Functions and Mechanics of Dynein Motor Proteins
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Mechanical Design of Translocating Motor Proteins
Cell Biochem Biophys (2009) 54:11–22 DOI 10.1007/s12013-009-9049-4 REVIEW PAPER Mechanical Design of Translocating Motor Proteins Wonmuk Hwang Æ Matthew J. Lang Published online: 19 May 2009 Ó Humana Press Inc. 2009 Abstract Translocating motors generate force and move Introduction along a biofilament track to achieve diverse functions including gene transcription, translation, intracellular cargo Motor proteins form distinct classes in the protein universe transport, protein degradation, and muscle contraction. as they can convert chemical energy directly into mechan- Advances in single molecule manipulation experiments, ical work. Among them, translocating motors move along structural biology, and computational analysis are making biopolymer tracks, such as nucleic acids, polypeptides, or it possible to consider common mechanical design princi- quaternary biofilament structures like F-actin or microtu- ples of these diverse families of motors. Here, we propose a bule (Kolomeisky and Fisher proposed the term ‘‘translo- mechanical parts list that include track, energy conversion case’’ for these motors [41]. However, we prefer to use machinery, and moving parts. Energy is supplied not just ‘‘translocating motor,’’ since translocase refers to mem- by burning of a fuel molecule, but there are other sources brane-bound motors such as SecA, whose function is to or sinks of free energy, by binding and release of a fuel or translocate a protein across the membrane [24]). Movement products, or similarly between the motor and the track. is an essential part of their function. For example, RNA Dynamic conformational changes of the motor domain can polymerase (RNAP) walks along the DNA molecule and be regarded as controlling the flow of free energy to and transcribes the genetic code into RNA [25]. -
Chloroplast Transit Peptides: Structure, Function and Evolution
reviews Chloroplast transit Although the first demonstration of precursor trans- port into chloroplasts was shown over two decades peptides: structure, ago3,4, only now is this area of cell biology becom- ing well understood. Many excellent reviews have been published recently on the evolution of plas- function and tids5, the evolution of organelle genomes6, the mechanism of gene transfer from organelles to the nucleus7 and the mechanism of protein import into evolution chloroplasts8,9. Proteins destined to plastids and other organ- elles share in common the requirement for ‘new’ Barry D. Bruce sequence information to facilitate their correct trafficking within the cell. Although in most cases this information resides in a cleavable, N-terminal sequence often collectively referred to as signal It is thought that two to three thousand different proteins are sequence, the different organelle-targeting se- targeted to the chloroplast, and the ‘transit peptides’ that act as quences have distinct properties and names: ‘signal peptides’ for the endoplasmic reticulum, chloroplast targeting sequences are probably the largest class of ‘presequences’ for the mitochondria and ‘transit peptides’ for chloroplasts and other plastids. This targeting sequences in plants. At a primary structural level, transit review focuses on recent progress in dissecting peptide sequences are highly divergent in length, composition and the role of the stromal-targeting domain of chloro- plast transit peptides. I will consider briefly the organization. An emerging concept suggests that transit peptides multitude of distinct functions that transit peptides contain multiple domains that provide either distinct or overlapping perform, provide an update on the limited struc- tural information of a number of transit peptides functions. -
The Wiskott-Aldrich Syndrome: the Actin Cytoskeleton and Immune Cell Function
Disease Markers 29 (2010) 157–175 157 DOI 10.3233/DMA-2010-0735 IOS Press The Wiskott-Aldrich syndrome: The actin cytoskeleton and immune cell function Michael P. Blundella, Austen Wortha,b, Gerben Boumaa and Adrian J. Thrashera,b,∗ aMolecular Immunology Unit, UCL Institute of Child Health, London, UK bDepartment of Immunology, Great Ormond Street Hospital NHS Trust, Great Ormond Street, London, UK Abstract. Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by immune dysregulation, microthrombocytopaenia, eczema and lymphoid malignancies. Mutations in the WAS gene can lead to distinct syndrome variations which largely, although not exclusively, depend upon the mutation. Premature termination and deletions abrogate Wiskott-Aldrich syndrome protein (WASp) expression and lead to severe disease (WAS). Missense mutations usually result in reduced protein expression and the phenotypically milder X-linked thrombocytopenia (XLT) or attenuated WAS [1–3]. More recently however novel activating mutations have been described that give rise to X-linked neutropenia (XLN), a third syndrome defined by neutropenia with variable myelodysplasia [4–6]. WASP is key in transducing signals from the cell surface to the actin cytoskeleton, and a lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration. Keywords: Wiskott-Aldrich syndrome, actin polymerization, lymphocytes, -
Unsheathing WASP's Sting
news and views required for Swallow-mediated localiza- Cytoplasmic dynein is implicated in Minneapolis 55455, Minnesota, USA tion within the oocyte. many biological processes, including ves- e-mail:[email protected] The interaction between Swallow and icle and organelle transport, mitotic- Roger Karess is at the CNRS Centre de Génétique Dlc is a significant finding and provides spindle function and orientation, and Moléculaire, Ave de la Terrasse, 91198 Gif-sur- the basis for a model in which the dynein- now RNA transport and localization. It is Yvette, France motor complex is responsible for the important to emphasize that a single iso- e-mail: [email protected] anterior localization of bicoid RNA within form of the dynein-motor subunit is 1. St Johnston, D. Cell 81, 167–170 (1995). the oocyte. Interestingly, the transient known to be targeted to several cellular 2. Bashirullah, A., Cooperstock, R. & Lipshitz, H. Annu. Rev. localization of Swallow to the oocyte functions and molecular cargoes within Biochem. 67, 335–394 (1998). anterior occurs at a time when most of the individual cells. Thus it is those molecules 3. Oleynikov, Y. & Singer, R. Trends Cell Biol. 8, 381–383 dynein-motor subunits are concentrated with adaptor functions, such as those pro- (1998). 13 4. Wilhelm, J. & Vale, R. J. Cell Biol. 123, 269–274 (1993). at the posterior of the oocyte . This raises posed here for Swallow, that must 5. Schnorrer, F., Bohmann, K. & Nusslein-Volhard, C. Nature Cell the possibility that at least two distinct account for the functional specificity of Biol. -
The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy
bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. The kinetics of nucleotide binding to isolated Chlamydomonas axonemes using UV-TIRF microscopy M. Feofilova, M. Mahamdeh, J. Howard 1 bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Key words: dynein, axoneme, cilia, flagella, mantATP, fluorescent nucleotide Abbreviations ADP – adenosine diphosphate ATP – adenosine triphosphate MantATP - methylanthraniloyl adenosine triphosphate TIRF – total-internal-reflection fluorescence 2 bioRxiv preprint doi: https://doi.org/10.1101/547091; this version posted February 11, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract Cilia and flagella are long, slender organelles found in many eukaryotic cells where they have sensory, developmental and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. -
Actin Cytoskeleton of Spread Fibroblasts Appears to Assemble at the Cell Edges
J. Cell Sd. 82, 235-248 (1986) 235 Printed in Great Britain © The Company of Biologists Limited 1986 ACTIN CYTOSKELETON OF SPREAD FIBROBLASTS APPEARS TO ASSEMBLE AT THE CELL EDGES TATJANA M. SVITKINA, ALEXANDER A. NEYFAKH, JR Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, USSR AND ALEXANDER D. BERSHADSKY All-Union Cancer Research Center, Academy of Medical Sciences, Moscow 115478, USSR SUMMARY The action of metabolic inhibitors on actin cytoskeleton of cultured quail embryo fibroblasts has been studied using electron microscopy of platinum replicas and immunofluorescence microscopy. Sodium azide as well as other inhibitors (oligomycin and dinitrophenol) caused the disassembly of all types of actin structures: actin meshwork at the cell active edges, microfilament sheath underlying the cell surface, and microfilament bundles. Studying the time- and dose-dependence of the destruction process we have found that the active edge meshwork and microfilament sheath are much more labile than microfilament bundles. After the removal of metabolic inhibitors actin cytoskeleton restoration begins at the cell edges. The first sign of this process is the formation of actin meshwork along the whole cell perimeter (l-10min of recovery). Sometimes fragments of this meshwork bend upwards forming ruffles. Later (10-20 min of recovery) the microfilament sheath appears at the cell periphery as a narrow band. The sheath seems to be formed from the edge meshwork, since ruffles in the process of transformation to sheath could be seen. During the following restoration the microfilament sheath gradually expands towards the cell centre. The last step of actin cytoskeleton restoration (60—120 min of recovery) is the formation of bundles. -
HTS-Tubulin Polymerization Assay Biochem Kit™
The Protein Manual Experts Cytoskeleton, Inc. V 8.0 HTS-Tubulin Polymerization Assay Biochem Kit™ (>97% pure tubulin, Porcine Tubulin) Cat. # BK004P Phone: (303) 322.2254 Fax: (303) 322.2257 Customer Service: [email protected] cytoskeleton.com Technical Support: [email protected] cytoskeleton.com Page 2 Manual Contents Section I: Introduction About Tubulin -------------------------------------------------------------------------- 5 About the BK004P polymerization Assay -------------------------------------- 6-7 Comparison of Polymerization Assays ----------------------------------------- 8-9 Section II: Purchaser Notification ------------------------------------------------------------ 10 Section III: Kit Contents ------------------------------------------------------------------------- 11-12 Section V: Reconstitution and Storage of Components ----------------------------- 13 Section IV: Important Technical Notes Notes on Updated version --------------------------------------------------------- 14 Spectrophotometer settings ------------------------------------------------------- 14 Spectrophotometer pathlength---------------------------------------------------- 15 Temperature & time dependence of polymerization ------------------------ 15 Recommended pipetting technique --------------------------------------------- 15-16 Tubulin protein stability ------------------------------------------------------------- 16 Test compound or protein preparation ------------------------------------------ 16-17 Section VI: Assay Protocol -
Theory of Cytoskeletal Reorganization During Crosslinker-Mediated Mitotic Spindle Assembly
bioRxiv preprint doi: https://doi.org/10.1101/419135; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Theory of cytoskeletal reorganization during crosslinker-mediated mitotic spindle assembly A. R. Lamson, C. J. Edelmaier, M. A. Glaser, and M. D. Betterton Abstract Cells grow, move, and respond to outside stimuli by large-scale cytoskeletal reorganization. A prototypical example of cytoskeletal remodeling is mitotic spindle assembly, during which micro- tubules nucleate, undergo dynamic instability, bundle, and organize into a bipolar spindle. Key mech- anisms of this process include regulated filament polymerization, crosslinking, and motor-protein activity. Remarkably, using passive crosslinkers, fission yeast can assemble a bipolar spindle in the absence of motor proteins. We develop a torque-balance model that describes this reorganization due to dynamic microtubule bundles, spindle-pole bodies, the nuclear envelope, and passive crosslink- ers to predict spindle-assembly dynamics. We compare these results to those obtained with kinetic Monte Carlo-Brownian dynamics simulations, which include crosslinker-binding kinetics and other stochastic effects. Our results show that rapid crosslinker reorganization to microtubule overlaps facilitates crosslinker-driven spindle assembly, a testable prediction for future experiments. Combin- ing these two modeling techniques, we illustrate a general method for studying cytoskeletal network reorganization. 1 bioRxiv preprint doi: https://doi.org/10.1101/419135; this version posted March 1, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. -
Tracking Melanosomes Inside a Cell to Study Molecular Motors and Their Interaction
Tracking melanosomes inside a cell to study molecular motors and their interaction Comert Kural*, Anna S. Serpinskaya†, Ying-Hao Chou†, Robert D. Goldman†, Vladimir I. Gelfand†‡, and Paul R. Selvin*§¶ *Center for Biophysics and Computational Biology and §Department of Physics, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and †Department of Cell and Molecular Biology, Northwestern University School of Medicine, Chicago, IL 60611 Communicated by Gordon A. Baym, University of Illinois at Urbana–Champaign, Urbana, IL, January 9, 2007 (received for review June 4, 2006) Cells known as melanophores contain melanosomes, which are membrane organelles filled with melanin, a dark, nonfluorescent pigment. Melanophores aggregate or disperse their melanosomes when the host needs to change its color in response to the environment (e.g., camouflage or social interactions). Melanosome transport in cultured Xenopus melanophores is mediated by my- osin V, heterotrimeric kinesin-2, and cytoplasmic dynein. Here, we describe a technique for tracking individual motors of each type, both individually and in their interaction, with high spatial (Ϸ2 nm) and temporal (Ϸ1 msec) localization accuracy. This method enabled us to observe (i) stepwise movement of kinesin-2 with an average step size of 8 nm; (ii) smoother melanosome transport (with fewer pauses), in the absence of intermediate filaments (IFs); and (iii) motors of actin filaments and microtubules working on the same cargo nearly simultaneously, indicating that a diffusive step is not needed between the two systems of transport. In concert with our previous report, our results also show that dynein-driven retro- grade movement occurs in 8-nm steps. Furthermore, previous studies have shown that melanosomes carried by myosin V move 35 nm in a stepwise fashion in which the step rise-times can be as long as 80 msec. -
Construction and Loss of Bacterial Flagellar Filaments
biomolecules Review Construction and Loss of Bacterial Flagellar Filaments Xiang-Yu Zhuang and Chien-Jung Lo * Department of Physics and Graduate Institute of Biophysics, National Central University, Taoyuan City 32001, Taiwan; [email protected] * Correspondence: [email protected] Received: 31 July 2020; Accepted: 4 November 2020; Published: 9 November 2020 Abstract: The bacterial flagellar filament is an extracellular tubular protein structure that acts as a propeller for bacterial swimming motility. It is connected to the membrane-anchored rotary bacterial flagellar motor through a short hook. The bacterial flagellar filament consists of approximately 20,000 flagellins and can be several micrometers long. In this article, we reviewed the experimental works and models of flagellar filament construction and the recent findings of flagellar filament ejection during the cell cycle. The length-dependent decay of flagellar filament growth data supports the injection-diffusion model. The decay of flagellar growth rate is due to reduced transportation of long-distance diffusion and jamming. However, the filament is not a permeant structure. Several bacterial species actively abandon their flagella under starvation. Flagellum is disassembled when the rod is broken, resulting in an ejection of the filament with a partial rod and hook. The inner membrane component is then diffused on the membrane before further breakdown. These new findings open a new field of bacterial macro-molecule assembly, disassembly, and signal transduction. Keywords: self-assembly; injection-diffusion model; flagellar ejection 1. Introduction Since Antonie van Leeuwenhoek observed animalcules by using his single-lens microscope in the 18th century, we have entered a new era of microbiology. -
Review of Molecular Motors
REVIEWS CYTOSKELETAL MOTORS Moving into the cell: single-molecule studies of molecular motors in complex environments Claudia Veigel*‡ and Christoph F. Schmidt§ Abstract | Much has been learned in the past decades about molecular force generation. Single-molecule techniques, such as atomic force microscopy, single-molecule fluorescence microscopy and optical tweezers, have been key in resolving the mechanisms behind the power strokes, ‘processive’ steps and forces of cytoskeletal motors. However, it remains unclear how single force generators are integrated into composite mechanical machines in cells to generate complex functions such as mitosis, locomotion, intracellular transport or mechanical sensory transduction. Using dynamic single-molecule techniques to track, manipulate and probe cytoskeletal motor proteins will be crucial in providing new insights. Molecular motors are machines that convert free energy, data suggest that during the force-generating confor- mostly obtained from ATP hydrolysis, into mechanical mational change, known as the power stroke, the lever work. The cytoskeletal motor proteins of the myosin and arm of myosins8,11 rotates around its base at the catalytic kinesin families, which interact with actin filaments and domain11–17, which can cause the displacement of bound microtubules, respectively, are the best understood. Less cargo by several nanometres18 (FIG. 1B). In kinesins, the is known about the dynein family of cytoskeletal motors, switching of the neck linker (~13 amino acids connecting which interact with microtubules. Cytoskeletal motors the catalytic core to the cargo-binding stalk domain) from power diverse forms of motility, ranging from the move- an ‘undocked’ state to a state in which it is ‘docked’ to the ment of entire cells (as occurs in muscular contraction catalytic domain, is the equivalent of the myosin power or cell locomotion) to intracellular structural dynamics stroke10. -
Yuri Gagarin Is Required for Actin, Tubulin and Basal Body Functions in Drosophila Spermatogenesis
1926 Research Article yuri gagarin is required for actin, tubulin and basal body functions in Drosophila spermatogenesis Michael J. Texada, Rebecca A. Simonette, Cassidy B. Johnson, William J. Deery and Kathleen M. Beckingham* Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 South Main Street, Houston, TX 77005, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 March 2008 Journal of Cell Science 121, 1926-1936 Published by The Company of Biologists 2008 doi:10.1242/jcs.026559 Summary Males of the genus Drosophila produce sperm of remarkable the yuri mutant, late clusters of syncytial nuclei are deformed length. Investigation of giant sperm production in Drosophila and disorganized. The basal bodies are also mispositioned on melanogaster has demonstrated that specialized actin and the nuclei, and the association of a specialized structure, the microtubule structures play key roles. The gene yuri gagarin centriolar adjunct (CA), with the basal body is lost. Some of (yuri) encodes a novel protein previously identified through its these nuclear defects might underlie a further unexpected role in gravitaxis. A male-sterile mutation of yuri has revealed abnormality: sperm nuclei occasionally locate to the wrong ends roles for Yuri in the functions of the actin and tubulin structures of the spermatid cysts. The structure of the axonemes that grow of spermatogenesis. Yuri is a component of the motile actin cones out from the basal bodies is affected in the yuri mutant, that individualize the spermatids and is essential for their suggesting a possible role for the CA in axoneme formation. formation. Furthermore, Yuri is required for actin accumulation in the dense complex, a microtubule-rich structure on the sperm Key words: Drosophila, Spermatogenesis, Actin, Tubulin, Basal nuclei thought to strengthen the nuclei during elongation.