Activity Of Cytoplasmic Dynein Research Articles

LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.

Cargo-Mediated Activation of Cytoplasmic Dynein in vivo.

Cytoplasmic dynein-1 is a minus-end-directed microtubule motor that transports a variety of cargoes including early endosomes, late endosomes and other organelles. In many cell types, dynein accumulates at the microtubule plus end, where it interacts with its cargo to be moved toward the minus end. Dynein binds to its various cargoes via the dynactin complex and specific cargo adapters. Dynactin and some of the coiled-coil-domain-containing cargo adapters not only link dynein to cargo but also activate dynein motility, which implies that dynein is activated by its cellular cargo. Structural studies indicate that a dynein dimer switches between the autoinhibited phi state and an open state; and the binding of dynactin and a cargo adapter to the dynein tails causes the dynein motor domains to have a parallel configuration, allowing dynein to walk processively along a microtubule. Recently, the dynein regulator LIS1 has been shown to be required for dynein activation in vivo, and its mechanism of action involves preventing dynein from switching back to the autoinhibited state. In this review, we will discuss our current understanding of dynein activation and point out the gaps of knowledge on the spatial regulation of dynein in live cells. In addition, we will emphasize the importance of studying a complete set of dynein regulators for a better understanding of dynein regulation in vivo.

Microtubule-dependent pushing forces contribute to long-distance aster movement and centration in Xenopus laevis egg extracts.

During interphase of the eukaryotic cell cycle, the microtubule (MT) cytoskeleton serves as both a supportive scaffold for organelles and an arborized system of tracks for intracellular transport. At the onset of mitosis, the position of the astral MT network, specifically its center, determines the eventual location of the spindle apparatus and ultimately the cytokinetic furrow. Positioning of the MT aster often results in its movement to the center of a cell, even in large blastomeres hundreds of microns in diameter. This translocation requires positioning forces, yet how these forces are generated and then integrated within cells of various sizes and geometries remains an open question. Here we describe a method that combines microfluidics, hydrogels, and Xenopus laevis egg extract to investigate the mechanics of aster movement and centration. We determined that asters were able to find the center of artificial channels and annular cylinders, even when cytoplasmic dynein-dependent pulling mechanisms were inhibited. Characterization of aster movement away from V-shaped hydrogel barriers provided additional evidence for a MT-based pushing mechanism. Importantly, the distance over which this mechanism seemed to operate was longer than that predicted by radial aster growth models, agreeing with recent models of a more complex MT network architecture within the aster.

Order in the cytoplasm

Cell Biology Extracts of the very large eggs of the African clawed frog, Xenopus laevis , have proven a valuable model system for the study of cell division. Cheng and Ferrell found that after homogenization, such cytoplasm can reorganize back into cell-like structures and undergo multiple rounds of division (see the Perspective by Mitchison and Field). This reorganization apparently occurs without the usual factors that are known to lead to such structural changes during cell division, such as F-actin, myosin II, various individual kinesins, aurora kinase A, or DNA. What is required is energy from adenosine triphosphate, microtubule polymerization, cytoplasmic dynein activity, and a specific kinase-involved cell cycle progression. Nongenetic information in the cytoplasm is apparently sufficient for basic spatial organization of the cell. Science , this issue p. [631][1]; see also p. [569][2] [1]: /lookup/doi/10.1126/science.aav7793 [2]: /lookup/doi/10.1126/science.aaz5635

Spontaneous emergence of cell-like organization in Xenopus egg extracts.

Every daughter cell inherits two things from its mother: genetic information and a spatially organized complement of macromolecular complexes and organelles. The extent to which de novo self-organization, as opposed to inheritance of an already organized state, can suffice to yield functional cells is uncertain. We used Xenopus laevis egg extracts to show that homogenized interphase egg cytoplasm self-organizes over the course of ~30 minutes into compartments 300 to 400 micrometers in length that resemble cells. Formation of these cell-like compartments required adenosine triphosphate and microtubule polymerization but did not require added demembranated sperm nuclei with their accompanying centrosomes or actin polymerization. In cycling extracts with added sperm, the compartments underwent multiple cycles of division and reorganization, with mother compartments giving rise to two daughters at the end of each mitotic cycle. These results indicate that the cytoplasm can generate much of the spatial organization and cell cycle function of the early embryo.

Role of Dynactin in the Intracellular Localization and Activation of Cytoplasmic Dynein.

Cytoplasmic dynein, the major minus end-directed motor protein in several cell types, transports a variety of intracellular cargo upon forming a processive tripartite complex with its activator dynactin and cargo adaptors such as Hook3 and BicD2. Our current understanding of dynein regulation stems from a combination of in vivo studies of cargo movement upon perturbation of dynein activity, in vitro single-molecule experiments, and cryo-electron microscopy studies of dynein structure and its interaction with dynactin and cargo adaptors. In this Perspective, we first consolidate data from recent publications to understand how perturbations to the dynein-dynactin interaction and dynactin's in vivo localization alter the behavior of dynein-driven cargo transport in a cell type- and experimental condition-specific manner. In addition, we touch upon results from in vivo and in vitro studies to elucidate how dynein's interaction with dynactin and cargo adaptors activates dynein and enhances its processivity. Finally, we propose questions that need to be addressed in the future with appropriate experimental designs so as to improve our understanding of the spatiotemporal regulation of dynein's function in the context of the distribution and dynamics of dynactin in living cells.

GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells

The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.

Issue Cover (June 2015)

Cover legend: Cholera toxin is enriched in plasma membrane tubules formed by cytoplasmic dynein. COS-7 cells were ATP depleted prior to labeling with Alexa555-CTxB and imaged live at 37°C on a confocal microscope. ATP depletion inhibits endocytic scission, leading to the generation of microns long, nascent endocytic tubules that are continuous with the plasma membrane. These invaginations are microtubule-dependent and form through persistent microtubule motor activity of cytoplasmic dynein providing a pulling force on the cell surface. Using this assay, Day et al. define a novel role for microtubule motors in tubulating the plasma membrane during clathrin-independent endocytosis. See Day et al. Traffic 2015; 16(6):572–590. Read the full article on doi:10.1111/tra.12269

Isolation and analysis of microtubules and associated proteins.

Microtubules, microtubule-associated proteins (MAPs), and motor proteins are essential components of all eukaryotic cells. They are all involved in mitosis and in the movement of organelles, proteins, and vesicles in cells. MAPs act as structural elements of the microtubule component of the cytoskeleton, whereas molecular motors propel cargo along microtubule tracks or translocate microtubules in the cytoplasm. This introduction provides an overview of procedures developed by many labs to isolate microtubules from cell homogenates, purify tubulin, MAPs, and motor proteins from microtubules preparations, and analyze kinesin and cytoplasmic dynein activity by video-enhanced differential interference contrast microscopy and fluorescence microscopy. These ingenious microscope-based assays, which were developed to determine the motility characteristics of kinesin and dynein, reveal, in clear and dramatic fashion, the activity of these amazing nanomachines in real time.

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.

The Drosophila MAST kinase Drop out is required to initiate membrane compartmentalisation during cellularisation and regulates dynein-based transport.

Cellularisation of the Drosophila syncytial blastoderm embryo into the polarised blastoderm epithelium provides an excellent model with which to determine how cortical plasma membrane asymmetry is generated during development. Many components of the molecular machinery driving cellularisation have been identified, but cell signalling events acting at the onset of membrane asymmetry are poorly understood. Here we show that mutations in drop out (dop) disturb the segregation of membrane cortical compartments and the clustering of E-cadherin into basal adherens junctions in early cellularisation. dop is required for normal furrow formation and controls the tight localisation of furrow canal proteins and the formation of F-actin foci at the incipient furrows. We show that dop encodes the single Drosophila homologue of microtubule-associated Ser/Thr (MAST) kinases. dop interacts genetically with components of the dynein/dynactin complex and promotes dynein-dependent transport in the embryo. Loss of dop function reduces phosphorylation of Dynein intermediate chain, suggesting that dop is involved in regulating cytoplasmic dynein activity through direct or indirect mechanisms. These data suggest that Dop impinges upon the initiation of furrow formation through developmental regulation of cytoplasmic dynein.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

3P154 細胞質ダイニンの自己阻害と協同的な活性化(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

3P154 細胞質ダイニンの自己阻害と協同的な活性化(11.分子モーター,ポスター,日本生物物理学会年会第51回(2013年度))

LIS1 Clamps Dynein to the Microtubule

Cytoplasmic dynein is a motor essential for numerous mechanical processes in eukaryotic cells. How its activity is regulated is largely unknown. By using a combination of approaches including single-molecule biophysics and electron microscopy, Huang et al. in this issue uncover the regulatory mechanism by which LIS1 controls the activity of cytoplasmic dynein.

Functional Dissection of LIS1 and NDEL1 for Understanding the Molecular Mechanism of Cytoplasmic Dynein Regulation

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected regulatory functions of LIS1 and NDEL1 on dynein motility using full length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1 and the mechanisms of cytoplasmic dynein regulation. Furthermore, we performed EM study to investigate the binding sites of LIS1 and NDEL1 on dynein molecule and to examine the conformational changes induced by the binding of LIS1 or NDEL1. Our findings will provide the new insights into the molecular mechanism of the regulation of cytoplasmic dynein by LIS1 and NDEL1.

Functional Dissection of LIS1 and NDEL1 Towards Understanding the Molecular Mechanisms of Cytoplasmic Dynein Regulation

LIS1 and NDEL1 are known to be essential for the activity of cytoplasmic dynein in living cells. We previously reported that LIS1 and NDEL1 directly regulated the motility of cytoplasmic dynein in an in vitro motility assay. LIS1 suppressed dynein motility and inhibited the translocation of microtubules (MTs), while NDEL1 dissociated dynein from MTs and restored dynein motility following suppression by LIS1. However, the molecular mechanisms and detailed interactions of dynein, LIS1, and NDEL1 remain unknown. In this study, we dissected the regulatory effects of LIS1 and NDEL1 on dynein motility using full-length or truncated recombinant fragments of LIS1 or NDEL1. The C-terminal fragment of NDEL1 dissociated dynein from MTs, whereas its N-terminal fragment restored dynein motility following suppression by LIS1, demonstrating that the two functions of NDEL1 localize to different parts of the NDEL1 molecule, and that restoration from LIS1 suppression is caused by the binding of NDEL1 to LIS1, rather than to dynein. The truncated monomeric form of LIS1 had little effect on dynein motility, but an artificial dimer of truncated LIS1 suppressed dynein motility, which was restored by the N-terminal fragment of NDEL1. This suggests that LIS1 dimerization is essential for its regulatory function. These results shed light on the molecular interactions between dynein, LIS1, and NDEL1, and the mechanisms of cytoplasmic dynein regulation.

Caenorhabditis elegans ortholog of the p24/p22 subunit, DNC‐3, is essential for the formation of the dynactin complex by bridging DNC‐1/p150Glued and DNC‐2/dynamitin

Dynactin is a multisubunit protein complex required for the activity of cytoplasmic dynein. In Caenorhabditis elegans, although 10 of the 11 dynactin subunits were identified based on the sequence similarities to their orthologs, the p24/p22 subunit has not been detected in the genome. Here, we demonstrate that DNC-3 (W10G11.20) is the functional counterpart of the p24/p22 subunit in C. elegans. RNAi phenotypes and subcellular localization of DNC-3 in early C. elegans embryos were nearly identical to those of the known dynactin components. All other dynactin subunits were co-immunoprecipitated with DNC-3, indicating that DNC-3 is a core component of dynactin. Furthermore, the overall secondary structure of DNC-3 resembles to those of the mammalian and yeast p24/p22. We found that DNC-3 is required for the localization of the DNC-1/p150Glued and DNC-2/dynamitin, the two components of the projection arm of dynactin, to the nuclear envelope of meiotic nuclei in the adult gonad. Moreover, DNC-3 physically interacted with DNC-1 and DNC-2 and significantly enhanced the binding ability between DNC-1 and DNC-2 in vitro. These results suggest that DNC-3 is essential for the formation of the projection arm subcomplex of dynactin.

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.

Regulation of Cytoplasmic Dynein ATPase by Lis1

Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport.