Dynein heavy chain 64C: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Dynein heavy chain 64C

Synonyms -

Cytological map position - 64C1--9

Function - molecular motor

Keywords - cytoskeleton, mitosis, vesicular transport

Symbol - Dhc64C

FlyBase ID: FBgn0261797

Genetic map position - 3-[19]

Classification - dynein-(cytoplasmic)-heavy-chain

Cellular location - cytoplasmic

NCBI link: Entrez Gene
Dhc64C orthologs: Biolitmine
Recent literature
Zur Lage, P., Stefanopoulou, P., Styczynska-Soczka, K., Quinn, N., Mali, G., von Kriegsheim, A., Mill, P. and Jarman, A. P. (2018). Ciliary dynein motor preassembly is regulated by Wdr92 in association with HSP90 co-chaperone, R2TP. J Cell Biol. Pubmed ID: 29743191
The massive dynein motor complexes that drive ciliary and flagellar motility require cytoplasmic preassembly, a process requiring dedicated dynein assembly factors (DNAAFs). How DNAAFs interact with molecular chaperones to control dynein assembly is not clear. By analogy with the well-known multifunctional HSP90-associated cochaperone, R2TP, several DNAAFs have been suggested to perform novel R2TP-like functions. However, the involvement of R2TP itself (canonical R2TP) in dynein assembly remains unclear. This study shows that in Drosophila melanogaster, the R2TP-associated factor, Wdr92, is required exclusively for axonemal dynein assembly, likely in association with canonical R2TP. Proteomic analyses suggest that in addition to being a regulator of R2TP chaperoning activity, Wdr92 works with the DNAAF Spag1 at a distinct stage in dynein preassembly. Wdr92/R2TP function is likely distinct from that of the DNAAFs proposed to form dynein-specific R2TP-like complexes. These findings thus establish a connection between dynein assembly and a core multifunctional cochaperone.
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.
Melkov, A., Baskar, R., Shachal, R., Alcalay, Y. and Abdu, U. (2019). The organization of Golgi in Drosophila bristles requires microtubule motor protein function and a properly organized microtubule array. PLoS One 14(10): e0223174. PubMed ID: 31577833
This study used highly elongated Drosophila bristle cells to dissect the role of dynein heavy chain (Dhc64C) in Golgi organization. Whereas in the bristle "somal" region Golgi units are composed of cis-, medial, and trans-Golgi compartments ("complete Golgi"), the bristle shaft contains Golgi satellites that lack the trans-Golgi compartment (hereafter referred to as "incomplete Golgi") and which are static and localized at the base area. However, in Dhc64C mutants, the entire bristle shaft was filled with complete Golgi units containing ectopic trans-Golgi components. To further understand Golgi bristle organization, the roles of microtubule (MT) polarity and the Dhc-opposing motor, kinesin heavy chain (Khc), were tested. Surprisingly, in Khc and Ik2Dominant-negative (DN) flies in which the polarized organization of MTs is affected, the bristle shaft was filled with complete Golgi, similarly to what is seen in Dhc64C flies. Thus, this study demonstrated that MTs and the motor proteins Dhc and Khc are required for bristle Golgi organization. However, the fact that both Dhc64C and Khc flies showed similar Golgi defects calls for an additional work to elucidate the molecular mechanism describing why these factors are required for bristle Golgi organization.
Aguilar-Aragon, M., Fletcher, G. and Thompson, B. J. (2019). The cytoskeletal motor proteins Dynein and MyoV direct apical transport of Crumbs. Dev Biol. PubMed ID: 31881198
Crumbs (Crb in Drosophila; CRB1-3 in mammals) is a transmembrane determinant of epithelial cell polarity and a regulator of Hippo signalling. Crb is normally localized to apical cell-cell contacts, just above adherens junctions, but how apical trafficking of Crb is regulated in epithelial cells remains unclear. This study used the Drosophila follicular epithelium to demonstrate that polarized trafficking of Crb is mediated by transport along microtubules by the motor protein Dynein and along actin filaments by the motor protein Myosin-V (MyoV). Blocking transport of Crb-containing vesicles by Dynein or MyoV leads to accumulation of Crb within Rab11 endosomes, rather than apical delivery. The final steps of Crb delivery and stabilisation at the plasma membrane requires the exocyst complex and three apical FERM domain proteins - Merlin, Moesin and Expanded- whose simultaneous loss disrupts apical localization of Crb. Accordingly, a knock-in deletion of the Crb FERM-binding motif (FBM) also impairs apical localization. Finally, overexpression of Crb challenges this system, creating a sensitized background to identify components involved in cytoskeletal polarization, apical membrane trafficking and stabilisation of Crb at the apical domain.
Bauerly, E., Yi, K. and Gibson, M. C. (2020). Wampa is a dynein subunit required for axonemal assembly and male fertility in Drosophila. Dev Biol. PubMed ID: 32387369
In cilia and flagella, dyneins form complexes which give rise to the inner and outer axonemal arms. Defects in the dynein arms are the leading cause of primary ciliary dyskinesia (PCD), which is characterized by chronic respiratory infections, situs inversus, and sterility. While the pathological features associated with PCD are increasingly well characterized, many of the causative genetic lesions remain elusive. Using Drosophila, this study analyzed genetic requirements for wampa (wam; CG17083), a previously uncharacterized component of the outer dynein arm. While homozygous mutant animals are viable and display no morphological defects, loss of wam results in complete male sterility. Ultrastructural analysis further reveals that wam mutant spermatids lack the axonemal outer dynein arms, which leads to a complete loss of flagellar motility. In addition to a role in outer dynein arm formation, other novel microtubule-associated requirements were uncovered for wam during spermatogenesis, including the regulation of mitochondrial localization and the shaping of the nuclear head. Due to the conserved nature of dyneins, this study advances understanding of the pathology of PCD and the functional role of dyneins in axoneme formation and other aspects of spermatogenesis.
Kim, J. Y., Tsogtbaatar, O. and Cho, K. O. (2021). Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster. J Dev Biol 9(4). PubMed ID: 34698231
Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. This study identified Dhc64Cm115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. It is proposed that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development.
Baker, F. C., Neiswender, H., Veeranan-Karmegam, R. and Gonsalvez, G. B. (2021). In vivo proximity biotin ligation identifies the interactome of Egalitarian, a Dynein cargo adaptor. Development 148(22). PubMed ID: 35020877
Numerous motors of the Kinesin family contribute to plus-end-directed microtubule transport. However, almost all transport towards the minus-end of microtubules involves Dynein. Understanding the mechanism by which Dynein transports this vast diversity of cargo is the focus of intense research. In selected cases, adaptors that link a particular cargo with Dynein have been identified. However, the sheer diversity of cargo suggests that additional adaptors must exist. This study used the Drosophila egg chamber as a model to address this issue. Within egg chambers, Egalitarian is required for linking mRNA with Dynein. However, in the absence of Egalitarian, Dynein transport into the oocyte is severely compromised. This suggests that additional cargoes might be linked to Dynein in an Egalitarian-dependent manner. This study therefore used proximity biotin ligation to define the interactome of Egalitarian. This approach yielded several novel interacting partners, including P body components and proteins that associate with Dynein in mammalian cells. A nanobody-based proximity biotinylation strategy was devised and validated that can be used to define the interactome of any GFP-tagged protein.
Lu, W., Lakonishok, M., Serpinskaya, A. S. and Gelfand, V. I. (2022). A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary. Elife 11. PubMed ID: 35170428
Cytoplasmic dynein, a major minus-end directed microtubule motor, plays essential roles in eukaryotic cells. Drosophila oocyte growth is mainly dependent on the contribution of cytoplasmic contents from the interconnected sister cells, nurse cells. Previous work has shown that cytoplasmic dynein is required for Drosophila oocyte growth and has assumed that it simply transports cargoes along microtubule tracks from nurse cells to the oocyte. This study reports that instead of transporting individual cargoes along stationary microtubules into the oocyte, cortical dynein actively moves microtubules within nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. This robust microtubule movement is sufficient to drag even inert cytoplasmic particles through the ring canals to the oocyte. Furthermore, replacing dynein with a minus-end directed plant kinesin linked to the actin cortex is sufficient for transporting organelles and cytoplasm to the oocyte and driving its growth. These experiments show that cortical dynein performs bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. It is proposed that the dynein-driven microtubule flow could serve as a novel mode of fast cytoplasmic transport.
Gaspar, I., Phea, L. J., McClintock, M. A., Heber, S., Bullock, S. L. and Ephrussi, A. (2023). An RNA-based feed-forward mechanism ensures motor switching in oskar mRNA transport. J Cell Biol 222(7). PubMed ID: 37213090
Regulated recruitment and activity of motor proteins is essential for intracellular transport of cargoes, including messenger ribonucleoprotein complexes (RNPs). This study shows that orchestration of oskar RNP transport in the Drosophila germline relies on interplay between two double-stranded RNA-binding proteins, Staufen and the dynein adaptor Egalitarian (Egl). It was found that Staufen antagonizes Egl-mediated transport of oskar mRNA by dynein both in vitro and in vivo. Following delivery of nurse cell-synthesized oskar mRNA into the oocyte by dynein, recruitment of Staufen to the RNPs results in dissociation of Egl and a switch to kinesin-1-mediated translocation of the mRNA to its final destination at the posterior pole of the oocyte. It was additionally shown that Egl associates with staufen (stau) mRNA in the nurse cells, mediating its enrichment and translation in the ooplasm. These observations identify a novel feed-forward mechanism, whereby dynein-dependent accumulation of stau mRNA, and thus protein, in the oocyte enables motor switching on oskar RNPs by downregulating dynein activity.
Jejina, A., Ayala, Y., Hernandez, G. and Suter, B. (2023). Role of BicDR in bristle shaft construction, tracheal development, and support of BicD functions. bioRxiv. PubMed ID: 37398393
Cell polarization requires asymmetric localization of numerous mRNAs, proteins, and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors, which function as multiprotein complexes. In the dynein/dynactinBicaudal-D (DDB) transport machinery, Bicaudal-D (BicD) links the cargo to the motor. This study focused on the role of BicD-related (BicDR) and its contribution to microtubule-dependent transport processes. Drosophila BicDR is required for the normal development of bristles and dorsal trunk tracheae. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft and the localization of Spn-F and Rab6 at the distal tip. BicDR supports the function of BicD in bristle development and the results suggest that BicDR transports cargo more locally whereas BicD is more responsible for delivering functional cargo over the long distance to the distal tip. This study identified the proteins that interact with BicDR and appear to be BicDR cargo in embryonic tissues. For one of them, EF1γ, this study showed that EF1γ genetically interacts with BicD and BicDR in the construction of the bristles.
Lu, W., Lakonishok, M., Gelfand, V. I. (2023). The dynamic duo of microtubule polymerase Mini spindles/XMAP215 and cytoplasmic dynein is essential for maintaining Drosophila oocyte fate. Proc Natl Acad Sci U S A, 120(39):e2303376120 PubMed ID: 37722034
In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16 interconnected cells form a germline cyst, where one cell differentiates into an oocyte, while the rest become nurse cells that supply the oocyte with mRNAs, proteins, and organelles through intercellular cytoplasmic bridges named ring canals via microtubule-based transport. This study finds that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for maintenance of the oocyte specification. mRNA encoding Msps is transported and concentrated in the oocyte by dynein-dependent transport along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, causing more microtubule plus ends to grow from the oocyte through the ring canals into nurse cells, further enhancing nurse cell-to-oocyte transport by dynein. Knockdown of msps blocks the oocyte growth and causes gradual loss of oocyte determinants. Thus, the Msps-dynein duo creates a positive feedback loop, ensuring oocyte fate maintenance by promoting high microtubule polymerization activity in the oocyte, and enhancing dynein-dependent nurse cell-to-oocyte transport.
Salvador-Garcia, D., Jin, L., Hensley, A., Golcuk, M., Gallaud, E., Chaaban, S., Port, F., Vagnoni, A., Planelles-Herrero, V. J., McClintock, M. A., Derivery, E., Carter, A. P., Giet, R., Gur, M., Yildiz, A. and Bullock, S. L. (2023). A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression. bioRxiv. PubMed ID: 37577480
The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, gene editing was used to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). Mutations associated with human neurological disease were shown to cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. A novel mutation in the microtubule-binding domain (MTBD) of Dhc is described that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. Mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, these data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, these work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

Dynein is a member of the myosin family of molecular motors and is involved in transporting cargo from one part of a cell to another. Equally important, dynein plays a role in cytoskeletal dynamics during mitosis. Axonemal dynein was first discovered in the guise of an ATPase involved in ciliary and flagellar beating. A morphologically similar but distinctly different cytoplasmic form of dynein was subsequently discovered that could translocate organelles toward the minus ends of microtubules. Cytoplasmic dynein is a multisubunit protein (1.2 MDa) consisting of two heavy chains of ~ 500 kDa, each of which fold to form the two heads of the motor, as well as multiple intermediate chains, light intermediate chains, and light chains. Dynein is a retrograde motor, which means that Dynein transports cargo from the terminus of axons back to the cell body from which axons emerge (Karki, 1999 and references).

The protein complex Dynactin, initially identified as a co-purifying set of polypeptides in dynein preparations from rat liver and testis, is also a large multisubunit complex consisting of at least seven distinct polypeptides ranging in size from 22-150 kDa. Dynactin was first demonstrated to be required for dynein-mediated transport in vitro, and is now believed to be required for most, or all, of dynein-mediated cellular activities. A physical interaction between dynein and dynactin has been confirmed by biochemical analyses. Cytoplasmic dynein binds dynactin through a direct interaction between dynein intermediate chains and the p150Glued subunit of dynactin. Dynactin is a platform involved in linking dynein to its cargo or to a substratum. During mitosis, dynactin attaches to a substratum consisting of the kinetochore of each chromosome (Karki, 1999 and references).

Mutations in genes regulating components of the dynein-dynactin complex disrupt axon path finding and synaptogenesis during metamorphosis in the Drosophila central nervous system. In order to learn about the role of the dynein-dynactin complex in the development of the nervous system, the growth of mutant sensory axons during metamorphosis was analyzed. The adult sensory axons grow into the thoracic CNS during the first half of metamorphosis (Shepherd, 1996). The P[Gal4] targeting system can be used to label axons of the femoral chordotonal organ (feCO) in the leg as well as some haltere axons. Using this system, axons are marked on the basis of the expression of specific genes that generate these axons. While most larval sensory neurons degenerate during the first 24 hours of metamorphosis, a segmentally repeated array of 6 neurons per segment persists into the adult stages to become functional adult neurons. These sensory neurons retain their axonal projections in the central nervous system intact and unchanged throughout metamorphosis. The adult sensory neuron axons, originating in the leg or haltere, enter the central nervous system at around 12 hours after puparium formation (APF) (Murphey, 1999). Most of these axons grow along the pathways defined by the persistent larval sensory axons. The ordering of the adult sensory projections is, therefore, established upon the larval pattern of projections (Shepherd, 1996).

Axons entering the first thoracic neuromere were studied because the orientation of three collaterals in this segment allows for ease in scoring. In wild type, the leg chordotonal sensory neuron axons arrive at the CNS in what appears to be a single bundle of growth cones. The bundle grows directly towards the midline without branching and reaches the medial target area by about 24 h APF. At 24 hours APF there is often a detectable defasciculation of the axon bundle in the region that will give rise to the collaterals in the anterior and posterior domains of the first thoracic neuromere. The posterior collaterals (pLAC) are first detected at about 30 hours APF, while the anterior collaterals (sLAC) appear by about 42 hours APF (Murphey, 1999).

In order to better understand the functions of the dynein retrograde motor in nervous system assembly, the pathfinding and arborization of sensory axons during metamorphosis were analyzed in wild-type and mutant backgrounds. As an indicator of dynein function, mutations in two genes were analysed: Glued, coding for the heavy chain of dynactin, and Cytoplasmic dynein light (Cdlc1), coding for a light chain of the dynein protein complex. In Glued1 and Cdlc1 mutants, proprioceptive and tactile axons arrive at the CNS on time but exhibit defects in terminal arborizations that increase in severity up to 48 h after puparium formation. The results show that axon growth occurs on schedule in these mutants but the final process of terminal branching, synaptogenesis, and stabilization of these sensory axons requires the dynein-dynactin complex. Since this complex functions as a retrograde motor, it is suggested that a retrograde signal needs to be transported to the nucleus for the proper termination of some sensory neurons (Murphey, 1999).

Dynein is required in some sensory inputs and not in others. A distinction is made between proprioceptive and tactile axons. Proprioceptors are sensory nerves that convey information about muscle tension, providing input related to the position of an organism in space and to forces experienced by the musculature. Proprioceptive information is provided by chordotonal neurons. Tactile neurons provide information about tactile stimuli received by the organism. A lacZ construct fused to the engrailed promoter was used to examine tactile neurons. en-lacZ-positive sensory axons are located in a posterior bundle in each leg nerve and this bundle extends around the posterior border of each leg neuropil to arborize in a ventral domain (Murphey, 1989b, Phillis, 1996, and Murphey, 1999). en-lacZ expression in tactile axons is activated before the axons reach the CNS and therefore their progress can be followed as the axons enter (Murphey, 1999).

The first pupal sensory axons to be detected in the CNS are a small group of axons that reach and enter the main longitudinal tracts where (between 12 and 24 hours APF) they turn posteriorly in the first thoracic neuromere (T1) or anteriorly in the second and third thoracic neuromeres (T2 and T3). These appear to be the leg 'pioneers' (Bate, 1976). These are unaffected by mutations that affect the function of dynein. The main bundle of tactile axons is first detected at the edge of the ganglion between 12 and 18 hours APF. The bundle extends posteriorly and medially around the neuromere, reaching most of the target areas and exhibiting the mature form by 48 hours. In Gl1 heterozygotes, in approximately 30% of the neuromeres, some of the axons take an aberrant anterior pathway around the neuropil. These axons appear to grow along paths and arborize in domains normally innervated by axons from the posterior, en-lacZ-minus regions of the leg. Thus early axon growth is normal but later crucial events are compromised by the Gl1 mutation and cause defects in arborization in both the tactile and chordotonal sensory axons (Murphey, 1999).

Two groups of sensory axons are unaffected by the Gl1 mutation: the haltere axons and the axons of a group of leg afferents called hair plates. The axons of the haltere nerve enter the CNS, course anteriorly, and give off a group of collaterals near the border of the first and second thoracic neuromeres and project through the connective to the brain. Just as with the feCO chordotonal axons, the haltere axons arrive at the CNS at 6-12 hours APF, grow toward the midline, reach the main longitudinal tract at 18 hours, and grow into the connnective by 24 hours APF (Murphey, 1999).

The differential responses of the various sensory neurons to the Gl1 mutation led to an examination of the three-dimensional structure of the thoracic neuromere in a search for further evidence of the disruption in axon projections. It has been demonstrated that each thoracic neuromere of insects is partitioned into a few discrete domains that are the processing centers for different sensory modalities (Pfluger, 1988 and Murphey, 1989a). Receptors associated with proprioceptive, tactile, and multimodal hairs have been studied (Murphey, 1998a). Proprioceptive hairs occur in clusters, called hair plates, and are situated near joints. The neuron innervating each proprioceptive hair has a large axon and coarse arborization in the CNS in an intermediate neuropil. Tactile receptors have smaller arbors that terminate in a ventral region of the thoracic neuromere. Finally, the multimodal hairs are each innervated by one tactile and four chemosensory neurons. The single tactile neuron has a central arbor that is indistinguishable from those of the tactile hairs; the four chemosensory neurons project to yet a third region of neuropil near the ventral surface of each neuromere. Thus leg sensory axons define two domains in leg neuropil: the intermediate proprioceptive region and the ventral tactile region. For example, the hair plate axons clearly outline the proprioceptive domain in wild type. This domain appears normal in Gl1 mutants as evidenced by the normal projections of the hair plate axons that surround it. Since the hair plate axons exhibit no defects in the mutant background they also define this domain in mutant animals, indicating its relative normality. Despite this apparent normality of the domain structure based on the hair plate projections, the Gl1 mutation disrupts axonal projections from the feCO within the proprioceptive domain (Murphey, 1999 and Reddy, 1997). The tactile receptors project to a separate ventral domain as revealed by the en-lacZ construct (Phillis, 1996). These mutant axons often make pathway choice errors, such as taking an abnormal anterior pathway, but they continue to terminate in the appropriate ventral domain. The results for Cdlc1 mutants are very similar to those obtained with Gl1. It is concluded that four distinct groups of sensory axons give different responses to mutations that affect the function of dynein: the chordotonal and tactile axons are disrupted, while the hair plate and haltere axons are unaffected (Murphey, 1999).

The relatively late onset of defects in the mutant backgrounds, after the axons have reached the CNS, suggests that defects in the dynein-dynactin complex are compromising a retrograde signaling system. This interpretation is that a retrograde signal, received after the sensory growth cones reach the CNS, is crucial to synaptogenesis or synaptic stabilization. Sine the Gl1 gene product disrupts retrograde transport, one possibility is that a retrograde signal must travel from the axon terminal to the nucleus of the sensory neurons in order to have its effect. There appears to be adequate time for retrograde signaling to the cell body, since the round-trip from terminal to soma would take as little as 10 minutes (fast retrograde transport at 1 micrometer/second for a distance of 600 micrometers). The defects seem to arise much more slowly than this, over a time course of approximately 6 hours (Murphey, 1999).

Dynein is also implicated in establishing the orientation of the mitotic spindle early in oogenesis. The orientation of the mitotic spindle has long been known to be important in directing asymmetric cell divisions during embryogenesis. More recently, molecular genetic analyses in C. elegans and Drosophila have identified molecules that couple spindle orientation to the asymmetric localization of cell fate determinants. Members of the myosin family of actin-based molecular motors have been implicated in the asymmetric segregation of factors necessary for cell fate determination in yeast and C. elegans. Less is known about the pathways that establish the initial cellular asymmetry that directs spindle orientation and polarized transport. Given the importance of the microtubule cytoskeleton in cytoplasmic organization and intracellular transport in polarized cell types, microtubules and their associated motor molecules may be part of the primary mechanism that establishes this cytoplasmic asymmetry (McGrail, 1997 and references).

Within the germline cyst a polarity must be established in order to define the pro-oocyte (a single cell selected from 16 germline cells making up the oocytic cyst) as the site of microtubule nucleation in the assembly of the polarized microtubule array. This polarity may be reflected in the asymmetric segregation of the spectrin-rich, multivesicular fusome during the four mitotic divisions (Lin, 1995) that give rise to the oocytic cyst (see alpha Spectrin for more information). The polarity reflects the fixed pattern of cytoplasmic connections among the 16 cystocytes. The asymmetric cystocyte divisions result from the behavior of the fusome and its interaction with spindle poles during mitotic division. It remains unclear how anchorage of the mitotic spindles to the fusome ensures the stereotypical pattern of cyst cell divisions, which creates a polarized cyst leading to oocyte determination, reorganization of the microtubule cytoskeleton, and oocyte differentiation. The identification of mutations in the Drosophila cytoplasmic dynein heavy chain gene Dhc64C (Gepner, 1996) has allowed for an investigation of the role of cytoplasmic dynein in oocyte determination and differentiation. The results provide evidence of a novel role for the cytoplasmic dynein in asymmetric cell division and cellular differentiation (McGrail, 1997).

Mosaic egg chambers whose germline is homozygous for a strong Dhc64C allele fails to produce mature eggs. To understand how oocyte determination is blocked in dynein mutant egg chambers, the polarity of the egg chambers in germline clones was examined. An antibody against a phospho-tyrosine epitope present in ring canals was used to reveal the size and position of the intercellular bridges in wild-type and mutant egg chambers. In the mosaic egg chambers the pattern (e.g., size and location) of the ring canal connections appears abnormal. The most posterior cell in the egg chamber does not have the typical cluster of the 4 largest ring canals. One explanation of this phenotype is based on the disruption of spindle orientation and division pattern, but a cell containing four ring canals may be is mislocalized within the 16-cell cyst. Nonetheless, the mosaic egg chambers always lack an oocyte nucleus, and instead contain 16 polyploid nurse cell nuclei. By contrast, in wild-type egg chambers an oocyte invariably is positioned at the posterior of the egg chamber, and is easily distinguished by the four largest ring canals that reside at the anterior margin of the oocyte. It is also found that Bicaudal-D does not accumulate in a single cell (the oocyte) in dynein mutant cysts. These results suggest that dynein function is required early in germline cyst formation to establish cyst polarity (McGrail, 1997).

Spindle orientation during asymmetric cell division is shown to require cytoplasmic dynein. The orientation and attachment of mitotic spindles to the fusome during cystocyte divisions is thought to ensure the fixed pattern of cell divisions necessary to create the polarized cyst. A striking defect is found in spindle orientation in the dynein mutant cysts. In wild-type mitotic cysts the spindles are arranged in compact clusters with one spindle pole in close contact with a single lobe or branch of the fusome. In contrast, in the mutant cysts the spindles frequently did not appear to contact the fusome, but instead are randomly oriented in the cyst. The defect in spindle orientation suggests that dynein function might be directly involved in the mechanism of spindle orientation. Dynein localization was examined in wild-type mitotic cysts and an enriched, punctate pattern of dynein has been found to be centrally located in a region of the cyst that would correspond to the position of the fusome. No enrichment of dynein on a fusome-like structure is found in interphase cysts. In the 2-cell anaphase cyst and the 4-cell metaphase cyst, each spindle was oriented with one pole in close association to the area of dynein staining. These data suggest that spindle attachment to the fusome is mediated by cytoplasmic dynein. This mechanism would ensure the fixed pattern of cystocyte divisions that lead to a polarized germline cyst (McGrail, 1997).

In addition to its role in the early events of oocyte determination and differentiation, the microtubule cytoskeleton has been implicated in the establishment of axial polarity and the localization of morphogens within the developing oocyte. Female adults doubly heterozygous for the dynein alleles (Dhc64C 6-6 and Dhc64C 6-12) produce late stage egg chambers but are female sterile. The localization of dynein to the posterior of the stage 9 oocyte is disrupted in egg chambers derived from Dhc64C 6-6 ;Dhc64C 6-12 females. Instead, dynein accumulates in a punctate pattern at the anterior margin of the oocyte. The posterior accumulation of a dynein-activating complex, the Glued complex, is also dependent on cytoplasmic dynein. The wild-type Glued complex is mislocalized along with the dysfunctional dynein to the oocyte anterior (McGrail, 1995 and McGrail, 1997).

A role cannot be found for the cytoplasmic dynein motor in the positioning of known morphogens within the oocyte. However, the dynein alleles used in these studies are not null mutations. Therefore a residual level of dynein activity that is insufficient for the proper localization of the Glued complex, but which may be sufficient for directing other mRNA and protein localizations, cannot be excluded. It will be important to identify the cargoes transported by dynein at this later stage of oogenesis. In this regard, the posterior accumulation of a predicted plus-end motor molecule, the kinesin-beta-gal fusion protein, is markedly reduced. Moreover, the fragile nature of the egg shell and the variable defects in the egg shape and chorionic appendages of the mutant eggs suggest another potential role for dynein in the polarized secretion of the egg shell components by the somatic follicle cells (McGrail, 1997).

It has been proposed that spindle positioning ensures the unequal inheritance of the ring canals and associated fusome material at each round of mitosis. This mechanism is believed to account for the production of a branched chain of interconnected cystocytes, in which the two cells with four ring canals and the largest amount of fusome material become pro-oocytes. It is not clear how the selection is made for one of the two cells to continue to differentiate as the oocyte, while the other takes up a nurse cell fate. However, a recent model has addressed this issue and proposes that the future single pro-oocyte is initially determined by the asymmetric segregation of the spectrosome at the first cystoblast division. The identity of the pro-oocyte is maintained at each division by the asymmetric inheritance of the fusome material, which leads to a polarized fusome (Lin, 1995). The polarized fusome may provide the information necessary to subsequently assemble a polarized microtubule array and establish directed transport to the pro-oocyte. Mutations that disrupt the assembly of the fusome and oocyte differentiation, including mutations in the fusome components alpha-spectrin and the adducin-like hu-li tai shao gene product, foster the view that fusome function is critical in oocyte differentiation. This analysis of mitotic germline cysts in the dynein mutant germaria reveals that mitotic spindles fail to acquire the proper orientation with respect to the fusome, and the normal pattern of ring canal connections and cyst polarity is disrupted. These results support the hypothesis that spindle orientation and asymmetric cell division are critical for oocyte determination (McGrail, 1997).

The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes

Because of their functional polarity and elongated morphologies, microtubule-based transport of proteins and organelles is critical for normal neuronal function. The proteasome is required throughout the neuron for the highly regulated degradation of a broad set of protein targets whose functions underlie key physiological responses including synaptic plasticity and axonal degeneration. Molecularly, the relationship between proteasome transport and the transport of the targets of proteasomes is unclear. The dynein motor complex is required for the microtubule-based motility of numerous proteins and organelles in neurons. This study demonstrates that microtubule-based transport of proteasomes within the neuron utilizes a distinct dynein light chain compared to synaptic proteins. Live imaging of proteasomes and synaptic vesicle proteins in axons and synapses finds that these cargoes traffic independently and that proteasomes exhibit significantly reduced retrograde transport velocities compared to synaptic vesicle proteins. Genetic and biochemical analyses reveals that the Drosophila homologue of the LC8 dynein light chain Cut-up binds proteasomes and functions specifically during their transport. These data support the model that Cut-up functions to specify the dynein-mediated transport of neuronal proteasomes (Kreko-Pierce, 2017).

Proteasomes are large protein complexes responsible for degradation of normal short-lived ubiquitylated proteins as well as mutant, misfolded or damaged proteins. All cells require regulated protein degradation; however, nerve cells are of particular interest due to their complex compartmentalization and the requirement of protein degradation for normal neuronal function. In addition, a large number of neurological disorders are characterized by accumulations of proteinaceous aggregates, suggesting that impaired protein degradation is an important disease etiology of many neurodegenerative diseases. Because protein degradation in neurons occurs on short timescales and is highly compartment specific, neurons must possess molecular mechanisms that can precisely position proteasomes near to where they are uniquely required, but also maintain a physical separation between proteasomes and neuronal targets to preserve the efficacy of regulated protein turnover (Kreko-Pierce, 2017).

Numerous studies have demonstrated the important physiological requirement of proteasome activity throughout the many compartments of the neuron. Pre-synaptically, proteasome-dependent protein degradation is critical for synapse formation, synaptic efficacy and neurotransmitter release. Post-synaptically, proteasomes have been implicated in regulating several forms of synaptic plasticity including long-term potentiation (LTP), long-term facilitation (LTF) and long-term depression (LTD). Furthermore, acute depolarization of neurons causes a global change in ubiquitylated active zone proteins at the synapse, supporting the role of proteasomes in the rapid turnover of proteins in response to neuronal activity. Collectively, these data suggest that proteasomes function locally at pre- and post-synaptic sites where they act as an important modulators of synaptic structure, function and plasticity (Kreko-Pierce, 2017).

In addition to the synaptic compartments, there is evidence that proteasome function also plays an important role in the growth, development and regeneration of axons. Recent work on neuronal development has shown that changes in retrograde axonal transport of proteasomes are critical during the specification and growth of the axon. Studies in Drosophila provide evidence that the degeneration of axons that occurs during developmental pruning or in response to injury requires the ubiquitin-proteasome system (UPS). Consistent with these observations in flies, inhibition of the UPS in rodent models delays the axonal die-back observed during Wallerian axonal degeneration demonstrating a role for protein degradation during programmed axonal degeneration in mammals. These data provide strong evidence for the evolutionarily conserved requirement of proteasome activity within the axon under both normal and pathological conditions (Kreko-Pierce, 2017).

Despite the critical compartment-specific requirements for proteasome function in neurons, little is known about the molecular mechanisms that govern proteasome transport and their targets within neurons. The trafficking of organelles and transport vesicles within all cells is predominantly mediated by microtubule (MT)-based transport mechanisms utilizing two distinct molecular motor proteins, kinesins and cytoplasmic dyneins. Kinesins mostly mediate MT plus-end-directed transport, including anterograde axonal transport in neurons. In the human genome, 45 genes code for the kinesin superfamily, supporting a genetic basis for the large diversity of cargo-specific kinesin-based transport events. Cytoplasmic dynein mediates MT minus-end-directed transport including retrograde axonal transport. However, unlike for the kinesin motor, cytoplasmic dynein is encoded by relatively few genes leading to the hypothesis that the cargo specificity of the dynein motor complex is accomplished by the heterogeneneity of dynein complex subunits and various dynein-associated accessory proteins. The LC8 dynein light chains (DYNLL1 and DYNLL2 in mammals), have been proposed as cargo-adaptors potentially providing specificity for the minus-end-directed MT transport of vesicles and organelles. This notion was supported by studies that found LC8 to simultaneously associate with the dynein motor and with a number of cargos that undergo MT-mediated transport (Kreko-Pierce, 2017).

This study used a combination of genetics, biochemistry and in vivo imaging to compare the MT-based transport of proteasomes and synaptic proteins in Drosophila motor neurons. These analyses found that proteasomes use MT-based axonal transport in axons and that the axonal transport is qualitatively similar to that of synaptic proteins. However, quantitative analysis of proteasome trafficking reveals significant differences in the retrograde transport of proteasomes compared to that of synaptic proteins. These data suggests potential molecular differences in the dynein motor complexes utilized by these two distinct cargo types. In support of this idea, a forward genetic screen identified the cut up (ctp) gene, a Drosophila homolog of LC8, as being required specifically for the axonal transport of proteasomes but not synaptic proteins. These results provide molecular evidence that proteasomes and their targets utilize specific dynein motor components during MT-based transport in neurons (Kreko-Pierce, 2017).

This study used fluorescence time-lapse imaging and single-particle tracking in Drosophila third-instar larvae to investigate trafficking of proteasomes in motor neurons. The data demonstrate that proteasomes use fast MT-based axonal transport to traffic in Drosophila motor neurons, including within the presynaptic nerve terminal. The quantitative analyses of proteasome trafficking in axons of motor neurons revealed that the velocity of retrograde transport of proteasomes is significantly slower than the velocity of anterograde transport. This is similar to what has been observed in developing hippocampal cultures derived from mouse brains, supporting the idea of conserved transport mechanisms. Furthermore, it was found that the values for retrograde velocity and run length of proteasomes are significantly less than that of synaptic vesicle proteins, and that mutations in the Klc gene had a much stronger effect on the retrograde transport velocities of synaptobrevin than they did on proteasomes. Finally, genetic analysis of proteasome transport revealed that the Drosophila homolog of the mammalian LC8 dynein light chain, cut up (ctp), is required for retrograde axonal transport of proteasomes but is dispensable for the retrograde axonal transport of synaptic vesicle proteins. These analyses strongly support the model that proteasomes utilize a different dynein motor complex for transport to that used by other synaptic cargo. It is interesting to note that synaptotagmin has been identified in both proteome studies of proteasome-dependent protein degradation and observed in polyQ-induced protein aggregates. These data support the idea that synaptotagmin is likely degraded by the proteasome, perhaps in the synapse. Given the physiological significance of changes in the abundance of key synaptic molecules such as synaptotagmin, perhaps it is not surprising that the proteasome and its synaptic targets utilize distinct transport mechanisms. It would be predictrf that this arrangement would protect against inadvertent interactions between key substrates and the proteasomes, preserving the physiological efficacy of regulated changes in protein abundance (Kreko-Pierce, 2017).

The model that proteasomes and synaptic proteins are trafficked independently is seemingly in conflict with data from a recent study of axonal transport of proteasomes from cultured hippocampal neurons that suggested that proteasomes are co-transported with various membrane-associated cargos, including the synaptic vesicle protein synaptophysin. This study specifically addressed this possibility by simultaneously co-expressing proteasomes and synaptobrevin in the same motor neuron and analyzing transport. These analyses of co-transport revealed that only 8% of the synaptobrevin transport vesicles co-transported with proteasomes, despite the comparatively large number of proteasome particles. It was also found that in the present study that most of the proteasomes (~65%) are moving, whereas another study reported a relatively small population of mobile proteasomes (~20%) with majority of particles exhibiting a random-like, reversing motion (~80%). Furthermore, the velocities of both retrograde and anterograde proteasome transport reported previously are much slower than what was observed in the current study. It should be noted that the velocities that are reported for both anterograde and retrograde transport of proteasomes are consistent with velocities of proteasomes observed in cultured hippocampal neurons during axonal differentiation. These inconsistencies between studies may reflect differences due to different neuronal cell types or reporter expression, or differences between the in situ model and cultured primary neurons (Kreko-Pierce, 2017).

Cytoplasmic dynein is a multi-subunit motor protein responsible for the MT-based transport of a wide range of cargos. The current data suggests that the Drosophila DLC Ctp can specify the axonal transport of distinct cargoes. Consistent with this role, the mammalian LC8 (DYNLL1 and DYNLL2) family of DLCs have been shown to simultaneously associate with the dynein motor and a range of cargo proteins including active zone components and proteins involved in mRNA localization during embryogenesis. Importantly, mutations that disrupt the interaction of these proteins with LC8 have been shown to disrupt their dynein-mediated MT transport, providing a link between LC8-binding partners and MT-dependent trafficking. In addition to demonstrating that ctp was necessary for the normal axonal transport of proteasomes, biochemical evidence is provided that Ctp physically interacts with the 20S and 19S proteasome subunits. It is not clear from these co-immunoprecipitation studies if Ctp directly binds to these specific subunits or perhaps some other proteasome subunit. Structural studies have indicated that a large number of diverse cargoes bind to the same groove in the LC8/Ctp dimer and in certain cases can either compete or facilitate binding with other cargoes including the intermediate chain of dynein. Based on the current data, it would be predicted that the binding of the 26S proteasome to Ctp would favor the association of Ctp with the intermediate chain of dynein and facilitate dynein-dependent transport. Neither synaptobrevin nor synaptotagmin would be predicted to have this activity (Kreko-Pierce, 2017).

In addition to providing cargo specificity, the results also suggest that Ctp can affect the processivity of the dynein motor. First, it was observed that proteasomes have a slower retrograde velocity of transport than does synaptobrevin. In addition, the retrograde velocities and run lengths of proteasome transport are reduced, but not absent, in ctp mutants. These results suggest that Ctp association with the dynein motor alters the biochemical function of the resulting motor complex. Currently, little is known about how DLCs participate in motor processivity in any system. Previous studies have demonstrated that the processivity and activity of the dynein motor can be altered by interactions with various regulatory proteins. For example, the dynactin complex has been shown to significantly increase dynein processivity similar to what the data shown for ctp. Interestingly, several recent studies suggest that the normal interaction between dynein and the dynactin complex requires an LC8 dynein light chain. In addition, mutations that disrupt Dyn2 function (a yeast homolog of LC8) also impaired the recruitment of the dynactin complex to the dynein motor complex. Further studies will be required to determine whether Ctp alters the processivity of proteasomes due to the recruitment of the dynactin complex (Kreko-Pierce, 2017).

Studies utilizing pharmacological inhibition of proteasome activity have shown that inhibition of proteasomes results in a rapid increase in synapse function, including neurotransmitter release, suggesting that proteasomes are localized near the neurotransmitter release site. Despite the evidence supporting the presence of proteasomes within the presynaptic nerve terminal, direct evidence for this is absent. This study is the first to visualize and study proteasome trafficking in the presynaptic nerve terminal. Furthermore, the consistency in transport dynamics between axon and synapse, the colocalization with Futsch and the lack of movement in ctp mutants support the idea that synaptic transport of proteasome is MT based. Furthermore, analysis of synapse morphology in ctp mutants demonstrates that proteasomes are required within the NMJ for normal synapse growth during larval development. Recent studies aimed at studying post-synaptic proteasomes have suggested that proteasomes undergo dynamic changes in their subcellular localization in response to depolarization suggesting a role for proteasome transport during synaptic plasticity. It will be important to investigate whether ctp mutations have effects on synaptic plasticity at the Drosophila NMJ (Kreko-Pierce, 2017).

Interestingly, this study also found that the trafficking behavior of proteasomes in the synapse has some important differences from trafficking in the axons. A substantial difference was observed between these two neuronal compartments in terms of the number of stationary particles, with the number of stationary proteasomes in the synapse increasing by ~400%. Additionally, it was found that synaptic proteasomes move, on average, more slowly than proteasomes in the axons. A similar change in the trafficking between the axon and nerve terminal has also been observed for neurexin (Nrxn) proteins, which are also transported more slowly in the synapse versus axon. The combination of these changes in trafficking behavior favor a longer residence of an individual proteasome in the synapse compared to in the axon. The distribution of stationary proteasomes throughout the NMJ could facilitate the local degradation of synaptic proteins within the bouton. This behavior is in contrast to studies of the trafficking of neuropeptide-containing dense core vesicles (DCVs) within the NMJ, which have few stationary vesicles and utilize a continuous 'conveyor belt' model transport at the synapses ensuring continuous source of DCVs. These differences suggest MT-based transport within the synapse is tailored to the specific cargo and their respective functions (Kreko-Pierce, 2017).

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).


Transcript length - 14381

Bases in 5' UTR - 162


Amino Acids - 4639

Structural Domains

The Drosophila cytoplasmic dynein heavy chain is most similar to the predicted rat cytoplasmic dynein heavy chain. Overall the two sequences share 84% similarity and 72% identity at the amino acid level. The homology is distributed across the entire length of the polypeptide. The conserved central domain encompasses a cluster of four phosphate-binding loop (P-loop) motifs. The predicted sequence of the first P-loop (residues 1895-1902) in Drosophila is totally conserved among all dynein sequences. The secondary structure prediction for the Drosophila cytoplasmic dynein heavy chain is similar to those obtained for other dynein sequences and shows that alpha helix, beta sheet, and beta turn conformations are predicted along the entire length of the protein. The prediction is consistent with the predominant globular shape of native dynein complexes as determined by electron microscopy (Li, 1994).

Dynein heavy chain 64C: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 1 March 2024

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