Project description:Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.
Project description:TRIM58 is an E3 ubiquitin ligase superfamily member implicated by genome-wide association studies to regulate human erythrocyte traits. Here, we show that Trim58 expression is induced during late erythropoiesis and that its depletion by small hairpin RNAs (shRNAs) inhibits the maturation of late-stage nucleated erythroblasts to anucleate reticulocytes. Imaging flow cytometry studies demonstrate that Trim58 regulates polarization and/or extrusion of erythroblast nuclei. In vitro, Trim58 directly binds and ubiquitinates the intermediate chain of the microtubule motor dynein. In cells, Trim58 stimulates proteasome-dependent degradation of the dynein holoprotein complex. During erythropoiesis, Trim58 expression, dynein loss, and enucleation occur concomitantly, and all are inhibited by Trim58 shRNAs. Dynein regulates nuclear positioning and microtubule organization, both of which undergo dramatic changes during erythroblast enucleation. Thus, we propose that Trim58 promotes this process by eliminating dynein. Our findings identify an erythroid-specific regulator of enucleation and elucidate a previously unrecognized mechanism for controlling dynein activity.
Project description:How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.
Project description:Cytoplasmic dynein mediates spindle orientation from the cell cortex through interactions with astral microtubules, but neither the mechanism governing its cortical targeting nor the regulation thereof is well understood. Here we show that yeast dynein offloads from microtubule plus ends to the daughter cell cortex. Mutants with an engineered peptide inserted between the tail domain and the motor head retain wild-type motor activity but exhibit enhanced offloading and cortical targeting. Conversely, shortening the "neck" sequence between the tail and motor domains precludes offloading from the microtubule plus ends. Furthermore, chimeric mutants with mammalian dynein "neck" sequences rescue targeting and function. These findings provide direct support for an active microtubule-mediated delivery process that appears to be regulated by a conserved masking/unmasking mechanism.
Project description:Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function.
Project description:Cortical dynein generates pulling forces via microtubule (MT) end capture-shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. We show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture-shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.
Project description:Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a 'cap' over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.
Project description:Cytoplasmic dynein participates in multiple aspects of neocortical development. These include neural progenitor proliferation, morphogenesis, and neuronal migration. The cytoplasmic dynein light intermediate chains (LICs) 1 and 2 are cargo-binding subunits, though their relative roles are not well understood. Here, we used in utero electroporation of shRNAs or LIC functional domains to determine the relative contributions of the two LICs in the developing rat brain. We find that LIC1, through BicD2, is required for apical nuclear migration in neural progenitors. In newborn neurons, we observe specific roles for LIC1 in the multipolar to bipolar transition and glial-guided neuronal migration. In contrast, LIC2 contributes to a novel dynein role in the little-studied mode of migration, terminal somal translocation. Together, our results provide novel insight into the LICs' unique functions during brain development and dynein regulation overall.
Project description:Cytoplasmic dynein is activated by the dynactin complex, cargo adapters and LIS1 (Lissencephaly 1). How this process is regulated in vivo remains unclear. The dynein motor ring contains six AAA+ (ATPases associated with diverse cellular activities) domains. Here, we used the filamentous fungus Aspergillus nidulans to examine whether ATP hydrolysis at AAA3 regulates dynein activation in the context of other regulators. In fungal hyphae, early endosomes undergo dynein-mediated movement away from the microtubule plus ends near the hyphal tip. Dynein normally accumulates at the microtubule plus ends. The early endosomal adaptor Hook protein, together with dynactin, drives dynein activation to cause its relocation to the microtubule minus ends. This activation process depends on LIS1, but LIS1 tends to dissociate from dynein after its activation. In this study, we found that dynein containing a mutation-blocking ATP hydrolysis at AAA3 can undergo LIS1-independent activation, consistent with our genetic data that the same mutation suppresses the growth defect of the A. nidulans LIS1-deletion mutant. Our data also suggest that blocking AAA3 ATP hydrolysis allows dynein activation by dynactin without the early endosomal adaptor. As a consequence, dynein accumulates at microtubule minus ends whereas early endosomes stay near the plus ends. Dynein containing a mutation-blocking ATP binding at AAA3 largely depends on LIS1 for activation, but this mutation abnormally prevents LIS1 dissociation upon dynein activation. Together, our data suggest that the AAA3 ATPase cycle regulates the coordination between dynein activation and cargo binding as well as the dynamic dynein-LIS1 interaction.
Project description:Microtubule arrays direct intracellular organization and define cellular polarity. Here, we show a novel function of glycogen synthase kinase-3beta (GSK-3beta) in the organization of microtubule arrays through the interaction with Bicaudal-D (BICD). BICD is known to form a complex with dynein-dynactin and to function in the intracellular vesicle trafficking. Our data revealed that GSK-3beta is required for the binding of BICD to dynein but not to dynactin. Knockdown of GSK-3beta or BICD reduced centrosomally focused microtubules and induced the mislocalization of centrosomal proteins. The unfocused microtubules in GSK-3beta knockdown cells were rescued by the expression of the dynein intermediate chain-BICD fusion protein. Microtubule regrowth assays showed that GSK-3beta and BICD are required for the anchoring of microtubules to the centrosome. These results imply that GSK-3beta may function in transporting centrosomal proteins to the centrosome by stabilizing the BICD1 and dynein complex, resulting in the regulation of a focused microtubule organization.