Kinesin heavy chain: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Kinesin heavy chain

Synonyms -

Cytological map position - 53A1--2

Function - vesicular transport motor protein

Keywords - cytoskeleton, axonal transport, mechanosensory bristles, oogenesis

Symbol - Khc

FlyBase ID: FBgn0001308

Genetic map position -

Classification - kinesin motor domain signature

Cellular location - cytoplasmic

NCBI link: Entrez Gene
Khc orthologs: Biolitmine
Recent literature
Veeranan-Karmegam, R., Boggupalli, D. P., Liu, G. and Gonsalvez, G. B. (2016). A new isoform of Drosophila non-muscle Tropomyosin 1 interacts with Kinesin-1 and functions in oskar mRNA localization. J Cell Sci 129: 4252-4264. PubMed ID: 27802167
Recent studies have revealed that diverse cell types use mRNA localization as a means to establish polarity. Despite the prevalence of this phenomenon, much less is known regarding the mechanism by which mRNAs are localized. The Drosophila melanogaster oocyte provides a useful model for examining the process of mRNA localization. oskar (osk) mRNA is localized at the posterior of the oocyte, thus restricting the expression of Oskar protein to this site. The localization of osk mRNA is microtubule dependent and requires the plus-end-directed motor Kinesin-1. Unlike most Kinesin-1 cargoes, localization of osk mRNA requires the Kinesin heavy chain (Khc) motor subunit, but not the Kinesin light chain (Klc) adaptor. This report, demonstrates that a newly discovered isoform of Tropomyosin 1, referred to as Tm1C, directly interacts with Khc and functions in concert with this microtubule motor to localize osk mRNA. Apart from osk mRNA localization, several additional Khc-dependent processes in the oocyte are unaffected upon loss of Tm1C. These results therefore suggest that the Tm1C-Khc interaction is specific for the osk localization pathway.
Kulkarni, A., Khan, Y. and Ray, K. (2016). Heterotrimeric kinesin-2, together with kinesin-1, steers vesicular acetylcholinesterase movements toward the synapse. Faseb j. [Epub ahead of print]. PubMed ID: 27920150
Acetylcholinesterase (AChE), which is implicated in the pathophysiology of neurological disorders, is distributed along the axon and enriched at the presynaptic basal lamina. It hydrolyses the neurotransmitter acetylcholine, which inhibits synaptic transmission. Aberrant AChE activity and ectopic axonal accumulation of the enzyme are associated with neurodegenerative disorders, such as Alzheimer's disease. The molecular mechanism that underlies AChE transport is still unclear. This study shows that expression of Drosophila AChE tagged with photoactivable green fluorescent protein and m-Cherry (GPAC) in cholinergic neurons compensates for the RNA interference-mediated knockdown of endogenous AChE activity. GPAC-AChE, which is enriched in the neuropil region of the brain, moves in the apparently vesicular form in axons with an anterograde bias in Drosophila larvae. Two anterograde motors, kinesin-1 and kinesin-2, propel distinct aspects of GPAC-AChE movements. Total loss of kinesin-2 reduces the density of anterograde traffic and increases bidirectional movements of GPAC-AChE vesicles without altering their speed. A partial loss of kinesin-1 reduces both the density and speed of anterograde GPAC-AChE traffic and enhances the pool of stationary vesicles. Together, these results suggest that combining activity of a relatively weak kinesin-2 with that of a stronger kinesin-1 motor could steer AChE-containing vesicles toward synapse, and provides a molecular basis for the observed subcellular distribution of the enzyme.
Gaspar, I., Sysoev, V., Komissarov, A. and Ephrussi, A. (2016). An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs. Embo J [Epub ahead of print]. PubMed ID: 28028052
Localization and local translation of oskar mRNA at the posterior pole of the Drosophila oocyte directs abdominal patterning and germline formation in the embryo. The process requires recruitment and precise regulation of motor proteins to form transport-competent mRNPs. The posterior-targeting kinesin-1 is loaded upon nuclear export of oskar mRNPs, prior to their dynein-dependent transport from the nurse cells into the oocyte. Kinesin-1 recruitment requires the DmTropomyosin1-I/C isoform, an atypical RNA-binding tropomyosin that binds directly to dimerizing oskar 3'UTRs. The isoform is on of 17 predicted mRNA isoforms and 13 distinct polypeptides encoded by the TM1 gene. Finally, a small but dynamically changing subset of oskar mRNPs gets loaded with inactive kinesin-1, and that the motor is activated during mid-oogenesis by the functionalized spliced oskar RNA localization element. This inefficient, dynamic recruitment of Khc decoupled from cargo-dependent motor activation constitutes an optimized, coordinated mechanism of mRNP transport, by minimizing interference with other cargo-transport processes and between the cargo-associated dynein and kinesin-1.
Lim, A., Rechtsteiner, A. and Saxton, W. M. (2017). Two kinesins drive anterograde neuropeptide transport. Mol Biol Cell [Epub ahead of print]. PubMed ID: 28904207
Motor-dependent anterograde transport, a process that moves cytoplasmic components from sites of biosynthesis to sites of use within cells, is crucial in neurons with long axons. Evidence has emerged that multiple anterograde kinesins can contribute to some transport processes. To test the multi-kinesin possibility for a single vesicle type, the functional relationships were studied of axonal kinesins to dense core vesicles (DCVs) that were filled with a GFP-tagged neuropeptide in the Drosophila nervous system. Past work showed that Unc-104 (a kinesin-3) is a key anterograde DCV motor. This study showed that anterograde DCV transport requires the well-known mitochondrial motor Khc (kinesin-1). The results indicate that this influence is direct. Khc mutations had specific effects on anterograde run parameters, neuron-specific inhibition of mitochondrial transport by Milton RNAi had no influence on anterograde DCV runs, and detailed co-localization analysis by super resolution microscopy revealed that Unc-104 and Khc co-associate with individual DCVs. DCV distribution analysis in peptidergic neurons suggest the two kinesins have compartment specific influences. A mechanism is suggested in which Unc-104 is particularly important for moving DCVs from cell bodies into axons, then Unc-104 and kinesin-1 function together to support fast, highly processive runs toward axon terminals.
Iacobucci, G. J. and Gunawardena, S. (2017). Ethanol stimulates the in vivo axonal movement of neuropeptide dense core vesicles in Drosophila motor neurons. J Neurochem [Epub ahead of print]. PubMed ID: 28960313
Proper neuronal function requires essential biological cargoes to be packaged within membranous vesicles and transported, intracellularly, through the extensive outgrowth of axonal and dendritic fibers. The precise spatiotemporal movement of these cargoes is vital for neuronal survival and, thus, is highly regulated. This study tested how the axonal movement of a neuropeptide containing dense core vesicle (DCV) responds to alcohol stressors. Ethanol was found to induce a strong anterograde bias in vesicle movement. Low doses of ethanol stimulate the anterograde movement of neuropeptide-DCV while high doses inhibit bidirectional movement. This process required the presence of functional kinesin-1 motors as reduction of kinesin prevented the ethanol-induced stimulation of the anterograde movement of neuropeptide-DCV. Furthermore, expression of inactive GSK-3beta also prevented ethanol-induced stimulation of neuropeptide-DCV movement, similar to pharmacological inhibition of GSK-3beta with lithium. Conversely, inhibition of PI3K/AKT signaling with wortmannin led to a partial prevention of ethanol-stimulated transport of neuropeptide-DCV. Taken together, it is concluded that GSK-3beta signaling mediates the stimulatory effects of ethanol. Therefore, this study provides new insight into the physiological response of the axonal movement of neuropeptide-DCV to exogenous stressors.
Herzmann, S., Gotzelmann, I., Reekers, L. F. and Rumpf, S. (2018). Spatial regulation of microtubule disruption during dendrite pruning in Drosophila. Development [Epub ahead of print]. PubMed ID: 29712642
Large scale neurite pruning is an important specificity mechanism during neuronal morphogenesis. Drosophila sensory neurons prune their larval dendrites during metamorphosis. Pruning dendrites are severed in their proximal regions, but how this spatial information is encoded is not clear. Dendrite severing is preceded by local breakdown of dendritic microtubules through PAR-1-mediated inhibition of Tau. This study investigated spatial aspects of microtubule breakdown during dendrite pruning. Live imaging of fluorescently tagged tubulin shows that microtubule breakdown first occurs at proximal dendritic branchpoints, followed by breakdown at more distal branchpoints, suggesting that the process is triggered by a signal emanating from the soma. In fly dendrites, microtubules are arranged in uniformly oriented arrays where all plus ends face towards the soma. Mutants in kinesin-1 and -2, which are required for uniform microtubule orientation, cause defects in microtubule breakdown and dendrite pruning. These data suggest that the local microtubule organization at branch points determines where microtubule breakdown occurs. Local microtubule organization may therefore contribute spatial information for severing sites during dendrite pruning.
Kelliher, M. T., Yue, Y., Ng, A., Kamiyama, D., Huang, B., Verhey, K. J. and Wildonger, J. (2018). Autoinhibition of kinesin-1 is essential to the dendrite-specific localization of Golgi outposts. J Cell Biol. PubMed ID: 29728423
Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. In vivo structure-function analysis of endogenous Drosophila melanogaster kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. These data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.
Lu, W., Lakonishok, M., Serpinskaya, A. S., Kirchenbuechler, D., Ling, S. C. and Gelfand, V. I. (2018). Ooplasmic flow cooperates with transport and anchorage in Drosophila oocyte posterior determination. J Cell Biol. PubMed ID: 30037924
The posterior determination of the Drosophila melanogaster embryo is defined by the posterior localization of oskar (osk) mRNA in the oocyte. Defects of its localization result in a lack of germ cells and failure of abdomen specification. A microtubule motor kinesin-1 is essential for osk mRNA posterior localization. Because kinesin-1 is required for two essential functions in the oocyte-transport along microtubules and cytoplasmic streaming-it is unclear how individual kinesin-1 activities contribute to the posterior determination. Staufen, an RNA-binding protein that is colocalized with osk mRNA, was examined as a proxy of posterior determination, and mutants were used that either inhibit kinesin-driven transport along microtubules or cytoplasmic streaming. Late-stage streaming is partially redundant with early-stage transport along microtubules for Staufen posterior localization. Additionally, an actin motor, myosin V, is required for the Staufen anchoring to the actin cortex. A model is proposed whereby initial kinesin-driven transport, subsequent kinesin-driven streaming, and myosin V-based cortical retention cooperate in posterior determination.
Oelz, D. B., Del Castillo, U., Gelfand, V. I. and Mogilner, A. (2018). Microtubule Dynamics, Kinesin-1 Sliding, and Dynein Action Drive Growth of Cell Processes. Biophys J. PubMed ID: 30268540
Recent experimental studies of the role of microtubule sliding in neurite outgrowth suggested a qualitative model, according to which kinesin-1 motors push the minus-end-out microtubules against the cell membrane and generate the early cell processes. At the later stage, dynein takes over the sliding, expels the minus-end-out microtubules from the neurites, and pulls in the plus-end-out microtubules that continue to elongate the nascent axon. This model leaves unanswered a number of questions: why is dynein unable to generate the processes alone, whereas kinesin-1 can? What is the role of microtubule dynamics in process initiation and growth? Can the model correctly predict the rates of process growth in control and dynein-inhibited cases? What triggers the transition from kinesin-driven to dynein-driven sliding? To answer these questions, computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors were combined with measurements of the process growth kinetics and pharmacological perturbations in Drosophila S2 cells. The results verify quantitatively the qualitative model of the microtubule polarity sorting and suggest that dynein-powered elongation is effective only when the processes are longer than a threshold length, which explains why kinesin-1 alone, but not dynein, is sufficient for the process growth. Furthermore, it was shown that the mechanism of process elongation depends critically on microtubule dynamic instability. Both modeling and experimental measurements show, surprisingly, that dynein inhibition accelerates the process extension. Implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion are discussed.
Russell, S. L., Lemseffer, N. and Sullivan, W. T. (2018). Wolbachia and host germline components compete for kinesin-mediated transport to the posterior pole of the Drosophila oocyte. PLoS Pathog 14(8): e1007216. PubMed ID: 30110391
Widespread success of the intracellular bacterium Wolbachia across insects and nematodes is due to efficient vertical transmission and reproductive manipulations. Many strains, including wMel from Drosophila melanogaster, exhibit a specific concentration to the germplasm at the posterior pole of the mature oocyte, thereby ensuring high fidelity of parent-offspring transmission. Transport of Wolbachia to the pole relies on microtubules and the plus-end directed motor kinesin heavy chain (KHC). However, the mechanisms mediating Wolbachia's association with KHC remain unknown. This study shows that reduced levels of the host canonical linker protein KLC results in dramatically increased levels of Wolbachia at the oocyte's posterior, suggesting that KLC and some key associated host cargos outcompete Wolbachia for association with a limited amount of KHC motor proteins. Consistent with this interpretation, over-expression of KHC causes similarly increased levels of posteriorly localized Wolbachia. However, excess KHC has no effect on levels of Vasa, a germplasm component that also requires KHC for posterior localization. Thus, Wolbachia transport is uniquely KHC-limited because these bacteria are likely outcompeted for binding to KHC by some host cargo/linker complexes. These results reveal a novel host-symbiont interaction that underscores the precise regulation required for an intracellular bacterium to co-opt, but not disrupt, vital host processes.
Metivier, M., Monroy, B. Y., Gallaud, E., Caous, R., Pascal, A., Richard-Parpaillon, L., Guichet, A., Ori-McKenney, K. M. and Giet, R. (2019). Dual control of Kinesin-1 recruitment to microtubules by Ensconsin in Drosophila neuroblasts and oocytes. Development 146(8). PubMed ID: 30936181
Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro. It is proposed that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.
Dimitrova-Paternoga, L., Jagtap, P. K. A., Cyrklaff, A., Vaishali, Lapouge, K., Sehr, P., Perez, K., Heber, S., Low, C., Hennig, J. and Ephrussi, A. (2021). Molecular basis of mRNA transport by a kinesin-1-atypical tropomyosin complex. Genes Dev 35(13-14): 976-991. PubMed ID: 34140355
Kinesin-1 carries cargos including proteins, RNAs, vesicles, and pathogens over long distances within cells. The mechanochemical cycle of kinesins is well described, but how they establish cargo specificity is not fully understood. Transport of oskar mRNA to the posterior pole of the Drosophila oocyte is mediated by Drosophila kinesin-1, also called kinesin heavy chain (Khc), and a putative cargo adaptor, the atypical tropomyosin, aTm1. How the proteins cooperate in mRNA transport is unknown. This study presents the high-resolution crystal structure of a Khc-aTm1 complex. The proteins form a tripartite coiled coil comprising two in-register Khc chains and one aTm1 chain, in antiparallel orientation. aTm1 binds to an evolutionarily conserved cargo binding site on Khc, and mutational analysis confirms the importance of this interaction for mRNA transport in vivo. Furthermore, this study demonstrates that Khc binds RNA directly and that it does so via its alternative cargo binding domain, which forms a positively charged joint surface with aTm1, as well as through its adjacent auxiliary microtubule binding domain. Finally, aTm1 was shown to plays a stabilizing role in the interaction of Khc with RNA, which distinguishes aTm1 from classical motor adaptors.
Hannaford, M. R., Liu, R., Billington, N., Swider, Z. T., Galletta, B. J., Fagerstrom, C. J., Combs, C., Sellers, J. R. and Rusan, N. M. (2022). Pericentrin interacts with Kinesin-1 to drive centriole motility. J Cell Biol 221(9). PubMed ID: 35929834
Centrosome positioning is essential for their function. Typically, centrosomes are transported to various cellular locations through the interaction of centrosomal microtubules (MTs) with motor proteins anchored at the cortex or the nuclear surface. However, it remains unknown how centrioles migrate in cellular contexts in which they do not nucleate MTs. This study demonstrates that during interphase, inactive centrioles move directly along the interphase MT network as Kinesin-1 cargo. Pericentrin-Like-Protein (PLP) as a novel Kinesin-1 interacting molecule essential for centriole motility. In vitro assays show that PLP directly interacts with the cargo binding domain of Kinesin-1, allowing PLP to migrate on MTs. Binding assays using purified proteins revealed that relief of Kinesin-1 autoinhibition is critical for its interaction with PLP. Finally, these studies of neural stem cell asymmetric divisions in the Drosophila brain show that the PLP-Kinesin-1 interaction is essential for the timely separation of centrioles, the asymmetry of centrosome activity, and the age-dependent centrosome inheritance.

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 Khcnull wing clones are sometimes kinked. To examine possible cuticle secretion defects in more detail, Khcnull 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. Khcnull 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 Khcnull 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 Khcnull 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 Khcnull 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).


cDNA clone length - 3547

Bases in 5' UTR - 320

Exons - 4

Bases in 3' UTR - 299


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).

Kinesin heavy chain: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 22 November 2022

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