Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.     

Early endosome motility spatially organizes polysome distribution

Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (D_c,60S = 0.311 µm²/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by “hitchhiking” on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that “off- and reloading” distributes the protein translation machinery.     

A Highlights from MBoC Selection: FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus

The minus end–directed microtubule motor cytoplasmic dynein transports various cellular cargoes, including early endosomes, but how dynein binds to its cargo remains unclear. Recently fungal Hook homologues were found to link dynein to early endosomes for their transport. Here we identified FhipA in Aspergillus nidulans as a key player for HookA (A. nidulans Hook) function via a genome-wide screen for mutants defective in early-endosome distribution. The human homologue of FhipA, FHIP, is a protein in the previously discovered FTS/Hook/FHIP (FHF) complex, which contains, besides FHIP and Hook proteins, Fused Toes (FTS). Although this complex was not previously shown to be involved in dynein-mediated transport, we show here that loss of either FhipA or FtsA (A. nidulans FTS homologue) disrupts HookA–early endosome association and inhibits early endosome movement. Both FhipA and FtsA associate with early endosomes, and interestingly, while FtsA–early endosome association requires FhipA and HookA, FhipA–early endosome association is independent of HookA and FtsA. Thus FhipA is more directly linked to early endosomes than HookA and FtsA. However, in the absence of HookA or FtsA, FhipA protein level is significantly reduced. Our results indicate that all three proteins in the FtsA/HookA/FhipA complex are important for dynein-mediated early endosome movement.     

Controlled and stochastic retention concentrates dynein at microtubule ends to keep endosomes on track

Bidirectional transport of early endosomes (EEs) involves microtubules (MTs) and associated motors. In fungi, the dynein/dynactin motor complex concentrates in a comet-like accumulation at MT plus-ends to receive kinesin-3-delivered EEs for retrograde transport. Here, we analyse the loading of endosomes onto dynein by combining live imaging of photoactivated endosomes and fluorescent dynein with mathematical modelling. Using nuclear pores as an internal calibration standard, we show that the dynein comet consists of ～55 dynein motors. About half of the motors are slowly turned over (T(1/2): ～98 s) and they are kept at the plus-ends by an active retention mechanism involving an interaction between dynactin and EB1. The other half is more dynamic (T(1/2): ～10 s) and mathematical modelling suggests that they concentrate at MT ends because of stochastic motor behaviour. When the active retention is impaired by inhibitory peptides, dynein numbers in the comet are reduced to half and ～10% of the EEs fall off the MT plus-ends. Thus, a combination of stochastic accumulation and active retention forms the dynein comet to ensure capturing of arriving organelles by retrograde motors.     

Peroxisomes,lipid droplets,and endoplasmic reticulum “hitchhike” on motile early endosomes

Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell.     

Early endosomes motility in filamentous fungi: How and why they move

Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to “walk” along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.     

Dynein Heavy Chain,Encoded by Two Genes in Agaricomycetes,Is Required for Nuclear Migration in Schizophyllum commune

The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.     

Microtubule acetylation promotes kinesin-1 binding and transport

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.     

beta III spectrin binds to the Arp1 subunit of dynactin.

Cytoplasmic dynein is an intracellular motor responsible for endoplasmic reticulum-to-Golgi vesicle trafficking and retrograde axonal transport. The accessory protein dynactin has been proposed to mediate the association of dynein with vesicular cargo. Dynactin contains a 37-nm filament made up of the actin-related protein, Arp1, which may interact with a vesicle-associated spectrin network. Here, we demonstrate that Arp1 binds directly to the Golgi-associated betaIII spectrin isoform. We identify two Arp1-binding sites in betaIII spectrin, one of which overlaps with the actin-binding site conserved among spectrins. Although conventional actin binds weakly to betaIII spectrin, Arp1 binds robustly in the presence of excess F-actin. Dynein, dynactin, and betaIII spectrin co-purify on vesicles isolated from rat brain, and betaIII spectrin co-immunoprecipitates with dynactin from rat brain cytosol. In interphase cells, betaIII spectrin and dynactin both localize to cytoplasmic vesicles, co-localizing most significantly in the perinuclear region of the cell. In dividing cells, betaIII spectrin and dynactin co-localize to the developing cleavage furrow and mitotic spindle, a novel localization for betaIII spectrin. We hypothesize that the interaction between betaIII spectrin and Arp1 recruits dynein and dynactin to intracellular membranes and provides a direct link between the microtubule motor complex and its membrane-bounded cargo.     

Motors in fungal morphogenesis: cooperation versus competition

Fungal tip growth underlies substrate invasion and is essential for fungal virulence. It requires the activity of molecular motors that deliver secretory vesicles to the growth region or which mediate bi-directional motility of early endosomes. Visualizing motors and their cargo in living fungal cells revealed unexpected cooperation between motors in membrane trafficking: (1) Class V chitin synthase, which has a class 17 myosin motor domain, moves bi-directionally, with myosin-5 and kinesin-1 cooperating in delivery to the growth region, and dynein taking it back to the cell centre. The myosin-17 motor domain competes with dynein by tethering the chitin synthase to the plasma membrane before exocytosis; (2) Long-range endosome motility is based on a cooperation of kinesin-3 and dynein, but towards the microtubule plus-end dynein competes with kinesin-3 to prevent the organelles from 'falling off the track'. These results reveal a fine-balanced network of cooperative and competitive motor activity, required for fungal morphogenesis.     

RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport

The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.     

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer''s disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.     

A Highlights from MBoC Selection: Discovery of a vezatin-like protein for dynein-mediated early endosome transport

Early endosomes are transported bidirectionally by cytoplasmic dynein and kinesin-3, but how the movements are regulated in vivo remains unclear. Here our forward genetic study led to the discovery of VezA, a vezatin-like protein in Aspergillus nidulans, as a factor critical for early endosome distribution. Loss of vezA causes an abnormal accumulation of early endosomes at the hyphal tip, where microtubule plus ends are located. This abnormal accumulation depends on kinesin-3 and is due to a decrease in the frequency but not the speed of dynein-mediated early endosome movement. VezA-GFP signals are enriched at the hypha tip in an actin-dependent manner but are not obviously associated with early endosomes, thus differing from the early endosome association of the cargo adapter HookA (Hook in A. nidulans). On loss of VezA, HookA associates normally with early endosomes, but the interaction between dynein-dynactin and the early-endosome-bound HookA is significantly decreased. However, VezA is not required for linking dynein-dynactin to the cytosolic ∆C-HookA, lacking the cargo-binding C-terminus. These results identify VezA as a novel regulator required for the interaction between dynein and the Hook-bound early endosomes in vivo.     

Opposite-polarity motors activate one another to trigger cargo transport in live cells

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.     

Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in Caenorhabditis elegans

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1–dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1–depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.     

Rab11-FIP3 binds dynein light intermediate chain 2 and its overexpression fragments the Golgi complex

The mechanochemical forces that move and position intracellular organelles and their intermediates in eukaryotic cells are provided by molecular motor proteins which include the cytoplasmic dynein-1 motor complex. Recently, we identified the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) as a novel binding-partner for dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1), a subunit of cytoplasmic dynein-1. Here, we show that FIP3 also binds the dynein light intermediate chain 2 subunit (DLIC-2, gene symbol DYNC1LI2). We show that like DLIC-1, DLIC-2 binds the amino-terminal 435 amino acids of FIP3 and that FIP3 links Rab11a to DLIC-2. We also show that FIP3 recruits DLIC-2 onto membranes and that DLIC-2 is necessary for the accumulation of endocytosed-transferrin (Tfn) at the pericentrosomal endosomal-recycling compartment (ERC). Finally, we demonstrate that overexpression of FIP3 fragments the Golgi complex by sequestering cytoplasmic dynein-1. In conclusion, we have identified FIP3 as the first membrane-associated interacting-partner for DLIC-2 and propose that this interaction serves to control endosomal trafficking from sorting endosomes to the ERC.     

Cortical dynein is critical for proper spindle positioning in human cells

Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–Gα in human cells and LIN-5–GPR-1/2–Gα in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of Gα in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning.     

Phosphorylation and Pin1 binding to the LIC1 subunit selectively regulate mitotic dynein functions

The dynein motor performs multiple functions in mitosis by engaging with a wide cargo spectrum. One way to regulate dynein’s cargo-binding selectivity is through the C-terminal domain (CTD) of its light intermediate chain 1 subunit (LIC1), which binds directly with cargo adaptors. Here we show that mitotic phosphorylation of LIC1-CTD at its three cdk1 sites is required for proper mitotic progression, for dynein loading onto prometaphase kinetochores, and for spindle assembly checkpoint inactivation in human cells. Mitotic LIC1-CTD phosphorylation also engages the prolyl isomerase Pin1 predominantly to Hook2-dynein-Nde1-Lis1 complexes, but not to dynein-spindly-dynactin complexes. LIC1-CTD dephosphorylation abrogates dynein-Pin1 binding, promotes prophase centrosome–nuclear envelope detachment, and impairs metaphase chromosome congression and mitotic Golgi fragmentation, without affecting interphase membrane transport. Phosphomutation of a conserved LIC1-CTD SP site in zebrafish leads to early developmental defects. Our work reveals that LIC1-CTD phosphorylation differentially regulates distinct mitotic dynein pools and suggests the evolutionary conservation of this phosphoregulation.     

PxdA interacts with the DipA phosphatase to regulate peroxisome hitchhiking on early endosomes

In canonical microtubule-based transport, adaptor proteins link cargoes to dynein and kinesin motors. Recently, an alternative mode of transport known as “hitchhiking” was discovered, where cargoes achieve motility by hitching a ride on already-motile cargoes, rather than attaching to a motor protein. Hitchhiking has been best studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets (LDs), and endoplasmic reticulum hitchhike on early endosomes (EEs). In A. nidulans, peroxisomes hitchhike using a putative molecular linker, peroxisome distribution mutant A (PxdA), which associates with EEs. However, whether other organelles use PxdA to hitchhike on EEs is unclear, as are the molecular mechanisms that regulate hitchhiking. Here we find that the proper distribution of LDs, mitochondria, and preautophagosomes do not require PxdA, suggesting that PxdA is a peroxisome-specific molecular linker. We identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdA’s ability to associate with EEs and reduces peroxisome movement. We also identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA colocalizes with EEs and its association with EEs relies on PxdA. Together, our data suggest that PxdA and the DipA phosphatase are specific regulators of peroxisome hitchhiking on EEs.     

Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine‐tuning the activity of the retrograde motor cytoplasmic dynein.