Biology subjects

Gelfand, V. I.

Publications and source records attributed to Gelfand, V. I..

16 recordsLinked to original sources

bioRxiv2026

An in vivo examination of Dynein-Cargo complex formation.

Long-range intracellular transport relies on microtubule motors. This process is particularly important in large cells such as neurons and oocytes. While transport towards the plus-end of microtubules utilizes many kinesins, minus-end transport is largely mediated by a single motor, cytoplasmic dynein. Activation of dynein requires the large dynactin complex as well as a cargo adaptor. How dynein, dynactin, and adaptors assemble in vivo, particularly within specialized tissues such as the Drosophila egg chamber remains unclear. In the current study, we defined the dynein interactome in Drosophila egg chambers using in vivo proximity biotin ligation. Our findings suggest that Bicaudal-D (BicD) is the principal adaptor responsible for activating dynein and linking it with cargo in this tissue. We also identified Centrocortin (Cen) as a dynein adaptor in egg chambers. However, unlike BicD, loss of Cen did not affect dynein localization or apparent activation. To more specifically analyze adaptor-dependent assembly and cargo transport, we examined dynein light intermediate chain (Dlic) mutants known to impair adaptor binding. As expected, these mutants disrupted the BicD-dynein interaction. However, Cen remained associated with the dynein/dynactin complex in the mutant background, suggesting that Cen engages the motor by a different mechanism. Finally, live imaging of microtubules revealed that even when adaptor binding is compromised, dynein-driven microtubule gliding can still deliver nurse cell-derived cargo into the oocyte, albeit with reduced efficiency. Collectively, our results reveal multiple, mechanistically distinct routes for adaptor association with dynein in vivo and indicate that redundant processes can sustain cargo transport during oogenesis.

cell biology↗

bioRxiv2026

To Be or Not to Be an Oocyte: Msps/XMAP215 Controls Oocyte Cell Fate in the Drosophila Ovary

Cell fate determination, the process by which cells commit to specific identities, is fundamental to development. Oocyte specification in Drosophila provides an excellent model to dissect these molecular mechanisms. In the Drosophila ovary, 16 interconnected germline cells arise through four mitotic divisions with incomplete cytokinesis. Among them, the two oldest cells become pro-oocytes, and one is selected to develop into the oocyte while maintaining diploidy; the remaining 15 differentiate into nurse cells and enter endoreplication. Previously, we showed that Mini spindles (Msps), a microtubule polymerase and homolog of XMAP215, is essential for oocyte maintenance. Here, we report that Msps/XMAP215 is expressed early in development, with both mRNA and protein enriched in the pro-oocytes prior to oocyte specification. Knockdown of Msps prevents oocyte specification, leading to egg chambers with 16 nurse cells. Loss of Msps also disrupts the accumulation of the oocyte marker Orb and the microtubule minus-end binding protein Patronin/CAMSAP, both critical for oocyte specification. Remarkably, optogenetic recruitment of Msps is sufficient to increase microtubule polymerization and promote Orb accumulation in nurse cells, suggesting that Msps activity is sufficient to drive oocyte fate determination. Furthermore, we show that Msps associates with the germline-specific organelles, spectrosome and fusome, and becomes asymmetrically distributed among sister cells, allowing the two pro-oocytes to inherit higher levels of Msps than their siblings. Together, we propose a model in which Msps-mediated microtubule polymerization provides the pro-oocyte with a competitive advantage, initiating a positive feedback loop that involves dynein-dependent transport of msps mRNA to reinforce oocyte specification. TeaserA self-reinforcing cytoskeletal feedback loop selects a single egg cell from a cluster of interconnected germ cells.

cell biology↗

bioRxiv2024

Developmentally regulated actin-microtubule crosstalk in Drosophila oogenesis

Actin-microtubules (MT) interactions are essential, but how those mechanisms are orchestrated in complex developing systems is poorly understood. Here we show that actin-MT crosstalk regulates actin cable assembly and the assembly and organization of MTs in Drosophila nurse cells during oogenesis. We found that a stable MT meshwork develops concurrently with actin cable initiation and requires acetylation for its maintenance. These {gamma}-tubulin nucleated MTs are cortically tethered via Patronin and Shortstop, extend into the cytoplasm, and coalign with the elongating actin cables. The MT network promotes the cortical enrichment of the actin assembly factors Diaphanous and Enabled and loss of MTs dramatically reduced actin cable density and growth rate. We further demonstrated that actin filament assembly promotes cortical tethering of MTs and that loss of the actin filament bundler Villin/Quail results in fewer, shorter, and more highly coaligned MTs. Together, our data reveal multiple modes of coordinated actin-MT crosstalk producing actin cable-MT network synergy that is instrumental for oogenesis. SummaryThis study demonstrates actin-microtubule crosstalk in Drosophila oogenesis, revealing coregulation between actin filament assembly/bundling and microtubule nucleation/organization, highlighting the coordinated regulation of the cytoskeleton required during development.

cell biology↗

bioRxiv2024

'Mitotic' kinesin-5 is a dynamic brake for axonal growth

During neuronal development, neurons undergo significant microtubule reorganization to shape axons and dendrites, establishing the framework for efficient wiring of the nervous system. Previous studies from our laboratory demonstrated the key role of kinesin-1 in driving microtubule-microtubule sliding, which provides the mechanical forces necessary for early axon outgrowth and regeneration in Drosophila melanogaster. In this study, we reveal the critical role of kinesin-5, a mitotic motor, in modulating the development of postmitotic neurons. Kinesin-5, a conserved homotetrameric motor, typically functions in mitosis by sliding antiparallel microtubules apart in the spindle. Here, we demonstrate that the Drosophila kinesin-5 homolog, Klp61F, is expressed in larval brain neurons, with high levels in ventral nerve cord (VNC) neurons. Knockdown of Klp61F using a pan-neuronal driver leads to severe locomotion defects and complete lethality in adult flies, mainly due to the absence of kinesin-5 in VNC motor neurons during early larval development. Klp61F depletion results in significant axon growth defects, both in cultured and in vivo neurons. By imaging individual microtubules, we observe a significant increase in microtubule motility, and excessive penetration of microtubules into the axon growth cone in Klp61F-depleted neurons. Adult lethality and axon growth defects are fully rescued by a chimeric human-Drosophila kinesin-5 motor, which accumulates at the axon tips, suggesting a conserved role of kinesin-5 in neuronal development. Altogether, our findings show that at the growth cone, kinesin-5 acts as a brake on kinesin-1-driven microtubule sliding, preventing premature microtubule entry into the growth cone. This regulatory role of kinesin-5 is essential for precise axon pathfinding during nervous system development.

cell biology↗

bioRxiv2024

EB-SUN, a New Microtubule Plus-End Tracking Protein in Drosophila.

Microtubule (MT) regulation is essential for oocyte development. In Drosophila, MT stability, polarity, abundance, and orientation undergo dynamic changes across developmental stages. In our effort to identify novel microtubule-associated proteins (MAPs) that regulate MTs in the Drosophila ovary, we identified a previously uncharacterized gene, CG18190, encoding a novel MT end-binding (EB) protein, which we propose to name EB-SUN. We show that EB-SUN colocalizes with EB1 at growing microtubule plus-ends in Drosophila S2 cells. Tissue-specific and developmental expression profiles from Paralog Explorer reveal that EB-SUN is predominantly expressed in the ovary and early embryos, while EB1 is ubiquitously expressed. Furthermore, as early as oocyte determination, EB-SUN comets are highly concentrated in oocytes during oogenesis. EB-SUN knockout (KO) results in a decrease in MT density at the onset of mid-oogenesis (Stage 7) and delays oocyte growth during late mid-oogenesis (Stage 9). Combining EB-SUN KO with EB1 knockdown (KD) in germ cells significantly further reduced MT density at Stage 7. Notably, all eggs from EB-SUN KO/EB1 KD females fail to hatch, unlike single gene depletion, suggesting a functional redundancy between these two EB proteins during embryogenesis. Our findings indicate that EB-SUN and EB1 play distinct roles during early embryogenesis. Significance StatementO_LIOur study identified a novel microtubule end-binding (EB) protein, which is highly expressed in the Drosophila ovary, and we propose to name it EB-SUN. C_LIO_LIWe demonstrate the functional redundancy of EB-SUN and EB1 during oocyte development, while highlighting their distinct roles in early embryogenesis. C_LIO_LIGiven the high conservation of MT-dependent mechanisms in oogenesis and EB proteins across species, these findings provide insights into the differential regulation and tissue-specific functions of the EB family, using Drosophila as a model system, potentially benefiting future research on MT regulation in higher organisms. C_LI

cell biology↗

bioRxiv2024

Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a [~]50 {micro}m3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time SummarySingle-particle tracking demonstrates that individual filaments in bundles of vimentin intermediate filaments are transported in the cytoplasm by motor proteins along microtubules. Furthermore, using 3D FIB-SEM the authors showed that vimentin filament bundles are loosely packed and co-aligned with microtubules.

cell biology↗

bioRxiv2023

Self-organized intracellular twisters

Life in complex systems, such as cities and organisms, comes to a standstill when global coordination of mass, energy, and information flows is disrupted. Global coordination is no less important in single cells, especially in large oocytes and newly formed embryos, which commonly use fast fluid flows for dynamic reorganization of their cytoplasm. Here, we combine theory, computing, and imaging to investigate such flows in the Drosophila oocyte, where streaming has been proposed to spontaneously arise from hydrodynamic interactions among cortically anchored microtubules loaded with cargo-carrying molecular motors. We use a fast, accurate, and scalable numerical approach to investigate fluid-structure interactions of 1000s of flexible fibers and demonstrate the robust emergence and evolution of cell-spanning vortices, or twisters. Dominated by a rigid body rotation and secondary toroidal components, these flows are likely involved in rapid mixing and transport of ooplasmic components.

biophysics↗

bioRxiv2023

Drosophila oocyte specification is maintained by the dynamic duo of microtubule polymerase Mini spindles/XMAP215 and dynein

In many species, only one oocyte is specified among a group of interconnected germline sister cells. In Drosophila melanogaster, 16-cell 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. In this study, we find that a microtubule polymerase Mini spindles (Msps), the Drosophila homolog of XMAP215, is essential for the oocyte fate determination. mRNA encoding Msps is 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, enhancing dynein-dependent nurse cell-to-oocyte transport and transforming a small stochastic difference in microtubule polarity among sister cells into a clear oocyte fate determination. Significance statementOocyte determination in Drosophila melanogaster provides a valuable model for studying cell fate specification. We describe the crucial role of the duo of microtubule polymerase Mini spindles (Msps) and cytoplasmic dynein in this process. We show that Msps is essential for oocyte fate determination. Msps concentration in the oocyte is achieved through dynein-dependent transport of msps mRNA along microtubules. Translated Msps stimulates microtubule polymerization in the oocyte, further enhancing nurse cell-to-oocyte transport by dynein. This creates a positive feedback loop that transforms a small stochastic difference in microtubule polarity among sister cells into a clear oocyte fate determination. Our findings provide important insights into the mechanisms of oocyte specification and have implications for understanding the development of multicellular organisms.

cell biology↗

bioRxiv2022

Gigaxonin is required for intermediate filament transport

Gigaxonin is an adaptor protein for E3 ubiquitin ligase substrates. It is necessary for ubiquitination and degradation of intermediate filament (IF) proteins. Giant axonal neuropathy is a pathological condition caused by mutations in the GAN gene that encodes gigaxonin. This condition is characterized by abnormal accumulation of IFs in both neuronal and non-neuronal cells; however, it is unclear what causes IF aggregation. In this work, we studied the dynamics of IFs using their subunits tagged with a photoconvertible protein mEOS 3.2. We have demonstrated that the loss of gigaxonin dramatically inhibited transport of IFs along microtubules by the microtubule motor kinesin-1. This inhibition was specific for IFs, as other kinesin-1 cargoes, with the exception of mitochondria, were transported normally. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytoplasm of gigaxonin knock-out cells. We speculate that these oligomers saturate a yet unidentified adapter that is required for kinesin-1 binding to IFs, which might inhibit IF transport along microtubules causing their abnormal accumulation.

cell biology↗

bioRxiv2021

A novel mechanism of bulk cytoplasmic transport by cortical dynein in Drosophila ovary

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. We have previously shown that cytoplasmic dynein is required for Drosophila oocyte growth, and assumed that it transports cargoes along microtubule tracks from nurse cells to the oocyte. Here we report that instead transporting cargoes along microtubules into the oocyte, cortical dynein actively moves microtubules in nurse cells and from nurse cells to the oocyte via the cytoplasmic bridges, the ring canals. We demonstrate this 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 can perform bulk cytoplasmic transport by gliding microtubules along the cell cortex and through the ring canals to the oocyte. We propose that the dynein-driven microtubule flow could serve as a novel mode of cargo transport for fast cytoplasmic transfer to support rapid oocyte growth.

cell biology↗

bioRxiv2021

Ataxin-2 is essential for neurodevelopment in Drosophila and is a major regulator of the cytoskeleton.

Ataxin-2 (Atx2) is a highly conserved RNA binding protein. Atx2 undergoes polyglutamine expansion leading to Amyotrophic Lateral Sclerosis (ALS) or Spinocerebellar Ataxia type 2 (SCA2). However, the physiological functions of Atx2 in neurons remain unknown. Here, using the powerful genetics of Drosophila, we show that Atx2 is essential for normal neuronal cytoskeletal dynamics and organelle trafficking. Upon neuron-specific Atx2 loss, the microtubule and actin networks were abnormally stabilized and cargo transport was drastically inhibited. Depletion of Atx2 caused multiple morphological defects in the nervous system of 3rd instar larvae. These include reduced brain size, impaired optic lobe innervation and decreased dendrite outgrowth. Defects in the nervous system caused loss of the ability to crawl and lethality at the pupal stage. Taken together, these data mark Atx2 as a major regulator of cytoskeletal dynamics and denote Atx2 as an essential gene in neurodevelopment, as well as a neurodegenerative factor.

cell biology↗

bioRxiv2020

Short stop is a gatekeeper at the ring canals of Drosophila ovary

Microtubules and actin filaments are two major cytoskeletal components essential for a variety of cellular functions. Spectraplakins are a family of large cytoskeletal proteins cross-linking microtubules and actin filaments among other components. In this study, we aim to understand how Short stop (Shot), the single Drosophila spectraplakin, coordinates microtubules and actin filaments for oocyte growth. The oocyte growth completely relies on the acquisition of cytoplasmic materials from the interconnected sister cells (nurse cells), through ring canals, cytoplasmic bridges that remained open after incomplete germ cell division. Given the open nature of the ring canals, it is unclear how the direction of transport through the ring canal is controlled. Here we show that Shot controls the directionality of flow of material from the nurse cells towards the oocyte. Knockdown of shot changes the direction of transport of many types of cargo through the ring canals from unidirectional (toward the oocyte) to bidirectional, resulting in small oocytes that fail to grow over time. In agreement with this flow-directing function of Shot, we find that it is localized at the asymmetric actin fibers adjacent to the ring canals at the nurse cell side, and controls the uniform polarity of microtubules located in the ring canals connecting the nurse cells and the oocyte. Together, we propose that Shot functions as a gatekeeper directing the material flow from the nurse cells to the oocyte, via organization of microtubule tracks.

developmental biology↗

bioRxiv2020

Kinetochore protein Spindly controls microtubule polarity in Drosophila axons

Microtubule polarity in axons and dendrites defines the direction of intracellular transport in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends facing the tips of the processes (plus-end-out), while dendrites contain microtubules with minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor required for dynein-dependent microtubule sorting in axons of Drosophila neurons. Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal cultures. In addition to these defects, depletion of Spindly in neurons causes major collapse of axonal patterning in the third-instar larval brain as well as severe coordination impairment in adult flies. These defects can be fully rescued by full-length Spindly, but not by variants with mutations in its dynein-binding site. Biochemical analysis demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between dynein and cortical actin in axons. Therefore, Spindly plays a critical role during neurodevelopment by mediating dynein-driven sorting of axonal microtubules. Significance StatementNeurons send and receive electrical signals through long microtubule-filled neurites called axons and dendrites. One of the main structural differences between axons and dendrites is how their microtubules are organized. Axons contains microtubules with their plus-ends out while microtubules in dendrites are organized with mixed or plus-end-in orientation. Dynein, the main minus-end microtubule motor, anchored to cortical actin filaments in the axons is responsible for the uniform microtubule polarity in axons. However, it is unknown how dynein is recruited to the actin cortex in axons. The major finding of this work is that Spindly, a protein involved in anchoring dynein to kinetochores during cell division, has a second important function in interphase cells recruiting dynein to the actin cortex in axons.

cell biology↗

bioRxiv2019

Competition between kinesin-1 and myosin-V define 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. In this study, we use Staufen, an RNA-binding protein that colocalizes with osk mRNA, as a proxy for posterior determination. We demonstrate that posterior localization of osk/Staufen is 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 it 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 oocyte is defined by kinesin-myosin competition, whose outcome is primarily determined by cortical microtubule density.

cell biology↗

bioRxiv2019

Ser/Thr kinase Trc controls neurite outgrowth in Drosophila by modulating microtubule-microtubule sliding

Correct neuronal development requires tailored neurite outgrowth. Neurite outgrowth is driven by microtubule sliding - the transport of microtubules along each other. We have recently demonstrated that a \"mitotic\" kinesin-6 (Pavarotti in Drosophila) effectively inhibits microtubule-sliding and neurite outgrowth. However, mechanisms of Pavarotti regulation in interphase cells and specifically in neurite outgrowth are unknown. Here, we use a combination of live imaging and biochemical methods to show that the inhibition of microtubule sliding by Pavarotti is controlled by phosphorylation. We identify the Ser/Thr NDR kinase Tricornered (Trc) as a Pavarotti-dependent regulator of microtubule sliding in neurons. Further, we show that Trc-mediated phosphorylation of Pavarotti promotes its interaction with 14-3-3 proteins. 14-3-3 binding is necessary for Pavarotti to interact with microtubules and inhibit sliding. Thus, we propose a pathway by which microtubule sliding can be up or down regulated in neurons to control neurite outgrowth, and establish parallels between microtubule sliding in mitosis and post-mitotic neurons.

cell biology↗

bioRxiv2018

The universal mechanism of intermediate filament transport

Intermediate filaments (IFs) are a major component of the cytoskeleton that regulates a wide range of physiological properties in eukaryotic cells. In motile cells, the IF network has to adapt to constant changes of cell shape and tension. In this study, we used two cell lines that express vimentin and keratins 8/18 to study the dynamic behavior of these IFs. We demonstrated that both IF types undergo extensive transport along microtubules. This was an unexpected result as keratin filament remodeling has been described to depend on actin dynamics. We established the role of kinesin-1 in vimentin and keratin IF transport by knocking out KIF5B, the ubiquitous isoform of kinesin-1. Futhermore, we demonstrated that unlike typical membrane cargoes, transport of both types of IFs does not involve kinesin light chains, but requires the presence of the same region of the kinesin-1 tail, suggesting a unified mechanism of IF transport.

cell biology↗