule plus end. A single motor domain is not processive. In contrast, dimerized motor domains are remarkably processive, making hundreds of steps before releasing from the microtubule. This feature is probably critical for the resolute transport of small organelles that can present only one or a few kinesin molecules to a microtubule (Brendza, 1999 and references therein).

The Kinesin heavy chain moves its cytoplasmic cargo toward the plus ends of microtubules. Microtubules are relatively long, straight polymers that course throughout the cytoplasm. In undifferentiated and in fibroblastoid cell types, the minus ends of microtubules are usually near the cell center, whereas the plus ends are usually near the periphery. This is consistent with the idea that most microtubule-based, outward vesicle movements (Golgi-to-ER or Golgi-to-plasma membrane) are driven by plus end-directed kinesins, and most inward movements are driven by cytoplasmic dyneins. In differentiated cells, a variety of microtubule orientations are seen. For instance, in the axons of neurons, almost all plus ends are away from the cell center and toward the terminal. In some but not all polarized epithelial cells, microtubules are oriented with their plus ends toward the basal pole and their minus ends near the apical pole. In such situations, in which the polarity is relatively uniform, models have been developed that invoke appropriate microtubule motors for various steps in vesicle or other organelle transport (R. P. Brendza, 2000a and references therein).

To address questions about kinesin function in motility processes, other than axonal transport, in an intact organism, an examination was carried out of the effects of recessive, Khc null mutations on various cell types in Drosophila. When the entire organism is homozygous for a null mutation, it becomes paralyzed and dies during the midlarval stage, preventing studies of Khc function during the remainder of development. To circumvent this lethality, a mitotic recombination strategy was used to generate chimeras with a few homozygous null cells in otherwise healthy heterozygous organisms. The fates of the descendants of such Khc null cells were then studied by light and electron microscopy. A focus was placed on two predictions of the hypothesis that kinesin is an important motor for vesicle transport in the late secretory and recycling pathways. (1) Because disruption of vesicle traffic should block membrane growth and thus cell proliferation, the proliferation of Khc null imaginal cells was assessed. Surprisingly, the null cells proliferate normally to produce large clones of adult cells. (2) Because disruption of vesicle traffic can cause striking changes in the organization of ER and Golgi as well as defective secretion, postmitotic cells that rely heavily on the secretory pathway were studied in detail. Defects consistent with a role for kinesin in the Golgi-to-ER recycling pathway are not seen. However, defects were seen consistent with a role for kinesin in long-distance late secretory vesicle transport (Golgi-to-plasma membrane) (R. P. Brendza, 2000a).

A mechanosensory bristle shaft forms as a fluted cylinder of cuticle around a long cytoplasmic extension that projects outward from a trichogen cell body. During bristle shaft differentiation, which occurs in pupae, the core of the extension contains many parallel microtubules running from the base toward the tip. Because the Golgi and nucleus of the trichogen are located in the cell body beneath the pupal epidermis, it is likely that secretion of the shaft cuticle components requires a great deal of vesicular traffic from the Golgi into and along the shaft-forming extension. The mechanosensory bristles in Khc^null wing clones are sometimes kinked. To examine possible cuticle secretion defects in more detail, Khc^null clones were examined throughout the adult epidermis. The longest null bristles had defects that were obvious even with a low-power light microscope. Some lay flat or twisted along the epidermal sheet rather than projecting outward from its surface. A number of those that did project outward were tested by direct mechanical manipulation. Beheaded flies will remain viable for several days in a moist chamber. They stand motionless but can respond to bristle deflections with reflex grooming behaviors (R. P. Brendza, 2000a).

Outside of test clones, the deflection of individual bristles with a tungsten needle caused normal pivoting at the base and elicited grooming reflexes. Inside test clones, bristles were so flaccid that attempted deflections usually caused a bend or kink rather than the pivoting needed to elicit a grooming reflex. Bristles from null and control clones were studied in detail by SEM. Khc^null bristles have a variety of structural defects. The longest test class bristles, the scutellar macrochaetae (300-400 µm), are usually ~20% shorter than the analogous wild-type bristles. This length defect is less evident in shorter macrochaetae and is not seen in microchaetae. The tips of test bristles are often contorted, and the contortions are most severe at the tips of long machrochaetae, which always exhibit flattened, flared, or twisted tips. Mutant microchaetae (65-70 µm) show less dramatic defects, such as bluntness or slight tip swelling. No defects are observed in the remainder of the integument, including the bristle sockets, the tiny (10-15 µm) nonsensory hairs of the epidermal cells, or the epidermal cuticle sheet. The severities of head, thoracic, and abdominal bristle defects were not detectably affected by clone size (R. P. Brendza, 2000a).

The evident weakness of Khc^null bristle shafts suggests that cuticle secretion from the shaft-forming extensions of trichogen cells is defective during differentiation. Consistent with this, SEM images showed defects in cuticle fluting. To study bristle cuticle pattern and thickness in more detail, serial cross-sections of wild-type and Khc^null scutellar bristles were compared by TEM. Overall, the cuticle layers of null bristles are quite thin. This effect is more pronounced at the tips of bristles than at their bases. These results suggest that KHC is critical for long-distance transport of secretory vesicles that bear cuticle precursors from the Golgi into and along the bristle shaft-forming extension. However, the fact that some cuticle secretion occurs even at the tips of the longest null bristles suggests that vesicle transport can continue at a low level despite the absence of KHC (R. P. Brendza, 2000a).

Conventional kinesin has long been suspected of being a vesicle motor. Initially this stemmed from its discovery in axoplasm, which is rich in Golgi-derived transport vesicles, and its co-localization with vesicles in cultured cells. A number of studies have focused on the identification of specific types of vesicles that kinesin might carry, but the results have not provided a consistent answer. For example, in a study of vesicle/tubule transport in the recycling pathway, antibody inhibition of KHC blocked brefeldin A-induced movement of Golgi membranes into the ER in cultured NRK cells. Conversely, antisense oligonucleotide inhibition of KHC in cultured rat astrocytes and gene disruption in cultured mouse extraembryonic cells does not prevent brefeldin A-induced Golgi-to-ER membrane transfer. With regard to Golgi-to-plasma membrane vesicle transport, antisense oligonucleotide inhibition of KHC in cultured vertebrate neurons impairs delivery of vesicles containing certain synaptic proteins to axon terminals. In contrast, Khc mutations in Drosophila (Gho, 1992) and Caenorhabditis elegans do not prevent the accumulation of normal levels of synaptic vesicles at axon terminals (R. P. Brendza, 2000a and references therein).

It has also been proposed that conventional kinesin is a motor for other elements of the cytoplasm, including mitochondria, lysosomes, ER, and intermediate filaments (Yabe, 1999 and reviews by Goodson, 1997; Lane, 1998; Goldstein, 1999). However, function disruption tests have again yielded conflicting data. Antisense oligonucleotide inhibition of KHC in cultured rat astrocytes causes a retraction of the ER network. In contrast, antibody inhibition of KHC in sea urchin embryonic cells or gene disruption in mouse extraembryonic cells has no dramatic effects on ER organization. Antibody inhibition of KHC in human fibroblasts and gene disruption in mouse extraembryonic cells causes mitochondria, which are normally dispersed throughout the cytoplasm, to cluster near the cell center. However, no effect on mitochondrial distribution is seen in rat astrocytes injected with Khc antisense oligonucleotides or in mutant strains of C. elegans and fungi (R. P. Brendza, 2000a and references therein).

Overall, in the context of the cells and processes examined in Drosophila, the results suggest (1) that conventional kinesin is not essential for vesicle movement from the Golgi to either the ER or the plasma membrane in most cells; (2) that it is important for proper long-range vesicle transport in some elongated cells, and (3) that it is not required for the proper distribution of ER or mitochondria, at least in photoreceptor cells. Comparisons of control and Khc^null clones indicate that in the undifferentiated cells of imaginal discs, neither the rates nor the extents of cell proliferation are affected by a depletion of KHC. This should put to rest any lingering suspicion that conventional kinesin might have an essential role in mitosis. Furthermore, this comparision shows that conventional kinesin is not essential for any of the interphase motility processes required for imaginal cell growth. Whether the positioning of ER, lysosomes, or mitochondria is critical for cell growth is not clear. However, it is clear that vesicle transport in the secretory pathway is essential. For instance, in S. cereviseae, mutations that inhibit the Golgi-to-ER recycling pathway or the Golgi-to-plasma membrane 'late' secretory pathway cause a rapid halt to cell growth and division. More directly relevant to the results of this study, when mitotic recombination was used in Drosophila to create cells null for Rop, a homolog of the late secretory gene SEC1 of yeast, imaginal cells could not proliferate sufficiently to produce clones of adult cells. Mutations in syntaxin, which encode a t-SNARE thought to interact with ROP protein, also block imaginal cell proliferation. Thus, the proliferation and development of Drosophila imaginal cells is indeed sensitive to disruption of known elements of the secretion pathway. The fact that imaginal cell proliferation is not affected by a loss of KHC suggests that both the recycling pathway and the late secretion pathway can operate at normal rates with little or no conventional kinesin in small cells (R. P. Brendza, 2000a and references therein).

Defects in eye differentiation caused by a loss of KHC were mild and again are not consistent with major secretory pathway defects. Previous work has shown that disruption of the secretory pathway in photoreceptors should cause easily recognized changes in ultrastructure. For example, expression in the adult eye of a dominant negative form of a protein thought to function early in the secretory pathway, Rab1, causes hypertrophy and swelling of the ER, vesiculation or absence of the Golgi, and dramatic shrinkage of rhabdomeres. Shrunken rhabdomeres are also caused by mutations that block the transport of vesicles bearing rhodopsin from the Golgi to the plasma membrane. Although a loss of KHC did cause some mild structural defects in a few photoreceptor cell bodies, it did not cause shrunken rhabdomeres or any major changes in the organization of cytoplasmic organelles, including mitochondria, the nucleus, ER, and Golgi (R. P. Brendza, 2000a and references therein).

If conventional kinesin is dispensable for the secretion pathway in imaginal and retinal cells, how is vesicle movement away from the Golgi accomplished? Four alternative and nonexclusive force-generating systems come to mind: diffusion, minus end-directed microtubule motors, cytoplasmic myosins, and plus end-directed kinesin-related proteins. In some differentiated vertebrate epithelial cells, microtubule arrays have mixed polarity with some minus ends near the apical cortex and some near the cell center. Therefore, minus end-directed motors such as cytoplasmic dynein might be sufficient for vesicle transport in both directions. The involvement of plus end-directed kinesin-related motors is an attractive possibility. There are several different types of motors in the kinesin superfamily that might function in the recycling and late secretion pathways (reviewed by Goldstein, 1999). Whether these motors are expressed in imaginal cells is uncertain. However, it is reasonable to think that one or more of them is present and active in vesicle transport (R. P. Brendza, 2000a and references therein).

The elongated trichogen cells that create mechanosensory bristles (~1-2 × 100-400 µm) clearly do require conventional kinesin for normal secretion of cuticle precursors. Previous studies of another elongated Drosophila cell type, the larval motor neuron (~0.3 × 300-2000 µm), have shown that conventional kinesin is important in axons for membrane excitability, terminal growth, neurotransmitter secretion, and fast organelle transport (Gho, 1992; Hurd, 1996a; Hurd, 1996b; Gindhart, 1998). In the shaft-forming extensions of trichogen cells, as in axons, parallel arrays of microtubules are prominent. In both motor axons and bristles, the loss of KHC has a graded effect; distal regions are most strongly affected, and the defects become more severe as cell length increases (Gho, 1992; Hurd, 1996a; R. P. Brendza, 2000a). These observations suggest that conventional kinesin function is especially important for long-range vesicle movements, processes that are likely to demand efficient, highly processive transport machinery (R. P. Brendza, 2000a).

It is possible that conventional kinesin is a 'specialty motor,' a major contributor only to long-distance transport in specialized cells, whereas more ordinary motility is accomplished by other motors, such as cytoplasmic myosins and kinesin-related proteins. The evolutionary conservation of KHC in metazoans and its ubiquitous expression in both undifferentiated and differentiated cell types argue against this hypothesis, but it remains possible. Alternatively, conventional kinesin could be a more general motor, combining with kinesin-related proteins and myosins as a contributor to both short- and long-range movement of a variety of organelles. In this case, the other organelle/vesicle motors could compensate for the absence of kinesin sufficiently to prevent dramatic defects in cells with transport tracks of normal length. However, as track length and the requirement for transport efficiency increases, the lack of kinesin would cause progressively more severe defects. That is consistent with what has been seen in the R. P. Brendza (2000a) study. Some of the conflicting results seen in KHC function disruption tests (Goodson, 1997; Lane, 1998) could then be due to differences in cell geometry and to variations in the sets of myosins and kinesin-related motors that are expressed in the different systems studied; that is, kinesin gets more or less support from other motors depending on cell size, cell type, and perhaps culture conditions. In either case, as has been found for mitotic chromosome movements, it is probable that interphase organelle/vesicle movements are driven by the cooperative activities of multiple types of motors and that monogamous motor-cargo relationships in common transport processes are rare. For both chromosomes and interphase organelles, a clear view of such cooperative or multilayered transport systems will require rigorous definitions of specific motor-cargo relationships, including linkage mechanisms and regulatory controls (R. P. Brendza, 2000a).

Kinesin heavy chain function in Drosophila glial cells controls neuronal activity

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. This study demonstrates that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood-brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, Khc-dependent Rab proteins were determined in glia, and evidence is presented that Neurexin IV, a well known blood-brain barrier constituent, is one of the relevant cargo proteins. This work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia (Schmidt, 2012).

It is well established that the modulation of neuronal functionality depends on the ability of glial cells to provide metabolic support, regulate neurotransmitter homeostasis and influence the electrical conductance. To decipher these processes a large-scale RNAi screen was performed and ~5000 genes were silenced specifically in glial cells. This study presents an unexpected glial function of Drosophila khc in regulating neuronal activity. The specificity of the phenotype was ascertained by interspecies rescue studies and cell type-specific rescue of the khc mutant. Furthermore, the fact that glial-specific knockdown of β3-tubulin results in identical neuronal phenotypes demonstrates the functional relevance of Khc-dependent transport along microtubules (Schmidt, 2012).

Drosophila Khc was identified more than 20 years ago. In khc mutant larvae, vesicles, lysosomes, and mitochondria accumulate in axons. This results in swollen axons and eventually in larval paralysis. This study has generated a number of UAS-driven khc constructs to examine cell type-specific requirements of Khc. Two different khc alleles, khc⁶ and khc⁸, were used in trans to a genomic deficiency to eliminate background effects. Surprisingly, ubiquitous expression of Khc is not able to rescue the hypomorphic allele khc⁶. In contrast the null mutation khc⁸ can be completely rescued by Khc expression. khc⁶ contains an alteration in coil 2 of the stalk domain, a region implemented to be important for Kinesin light chain and cargo linkage. Thus, khc⁶ may induce some antimorphic functions. But the introduction of a genomic rescue construct of khc (P-khc⁺) has been described as being able to rescue khc⁶-associated lethality, which points to very high levels of endogenous (Schmidt, 2012).

Using a null mutant background (khc⁸/Df(2R)Jp6), it was possible to perform cell type-specific rescue experiments. Following expression of khc in neurons using elavGal4 only 6% of the expected flies survived. These flies were very sluggish and died within 6 d. To analyze glial-specific requirements of Khc, the ubiquitous Gal4-dependent rescue was compare with and without a glial cell-specific Gal4 repressor (repoGal80). A significant difference was apparent, the repressor reduced viability down to ~60%, which further supports the crucial role of Khc in glial cells (Schmidt, 2012).

Glial-specific knockdown of khc revealed an adult phenotype, which resembled a hyperexcitation phenotype. khc knockdown flies have the ability to walk. However, to initiate flight, flies need to jump. Upon khc knockdown, flies fail to perform this jump but rather directly start with a wing beat. Such flies then turn on their backs or spin over the floor, which then results in the characteristic popcorn-like movements. Thus, khc knockdown delays or prevents the jumping response (Schmidt, 2012).

In larvae, glial knockdown of khc results in reduced and delayed evoked responses at the muscle. Concomitantly, swellings of the peripheral nerves were apparent. In the majority of the swellings (and only in the swellings) a mislocalization of the septate junction protein Neurexin IV was found. This indicates that knockdown of khc results in an opening of the blood-brain barrier. In turn, this results in influx of potassium into the nervous system. Such influx is known to induce changes in axonal physiology and conductance velocity. Recently the role of focal adhesion kinase 56 (fak56) in Drosophila nerves was reported. Focal adhesion kinase acts downstream of integrin receptors, which are also known to be expressed by Drosophila glial cells. Interestingly, loss of fak56 function in the subperineurial glial cells (which constitute the blood-brain barrier) lowers axonal conductance in larvae and also renders the larvae sluggish pointing toward the notion that adhesion is required for a full establishment of the blood-brain barrier (Schmidt, 2012).

The observed suppression of EJP could in principle also be due to presynaptic defects or defects in the glutamate receptors expressed by the muscle. However, in contrast to vertebrates, where terminal Schwann cells cover the NMJ, the NMJ of Drosophila larvae grows into the muscle cell, which forms a so called subsynaptic reticulum around the NMJ. In addition, only few (subperineurial) glial processes invade parts of the NMJ in a highly dynamic fashion and where they participate in the removal of presynaptic debris. To further discriminate between a possible synaptic or an axonal defect the latency of EJP suppression was compared by injecting the current in the nerve 200-300 μm distant from the NMJ or in the ventral nerve cord 1000 μm distant from the NMJ. In this setting the latency doubled suggesting that axonal conductance is impaired upon glial khc knockdown. Therefore, it is anticipated that the suppression of the EJPs caused by glial reduction of khc function is unlikely due to synaptic defects (Schmidt, 2012).

Moreover, if khc would modulate synaptic activities, it would be acting in adult flies. However, when khc function was silenced only during adult stages using the TARGET system, no abnormal locomotion was observed, demonstrating that Khc is needed during development and not during adult stages (Schmidt, 2012).

Several Rab proteins were identified as highly motile in glia and their motility was significantly reduced upon khc suppression. Rab30 has been identified as a downstream target of JNK signaling, which can control kinesin cargo linkage. In addition, JNK-interacting proteins (JIPs) link Kinesin with membrane vesicles and are involved in Khc activation and cargo release. For vertebrate Rab21 a possible role in targeted Integrin trafficking from and to the cleavage furrow during cell division and an involvement in the endocytic pathway have been described (Schmidt, 2012).

The swollen nerves are likely due to a defective blood-brain barrier. The integrity of septate junctions for normal locomotion has also been suggested by the analysis of mutants affecting several septate junction components. At the end of embryogenesis, lachesin, neurexin IV, and gliotactin mutant embryos are hyperactive before becoming immobile. Possibly, the influx of potassium and sodium ions into the nervous system affects conductivity. Interestingly, panglial downregulation of the Na-K-Cl cotransporter Ncc69 or the serine/threonine kinase Fray also results in locomotion defects characterized by jumping flies. Fray and Ncc69 act in the subperineurial glia but no affect on neuronal conductance had been determined in peripheral nerves (Leiserson, 2011a; Leiserson, 2011b). Thus, the disruption of septate junctions as seen following knockdown of khc likely affects ion homeostasis more dramatically (Schmidt, 2012).

Drosophila NrxIV is highly mislocalized upon glial khc suppression. Other examples of a role for kinesin in cell polarization have been described. KIF5B and KIF5C are involved in polarized transport of certain apically distributed proteins in cultured mammalian epithelial cells. Additionally, KIF13B has been shown to directly transport proteins like Dlg1 to myelinating sites in Schwann cells. Thus, a similar function might be performed by Drosophila Khc in subperineurial glial cells and Khc might be involved in the directed transport of transmembrane proteins to septate junctions (Schmidt, 2012).

Germ plasm anchoring is a dynamic state that requires persistent trafficking

Localized cytoplasmic determinants packaged as ribonucleoprotein (RNP) particles direct embryonic patterning and cell fate specification in a wide range of organisms. Once established, the asymmetric distributions of such RNP particles must be maintained, often over considerable developmental time. A striking example is the Drosophila germ plasm, which contains RNP particles whose localization to the posterior of the egg during oogenesis results in their asymmetric inheritance and segregation of germline from somatic fates in the embryo. Although actin-based anchoring mechanisms have been implicated, high-resolution live imaging revealed persistent trafficking of germ plasm RNP particles at the posterior cortex of the Drosophila oocyte. This motility relies on cortical microtubules, is mediated by kinesin and dynein motors, and requires coordination between the microtubule and actin cytoskeletons. Finally, RNP particle motility was shown to be required for long-term germ plasm retention. It is proposed that anchoring is a dynamic state that renders asymmetries robust to developmental time and environmental perturbations (Sinsimer, 2013).

To determine whether motors mediate microtubule-dependent germ plasm RNP particle motility in late-stage oocytes, advantage was taken of mutations that disrupt motor protein activity. The initial localization of osk mRNA during midoogenesis is mediated by the plus-end motor kinesin, and a null mutation in Kinesin heavy chain (Khc), the force-generating component of kinesin, causes mis-localization of germ plasm around the entire oocyte cortex during midoogenesis. Visualization of GFP-Vas in Khc mutant germline clones showed that this aberrant pattern persisted in late-stage oocytes. Despite the paucity of GFP-Vas particles at any one cortical location, examples were observed of dynamic behavior, and these occurred regardless of where on the cortex the particles were located. Quantification of the small population of GFP-Vas particles at the posterior cortex of Khc mutant oocytes showed a 1.7-fold reduction in the motile fraction as compared to wild-type oocytes and a 36% reduction in the median velocity of the motile particles (Sinsimer, 2013).

The finding that germ plasm RNP particle motility is reduced but not abolished in the complete absence of kinesin function suggested the involvement of a second motor. Therefore whether the minus-end motor dynein might be required was tested. Unlike kinesin, dynein is essential during early oogenesis, and complete abrogation of dynein activity precludes egg development. Consequently, the effect of hypomorphic mutations in Dynein heavy chain (Dhc) was examined; these eggs have reduced dynein activity but still complete oogenesis. Quantification of GFP-Vas in late-stage Dhc mutant oocytes showed a 1.6-fold decrease in the motile fraction (19%) as compared to wild-type. Moreover, the median velocity of motile particles in Dhc mutants was half that of wild-type oocytes. In a second approach, dynein activity was disrupted acutely in late-stage oocytes by heat shock-inducible expression of p50/Dynamitin (Dmn), a component of the dynactin complex that interferes with dynein activity when overexpressed. Although heat shock alone had a minor effect on GFP-Vas particle motility, there was a 2.4-fold decrease in the motile fraction when dynein was inactivated as compared to heat-shocked wild-type controls. Moreover, the median velocity of the motile population in late oocytes overexpressing Dmn was reduced by 30% compared to control oocytes. Thus, it is concluded that both kinesin and dynein contribute to germ plasm RNP motility. The comparable loss of motility in null kinesin and hypomorphic dynein mutants, however, suggests that dynein-mediated transport predominates (Sinsimer, 2013).

Both of these motors are also involved in transport events during midoogenesis: dynein for movement of mRNAs from nurse cells to oocyte and anteriorly directed transport within the oocyte, kinesin for posterior transport of osk. Previous work indicated that the bulk of germ plasm mRNA localization occurs not by motor-dependent transport, however, but by diffusion and entrapment of transcripts that enter the oocyte during nurse cell dumping. It has not been possible to resolve particles containing kinesin or dynein in late oocytes using current GFP fusions. Thus, the important question of when and where the association of motors with germ plasm RNP particles occurs awaits the development of new methods for visualization of motors in Drosophila oocytes (Sinsimer, 2013).

The association of localized germ plasm RNP complexes with dynein in late-stage oocytes may serve a second purpose, providing preassembled transport particles for germ cell inheritance in the early embryo. The process of germ cell formation initiates when centrosomes and/or astral microtubules associated with nuclei that migrate to the posterior of the syncytial embryo induce release of germ plasm from the posterior cortex. Recruitment of germ plasm to the centrosomes by dynein-dependent transport on astral microtubules is required for these nuclei to induce germ cell formation and for the inheritance of the germ plasm by the newly formed germ cells. The prior coupling of germ plasm RNP particles to dynein in the oocyte may allow their rapid accumulation on astral microtubules upon release from the cortex (Sinsimer, 2013).

Under conditions of stress such as nutrient deprivation or in the absence of potential mates, female flies will hold mature eggs until conditions improve to increase the likelihood of survival for their progeny. Notably, females can hold mature eggs for at least 15 days without consequence to the viability or fertility of their progeny). Thus, sustaining germ plasm localization through such a delay of fertilization and the onset of embryogenesis is biologically crucial. It is hypothesized that the persistent trafficking of germ plasm might provide a mechanism for retaining germ plasm at the posterior over long periods of time (Sinsimer, 2013).

Because dynein is a major mediator of germ plasm RNP motility and can be manipulated acutely, whether compromising dynein function in held eggs by inducible Dmn overexpression would lead to a progressive loss of germ plasm from the posterior was tested. Advantage was taken of a single-molecule fluorescent in situ hybridization (smFISH) method to detect endogenous nos and osk mRNAs in mature oocytes. smFISH provides a major advance for mRNA analyses during the vitellogenic stages of Drosophila oocytes, which are largely impervious to standard molecular probes. The amount of localized germ plasm was quantified by measuring fluorescence intensity for each probe (Sinsimer, 2013).

More than 70% of mature oocytes from wild-type control or Dmn-overexpressing females, dissected immediately following heat shock, exhibited robust germ plasm accumulation. A spreading of nos and osk along the cortex was observed in some oocytes overexpressing Dmn, likely due to an immediate effect of dynein inhibition on germ plasm retention following the 2 hr heat shock regimen. Examination of wild-type control oocytes held for 18 hr showed little effect of the holding period alone on localization of the germ plasm RNAs. In contrast, inhibition of dynein function in held oocytes led to a dramatic loss of both nos and osk from the posterior cortex, with robust localization persisting in fewer than 30%. Thus, it is concluded that dynein-mediated motility is required for long-term retention of germ plasm at the posterior cortex of the oocyte (Sinsimer, 2013).

Similarly, the effect of MyoV loss on germ plasm components was tested in held oocytes. In mature didum mutant oocytes, nos mRNA was properly localized to the posterior, and unlike the case for Dmn overexpression, nos persisted during the holding period. Together, these data support a model in which enhanced dynein-mediated motility facilitates nos RNP particle accumulation at the posterior. In contrast, osk mRNA was no longer confined to the posterior cortex in didum mutant oocytes but often had a graded or diffuse distribution. Thus, a requirement for MyoV function in entrapment and/or retention of osk extends into late oogenesis. Strikingly, a tight cortical distribution of osk was largely restored in didum mutant oocytes held for 18 hr. This suggests that given sufficient time, microtubule-based motility of osk RNP particles allows localization to recover in the absence of MyoV (Sinsimer, 2013).

Competition between kinesin-1 and myosin-V defines Drosophila posterior determination

Local accumulation of oskar (osk) mRNA in the Drosophila oocyte determines the posterior pole of the future embryo. Two major cytoskeletal components, microtubules and actin filaments, together with a microtubule motor, kinesin-1, and an actin motor, myosin-V, are essential for osk mRNA posterior localization. This study used Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for osk mRNA. Posterior localization of osk/Staufen was shown to be determined by competition between kinesin-1 and myosin-V. While kinesin-1 removes osk/Staufen from the cortex along microtubules, myosin-V anchors osk/Staufen at the cortex. Myosin-V wins over kinesin-1 at the posterior pole due to low microtubule density at this site, while kinesin-1 wins at anterior and lateral positions because they have high density of cortically-anchored microtubules. As a result, posterior determinants are removed from the anterior and lateral cortex but retained at the posterior pole. Thus, posterior determination of Drosophila oocytes is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density (Lu, 2020).

It is well established that kinesin-1 is essential for localization of osk/Staufen particles at the posterior pole of the Drosophila oocyte. However, it remained unclear how the compact posterior cap is anchored and retained over time. Cortical F-actin remodeling and Myosin-V, as well as the Arp1 subunit of the dynactin complex, have been all implicated in the osk/Staufen cortical localization. This study combined genetic and optogenetic tools to demonstrate that direct competition between two motors, kinesin-1 and myosin-V, ensures the posterior anchorage of osk/Staufen. Notably, it was demonstrated that the outcome of the competition is primarily determined by the density of cortical microtubules. High microtubule density at the anterior and lateral cortex favors kinesin-driven osk/Staufen cortical exclusion, while low microtubule density at the posterior pole favors myosin-driven cortical retention. Therefore, the kinesin-myosin competition and cortical microtubule density together determine the initial accumulation of osk mRNA at the posterior pole (Lu, 2020).

The cortical exclusion model was first proposed after uniform cortical localization of osk mRNA was observed in the kinesin-null oocytes. In agreement with this model, this study shows that constitutively active kinesin-1 causes osk/Staufen mislocalization in the cytoplasm of the oocyte, whereas reducing microtubule density at the lateral cortex leads to ectopic accumulation of Staufen at the cortex. These data support the model that kinesin-driven cortical exclusion along cortically-attached microtubules plays an essential role in restricting osk/Staufen to the posterior pole (Lu, 2020).

Previously, several groups have proposed that kinesin-1 transports osk/Staufen particles along slightly biased cortical microtubules, resulting in net movement of osk/Staufen from the anterior side to the posterior pole in stage 8-9 oocytes. In fact, kinesin-driven cortical exclusion and kinesin-driven transport towards the posterior pole are not mutually exclusive; they describe the same event of osk/Staufen movement. Within the oocyte, cortical microtubules are anchored to the cortex by their minus-ends while their plus-ends face towards the cytoplasm. Due to the anterior-posterior gradient of cortical microtubule density, more microtubule plus ends are oriented towards the posterior pole. Thus, kinesin-1-driven transport along microtubules is a prerequisite for kinesin-1-driven cortical exclusion. Cortical exclusion of osk/Staufen by kinesin-1 results in biased transport of osk/Staufen towards the posterior pole (Lu, 2020).

This kinesin-myosin competition model is suggested by genetic interaction data from a previous study. Specifically, increasing KHC dosage enhances osk/Staufen mislocalization phenotypes in myosin-V loss-of-function mutants, while reducing KHC dosage by half partially suppresses myosin loss-of-function phenotypes. Furthermore, double MyoV and Khc mutant clones have diffuse cytoplasmic localization of osk mRNA, compared to uniform cortical localization of osk mRNA in Khc single mutant clones. These data strongly imply that in the absence of kinesin-1, myosin-V promiscuously anchors osk/Staufen everywhere in the cortex. This study manipulated the activity of either kinesin-1 or myosin-V and found that proper balance between the activities of these two motors is critical for correct osk/Staufen localization, supporting the model in which kinesin-myosin competition is key to the correct posterior determination in the Drosophila oocyte (Lu, 2020).

The competition between kinesin-1 and myosin-V described in this study is not the first example of such a mechanism for cargo transport and localization. For instance, myosin-V opposes microtubule-dependent transport and provides a dynamic anchor for melanosomes, peroxisomes , recycling endosomes, mitochondria, and synaptic vesicles at sites of local accumulation of F-actin. At these sites, the abundance of F-actin tracks provides an upper hand for myosin-V to win the tug-of-wars over microtubule motors. Kinesin-myosin competition appears to be an evolutionarily conserved mechanism to allow flexible refinement and/or error correction, as motors constantly undergo reversible binding and releasing activity on cytoskeletal filaments (Lu, 2020).

In the oocytes, the machinery responsible for osk/Staufen localization contains the same basic building blocks; however, unlike the other systems, the outcome of the competition is not determined by actin filament density, as F-actin density is uniform along the oocyte cortex. Instead, the outcome of this competition is decided by abundance of microtubule tracks. Higher microtubule density at the anterior and lateral cortex favors kinesin-mediated cortical removal of osk/Staufen, while scarcity of microtubule tracks at the posterior pole favors myosin-V-dependent anchoring. To confirm this model, optogenetic tools were used to recruit a microtubule-depolymerizing kinesin, kinesin-13/Klp10A, to actin cortex, and thus locally modulate cortical microtubule density. Locally decreasing cortical microtubule density causes ectopic accumulation of Staufen at the cortex. The loss of microtubules prevents kinesin-driven cortical exclusion, which allows myosin-V to win the competition and form a patch of cortically localized Staufen. This recruitment of Staufen is reversible and repeatable, indicating this kinesin-myosin competition is continuous, and the outcome of this never-ending battle is decided by the local microtubule concentration (Lu, 2020).

Competition between kinesin-1 and myosin-V is sufficient for initial anchoring at the posterior pole Previously, synthetic motor domains of a plus-end motor, kinesin-1, and a minus-end motor, kinesin-14/Nod, were used to label overall microtubule polarity in Drosophila oocytes and neurons (Kin:βGal and Nod:βGal). As myosin-V is essential for osk/Staufen localization, in this study two synthetic motor constructs (KHC576 and MyoVHMM) were expressed in the oocyte, and their dimerization was induced using a rapalog-dependent dimerization system. Dimerized motors accumulate at the posterior pole, highly resembling the osk/Staufen localization. This posterior accumulation is dependent on the anterior-posterior microtubule gradient; dimerized motors fail to localize at the posterior pole after microtubule depolymerization. Together, using dimerized synthetic motors, this study demonstrated that direct competition (without any cargo binding) between a microtubule motor, kinesin-1, and an actin motor, myosin-V, is sufficient for initial posterior localization in a Drosophila oocyte (Lu, 2020).

In summary, this study has elucidated the anchorage mechanism for initial posterior localization of osk/Staufen during mid-oogenesis. Kinesin-1 competes with myosin-V to control osk/Staufen localization. The outcome of this kinesin-myosin competition is primarily determined by cortical microtubule density. Higher microtubule density at anterior-lateral cortex allows kinesin-1 to win and cortically exclude osk/Staufen, while lower microtubule density at posterior pole favors myosin-V-mediated anchorage at the cortex. Together, two cytoskeletal components (microtubules and F-actin) and two molecular motors (kinesin-1 and myosin-V) govern the posterior determination for future Drosophila embryos (Lu, 2020).

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

cDNA clone length - 3547

Bases in 5' UTR - 320

Exons - 4

Bases in 3' UTR - 299

PROTEIN STRUCTURE

Amino Acids - 975

Structural Domains

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a 'motor' domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative 'motor' domain, it is proposed that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules (Yang, 1989).

date revised: 25 October 2023

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