The dynamic duo of microtubule polymerase Mini spindles/XMAP215 and cytoplasmic dynein is essential for maintaining Drosophila oocyte fate.

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. In this study, we find 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.

Go with the flow - bulk transport by molecular motors.

Cells are the smallest building blocks of all living eukaryotic organisms, usually ranging from a couple of micrometers (for example, platelets) to hundreds of micrometers (for example, neurons and oocytes) in size. In eukaryotic cells that are more than 100 µm in diameter, very often a self-organized large-scale movement of cytoplasmic contents, known as cytoplasmic streaming, occurs to compensate for the physical constraints of large cells. In this Review, we discuss cytoplasmic streaming in multiple cell types and the mechanisms driving this event. We particularly focus on the molecular motors responsible for cytoplasmic movements and the biological roles of cytoplasmic streaming in cells. Finally, we describe bulk intercellular flow that transports cytoplasmic materials to the oocyte from its sister germline cells to drive rapid oocyte growth.

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. Abnormal distribution of IFs in the cytoplasm can be rescued by direct binding of kinesin-1 to IFs, demonstrating that transport inhibition is the primary cause for the abnormal IF distribution. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytosol 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.

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 a 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.

Conserved role for Ataxin-2 in mediating endoplasmic reticulum dynamics.

Ataxin-2, a conserved RNA-binding protein, is implicated in the late-onset neurodegenerative disease Spinocerebellar ataxia type-2 (SCA2). SCA2 is characterized by shrunken dendritic arbors and torpedo-like axons within the Purkinje neurons of the cerebellum. Torpedo-like axons have been described to contain displaced endoplasmic reticulum (ER) in the periphery of the cell; however, the role of Ataxin-2 in mediating ER function in SCA2 is unclear. We utilized the Caenorhabditis elegans and Drosophila homologs of Ataxin-2 (ATX-2 and DAtx2, respectively) to determine the role of Ataxin-2 in ER function and dynamics in embryos and neurons. Loss of ATX-2 and DAtx2 resulted in collapse of the ER in dividing embryonic cells and germline, and ultrastructure analysis revealed unique spherical stacks of ER in mature oocytes and fragmented and truncated ER tubules in the embryo. ATX-2 and DAtx2 reside in puncta adjacent to the ER in both C. elegans and Drosophila embryos. Lastly, depletion of DAtx2 in cultured Drosophila neurons recapitulated the shrunken dendritic arbor phenotype of SCA2. ER morphology and dynamics were severely disrupted in these neurons. Taken together, we provide evidence that Ataxin-2 plays an evolutionary conserved role in ER dynamics and morphology in C. elegans and Drosophila embryos during development and in fly neurons, suggesting a possible SCA2 disease mechanism.

Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.

Keratin intermediate filaments (IFs) are the major cytoskeletal component in epithelial cells. The dynamics of keratin IFs have been described to depend mostly on the actin cytoskeleton, but the rapid transport of fully polymerized keratin filaments has not been reported. In this work, we used a combination of photoconversion experiments and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing to study the role of microtubules and microtubule motors in keratin filament transport. We found that long keratin filaments, like other types of IFs, are transported along microtubules by kinesin-1. Our data revealed that keratin and vimentin are nonconventional kinesin-1 cargoes because their transport did not require kinesin light chains, which are a typical adapter for kinesin-dependent cargo transport. Furthermore, we found that the same domain of the kinesin heavy chain tail is involved in keratin and vimentin IF transport, strongly suggesting that multiple types of IFs move along microtubules using an identical mechanism.-Robert, A., Tian, P., Adam, S. A., Kittisopikul, M., Jaqaman, K., Goldman, R. D., Gelfand, V. I. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.

Microtubule-microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes.

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.

Role of kinesin-1-based microtubule sliding in Drosophila nervous system development.

The plus-end microtubule (MT) motor kinesin-1 is essential for normal development, with key roles in the nervous system. Kinesin-1 drives axonal transport of membrane cargoes to fulfill the metabolic needs of neurons and maintain synapses. We have previously demonstrated that kinesin-1, in addition to its well-established role in organelle transport, can drive MT-MT sliding by transporting "cargo" MTs along "track" MTs, resulting in dramatic cell shape changes. The mechanism and physiological relevance of this MT sliding are unclear. In addition to its motor domain, kinesin-1 contains a second MT-binding site, located at the C terminus of the heavy chain. Here, we mutated this C-terminal MT-binding site such that the ability of kinesin-1 to slide MTs is significantly compromised, whereas cargo transport is unaffected. We introduced this mutation into the genomic locus of kinesin-1 heavy chain (KHC), generating the Khc(mutA) allele. Khc(mutA) neurons displayed significant MT sliding defects while maintaining normal transport of many cargoes. Using this mutant, we demonstrated that MT sliding is required for axon and dendrite outgrowth in vivo. Consistent with these results, Khc(mutA) flies displayed severe locomotion and viability defects. To test the role of MT sliding further, we engineered a chimeric motor that actively slides MTs but cannot transport organelles. Activation of MT sliding in Khc(mutA) neurons using this chimeric motor rescued axon outgrowth in cultured neurons and in vivo, firmly establishing the role of sliding in axon outgrowth. These results demonstrate that MT sliding by kinesin-1 is an essential biological phenomenon required for neuronal morphogenesis and normal nervous system development.

Microtubule Dynamics, Kinesin-1 Sliding, and Dynein Action Drive Growth of Cell Processes.

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, we combine computational modeling of a network of elastic dynamic microtubules and kinesin-1 and dynein motors 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, we show 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. We discuss implications of the model for the general problems of cell polarization, cytoskeletal polarity emergence, and cell process protrusion.

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein.

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

Intermediate filament dynamics: What we can see now and why it matters.

The mechanical properties of vertebrate cells are largely defined by the system of intermediate filaments (IF). As part of a dense network, IF polymers are constantly rearranged and relocalized in the cell to fulfill their duty as cells change shape, migrate, or divide. With the development of new imaging technologies, such as photoconvertible proteins and super-resolution microscopy, a new appreciation for the complexity of IF dynamics has emerged. This review highlights new findings about the transport of IF, the remodeling of filaments by a process of severing and re-annealing, and the subunit exchange that occurs between filament precursors and a soluble pool of IF. We will also discuss the unique dynamic features of the keratin IF network. Finally, we will speculate about how the dynamic properties of IF are related to their functions.

Vimentin filament precursors exchange subunits in an ATP-dependent manner.

Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentin(Y117L)). By tagging vimentin(Y117L) with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentin(Y117L) contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks.

Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks.

Mitochondrial membrane potential is regulated by vimentin intermediate filaments.

This study demonstrates that the association of mitochondria with vimentin intermediate filaments (VIFs) measurably increases their membrane potential. This increase is detected by quantitatively comparing the fluorescence intensity of mitochondria stained with the membrane potential-sensitive dye tetramethylrhodamine-ethyl ester (TMRE) in murine vimentin-null fibroblasts with that in the same cells expressing human vimentin (∼35% rise). When vimentin expression is silenced by small hairpin RNA (shRNA) to reduce vimentin by 90%, the fluorescence intensity of mitochondria decreases by 20%. The increase in membrane potential is caused by specific interactions between a subdomain of the non-α-helical N terminus (residues 40 to 93) of vimentin and mitochondria. In rho 0 cells lacking mitochondrial DNA (mtDNA) and consequently missing several key proteins in the mitochondrial respiratory chain (ρ(0) cells), the membrane potential generated by an alternative anaerobic process is insensitive to the interactions between mitochondria and VIF. The results of our studies show that the close association between mitochondria and VIF is important both for determining their position in cells and their physiologic activity.

Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

Tubulin CFEOM mutations both inhibit or activate kinesin motor activity.

Kinesin-mediated transport along microtubules is critical for axon development and health. Mutations in the kinesin Kif21a, or the microtubule subunit ß-tubulin, inhibit axon growth and/or maintenance resulting in the eye-movement disorder congenital fibrosis of the extraocular muscles (CFEOM). While most examined CFEOM-causing ß-tubulin mutations inhibit kinesin-microtubule interactions, Kif21a mutations activate the motor protein. These contrasting observations have led to opposed models of inhibited or hyperactive Kif21a in CFEOM. We show that, contrary to other CFEOM-causing ß-tubulin mutations, R380C enhances kinesin activity. Expression of ß-tubulin-R380C increases kinesin-mediated peroxisome transport in S2 cells. The binding frequency, percent motile engagements, run length and plus-end dwell time of Kif21a are also elevated on ß-tubulin-R380C compared with wildtype microtubules in vitro. This conserved effect persists across tubulins from multiple species and kinesins from different families. The enhanced activity is independent of tail-mediated kinesin autoinhibition and thus utilizes a mechanism distinct from CFEOM-causing Kif21a mutations. Using molecular dynamics, we visualize how ß-tubulin-R380C allosterically alters critical structural elements within the kinesin motor domain, suggesting a basis for the enhanced motility. These findings resolve the disparate models and confirm that inhibited or increased kinesin activity can both contribute to CFEOM. They also demonstrate the microtubule's role in regulating kinesins and highlight the importance of balanced transport for cellular and organismal health.

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

Single-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 coaligned with microtubules. 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 µm3 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.

Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.

Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function.

Nanoscale architect: Illuminating the key organizer of the fruit fly's sensory world.

Mechanosensory neurons utilize specialized compartments called mechanosensory organelles (MOs) to process external forces, yet the MO organization mechanisms remained unclear. In this issue, Song et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202209116) discovered that a microtubule-binding protein, DCX-EMAP, is the key organizer of fly MOs.