DOCSLIB.ORG

Insight Into Microtubule Nucleation from Tubulin-Capping Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Insight Into Microtubule Nucleation from Tubulin-Capping Proteins Insight into microtubule nucleation from tubulin-capping proteins Valérie Campanaccia, Agathe Urvoasa, Soraya Cantos-Fernandesa, Magali Aumont-Nicaisea, Ana-Andreea Artenia, Christophe Veloursa, Marie Valerio-Lepinieca, Birgit Dreierb, Andreas Plückthunb, Antoine Pilonc,d, Christian Poüsc,e,1, Philippe Minarda, and Benoît Giganta,1 aInstitute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette Cedex, France; bDepartment of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland; cINSERM UMR-S 1193, Université Paris-Sud, Université Paris-Saclay, 92296 Châtenay-Malabry, France; dBiochimie, APHP, Hôpitaux Universitaires de l’Est Parisien, 75571 Paris Cedex 12, France; and eBiochimie-Hormonologie, APHP, Hôpitaux Universitaires Paris-Sud, 92141 Clamart, France Edited by Antonina Roll-Mecak, National Institute of Neurological Disorders and Stroke, Bethesda, MD, and accepted by Editorial Board Member Sue Biggins April 5, 2019 (received for review August 7, 2018) Nucleation is one of the least understood steps of microtubule solutions in the early steps of assembly (17, 18). However, the dynamics. It is a kinetically unfavorable process that is templated exact pathway of microtubule nucleation is not understood. in the cell by the γ-tubulin ring complex or by preexisting micro- Here we link the binding mode of proteins targeting tubulin tubules; it also occurs in vitro from pure tubulin. Here we study the surfaces involved in longitudinal contacts to their ability to in- nucleation inhibition potency of natural or artificial proteins in hibit microtubule nucleation, focusing on the CopN protein from connection with their binding mode to the longitudinal surface Chlamydia pneumoniae. CopN has been shown to interfere with of α-orβ-tubulin. The structure of tubulin-bound CopN, a Chlamydia microtubule growth (19, 20). While a mechanism of tubulin protein that delays nucleation, suggests that this protein may inter- fere with two protofilaments at the (+) end of a nucleus. Designed sequestration has been proposed for the inhibition of micro- ankyrin repeat proteins that share a binding mode similar to that of tubule elongation (20, 21), CopN also delays nucleation, sug- CopN also impede nucleation, whereas those that target only one gesting direct interference with formation of the nucleus (21). protofilament do not. In addition, an αRep protein predicted to tar- We have determined the structure of CopN bound to tubulin. CELL BIOLOGY get two protofilaments at the (−) end does not delay nucleation, Modeling indicates that CopN would cap a protofilament at the pointing to different behaviors at both ends of the nucleus. Our (+) end of a microtubule in a way that would also prevent the results link the interference with protofilaments at the (+)endand elongation of a neighboring protofilament. Among various the inhibition of nucleation. possible mechanisms, this model and the comparison with β-tubulin-specific designed ankyrin repeat proteins (DARPins) cytoskeleton | microtubule nucleation | structural biology | CopN | (22, 23) raise the hypothesis that microtubule nucleation is artificial binding proteins inhibited by the simultaneous destabilization of two adjacent protofilaments. n eukaryotic cells, microtubules form different types of arrays to fulfill different functions. For instance, a microtubule aster I Significance organizes the cytoplasm in interphase, whereas the mitotic spindle of dividing cells ensures faithful chromosome segrega- tion. Generating and maintaining these arrays require that both Microtubules are involved in many key functions of eukaryotic the formation and the length of microtubules be controlled in cells, including cell division, intracellular transport, and cell space and time (1, 2). Microtubule assembly proceeds in two shape. They are hollow tubes made of parallel filaments, themselves formed by the self-assembly of αβ-tubulin mole- main steps. First a nucleus forms, and then it elongates at its free cules. Whereas microtubules lengthen and shorten from their ends. The microtubule elongation phase and the subsequent ends dynamically, their birth, called nucleation, remains poorly behavior of microtubules have been characterized mainly by the understood. To gain information on this process, we have de- description of a dynamic instability mechanism, with alternating termined the structure of tubulin bound to CopN, a bacterial periods of slow growth and faster shortening (3). In comparison, protein that delays nucleation. Together with the behavior of microtubule nucleation has remained far less well described (4, 5). artificial tubulin-binding proteins, our results lead to the hy- Although the issue is debated (6), nucleation is generally consid- pothesis that targeting two filaments at the fast-growing end ered a kinetically unfavorable process. To overcome this kinetic of the microtubule inhibits nucleation. They also suggest dif- γ barrier, in the cell, nucleation is templated by the -tubulin ring ferent dynamics at both ends of the nucleus. complex (γ-TuRC) (7) in combination with, for instance, XMAP215 family proteins (8, 9), but also by preexisting mi- Author contributions: V.C., C.P., P.M., and B.G. designed research; V.C., A.U., S.C.-F., crotubules (1, 10). Nucleation is further assisted by microtubule- M.A.-N., A.-A.A., C.V., A. Pilon, C.P., and B.G. performed research; B.D. and A. Plückthun associated proteins (2, 11, 12). contributed new reagents/analytic tools; V.C., A.U., M.V.-L., A. Pilon, C.P., P.M., and B.G. analyzed data; and C.P. and B.G. wrote the paper. Whereas several models for in vitro spontaneous nucleation (from a pure tubulin solution) have been proposed (summarized The authors declare no conflict of interest. This article is a PNAS Direct Submission. A.R.-M. is a guest editor invited by the in ref. 6), recent characterizations of the interaction between Editorial Board. tubulin molecules in the microtubule have allowed narrowing Published under the PNAS license. down the possible nucleation process. Indeed, longitudinal con- Data deposition: The atomic coordinates and structure factors have been deposited in the tacts (between tubulins within a protofilament) have been shown Protein Data Bank, www.wwpdb.org (PDB ID codes 6GX7, 6GVM, and 6GVN). to be stronger that lateral contacts (between adjacent protofila- 1To whom correspondence may be addressed. Email: [email protected] or benoit. ments) in the core of the microtubule (13–15) and at its growing [email protected]. end (16). Building on these results, it is also likely that lateral This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. contacts in the nucleus are weaker that longitudinal ones (5), in 1073/pnas.1813559116/-/DCSupplemental. agreement with electron microscopy experiments from tubulin Published online April 29, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1813559116 PNAS | May 14, 2019 | vol. 116 | no. 20 | 9859–9864 Downloaded by guest on September 30, 2021 Results turbidity plateau level was obtained with tubulin and tubu- CopN Inhibits Microtubule Nucleation. In the presence of CopN, the lin:CopN (Fig. 1A), and because fewer microtubules were C assembly of tubulin in microtubules as monitored by turbidity is formed in this latter case (Fig. 1 ), we expected that they grew delayed compared with the control (21). This observation led us longer. To verify this hypothesis, we recorded the distributions of to propose that CopN interferes with microtubule nucleation. To microtubule lengths formed in these conditions. We found that microtubules grew longer along with the increase in turbidity in confirm this hypothesis, we directly counted the number of mi- the presence of CopN, whereas microtubule length did not vary crotubules obtained from tubulin:CopN and tubulin-alone sam- D E “ ” significantly in the tubulin control (Fig. 1 and ). This ob- ples, setting the concentration of free tubulin (not bound to servation is in agreement with the high nucleation efficiency of CopN) constant. For convenience, this experiment was first GMPCPP-tubulin (24) at the expense of elongation (25). In the performed with GMPCPP-tubulin, which leads to stable micro- presence of CopN, fewer nuclei being formed, microtubule tubules (24). As observed with GTP-tubulin (21), CopN also elongation is favored. increased the lag phase of the GMPCPP-tubulin assembly (Fig. 1A) and consistently decreased the nucleation rate (Fig. 1 B and CopN Binds to the Longitudinal Surface of the Tubulin β Subunit. C). A similar trend was observed with microtubules assembled Having established that CopN interferes with microtubule nu- from GTP-tubulin (SI Appendix, Fig. S1). Because a similar cleation, to gain insight into this mechanism, we aimed to de- termine the structure of CopN bound to tubulin. Because our attempts to crystallize this binary complex were unsuccessful, we considered using a tubulin-stabilizing protein as a crystallization chaperone. Because the two most commonly used tubulin crys- tallization helpers—stathmin-like domain (SLD) proteins and β-tubulin targeting DARPins—compete with CopN for tubulin binding (20, 21), we instead used αRep proteins (26, 27) specific for α-tubulin (28). Among these proteins, the iiiA5 αRep inter- acted with tubulin with a dissociation constant (KD) in the nanomolar range and made a ternary CopN-tubulin-iiiA5 com- plex (SI Appendix, Fig. S2). Using the Δ84 CopN construct (20) (Fig. 2A),
Load more

Recommended publications
  • Conserved Microtubule–Actin Interactions in Cell Movement and Morphogenesis
    REVIEW Conserved microtubule–actin interactions in cell movement and morphogenesis Olga C. Rodriguez, Andrew W. Schaefer, Craig A. Mandato, Paul Forscher, William M. Bement and Clare M. Waterman-Storer Interactions between microtubules and actin are a basic phenomenon that underlies many fundamental processes in which dynamic cellular asymmetries need to be established and maintained. These are processes as diverse as cell motility, neuronal pathfinding, cellular wound healing, cell division and cortical flow. Microtubules and actin exhibit two mechanistic classes of interactions — regulatory and structural. These interactions comprise at least three conserved ‘mechanochemical activity modules’ that perform similar roles in these diverse cell functions. Over the past 35 years, great progress has been made towards under- crosstalk occurs in processes that require dynamic cellular asymme- standing the roles of the microtubule and actin cytoskeletal filament tries to be established or maintained to allow rapid intracellular reor- systems in mechanical cellular processes such as dynamic shape ganization or changes in shape or direction in response to stimuli. change, shape maintenance and intracellular organelle movement. Furthermore, the widespread occurrence of these interactions under- These functions are attributed to the ability of polarized cytoskeletal scores their importance for life, as they occur in diverse cell types polymers to assemble and disassemble rapidly, and to interact with including epithelia, neurons, fibroblasts, oocytes and early embryos, binding proteins and molecular motors that mediate their regulated and across species from yeast to humans. Thus, defining the mecha- movement and/or assembly into higher order structures, such as radial nisms by which actin and microtubules interact is key to understand- arrays or bundles.
    [Show full text]
  • Absence of NEFL in Patient-Specific Neurons in Early-Onset Charcot-Marie-Tooth Neuropathy Markus T
    ARTICLE OPEN ACCESS Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy Markus T. Sainio, MSc, Emil Ylikallio, MD, PhD, Laura M¨aenp¨a¨a, MSc, Jenni Lahtela, PhD, Pirkko Mattila, PhD, Correspondence Mari Auranen, MD, PhD, Johanna Palmio, MD, PhD, and Henna Tyynismaa, PhD Dr. Tyynismaa [email protected] Neurol Genet 2018;4:e244. doi:10.1212/NXG.0000000000000244 Abstract Objective We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot- Marie-Tooth (CMT) disease. Methods Motor neurons were differentiated from induced pluripotent stem cells of a patient with early- onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient’s cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neuro- filaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most down- regulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions The strict neuronal specificity of neurofilament has hindered the mechanistic studies of re- cessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism.
    [Show full text]
  • 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.
    [Show full text]
  • Microtubule Nucleation Remote from Centrosomes May Explain
    Microtubule nucleation remote from centrosomes may INAUGURAL ARTICLE explain how asters span large cells Keisuke Ishiharaa,b,1, Phuong A. Nguyena,b, Aaron C. Groena,b, Christine M. Fielda,b, and Timothy J. Mitchisona,b,1 aDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; and bMarine Biological Laboratory, Woods Hole, MA 02543 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2014. Edited by Ronald D. Vale, Howard Hughes Medical Institute and University of California, San Francisco, CA, and approved November 13, 2014 (received for review October 6, 2014) A major challenge in cell biology is to understand how nanometer- aster growth in large cells, such as microtubule sliding, tread- sized molecules can organize micrometer-sized cells in space and milling, or nucleation remote from centrosomes. time. One solution in many animal cells is a radial array of Previously we developed a cell-free system to reconstitute microtubules called an aster, which is nucleated by a central cleavage furrow signaling where growing asters interacted (5, organizing center and spans the entire cytoplasm. Frog (here 12). Here, we combine cell-free reconstitution and quantitative Xenopus laevis) embryos are more than 1 mm in diameter and imaging to identify microtubule nucleation away from the cen- divide with a defined geometry every 30 min. Like smaller cells, trosome as the key biophysical mechanism underlying aster they are organized by asters, which grow, interact, and move to growth. We propose that aster growth in large cells should be precisely position the cleavage planes.
    [Show full text]
  • Microtubule-Associated Protein Tau (Molecular Pathology/Neurodegenerative Disease/Neurofibriliary Tangles) M
    Proc. Nati. Acad. Sci. USA Vol. 85, pp. 4051-4055, June 1988 Medical Sciences Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: Identification as the microtubule-associated protein tau (molecular pathology/neurodegenerative disease/neurofibriliary tangles) M. GOEDERT*, C. M. WISCHIK*t, R. A. CROWTHER*, J. E. WALKER*, AND A. KLUG* *Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; and tDepartment of Psychiatry, University of Cambridge Clinical School, Hills Road, Cambridge CB2 2QQ, United Kingdom Contributed by A. Klug, March 1, 1988 ABSTRACT Screening of cDNA libraries prepared from (21). This task is made all the more difficult because there is the frontal cortex ofan zheimer disease patient and from fetal no functional or physiological assay for the protein(s) of the human brain has led to isolation of the cDNA for a core protein PHF. The only identification so far possible is the morphol- of the paired helical fiament of Alzheimer disease. The partial ogy of the PHFs at the electron microscope level, and here amino acid sequence of this core protein was used to design we would accept only experiments on isolated individual synthetic oligonucleotide probes. The cDNA encodes a protein of filaments, not on neurofibrillary tangles (in which other 352 amino acids that contains a characteristic amino acid repeat material might be occluded). One thus needs a label or marker in its carboxyl-terminal half. This protein is highly homologous for the PHF itself, which can at the same time be used to to the sequence ofthe mouse microtubule-assoiated protein tau follow the steps of the biochemical purification.
    [Show full text]
  • Role of Nucleation in Cortical Microtubule Array Organization: Variations on a Theme Erica A
    Washington University in St. Louis Washington University Open Scholarship Biology Faculty Publications & Presentations Biology 7-2013 Role of nucleation in cortical microtubule array organization: variations on a theme Erica A. Fishel Washington University in St Louis Ram Dixit Washington University in St Louis, [email protected] Follow this and additional works at: https://openscholarship.wustl.edu/bio_facpubs Part of the Biochemistry Commons, Biology Commons, and the Plant Biology Commons Recommended Citation Fishel, Erica A. and Dixit, Ram, "Role of nucleation in cortical microtubule array organization: variations on a theme" (2013). Biology Faculty Publications & Presentations. 37. https://openscholarship.wustl.edu/bio_facpubs/37 This Article is brought to you for free and open access by the Biology at Washington University Open Scholarship. It has been accepted for inclusion in Biology Faculty Publications & Presentations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. 1 Title: Role of nucleation in cortical microtubule array organization: variations on a theme Authors: Erica A. Fishel and Ram Dixit Running title: Microtubule nucleation in the CMT array Key words: Noncentrosomal microtubules, γ-tubulin, Arabidopsis thaliana, Branching microtubules, Computer simulations, Interphase Corresponding Author: Ram Dixit Biology Department Washington University in St. Louis One Brookings Drive, CB 1137 St. Louis, MO 63130. Phone: (314) 935-8823 Fax: (314) 935-4432 Email: [email protected] 2 Abstract The interphase cortical microtubules (CMTs) of plant cells form strikingly ordered arrays in the absence of a dedicated microtubule-organizing center. Considerable effort has focused on activities such as bundling and severing that occur after CMT nucleation and are thought to be important for generating and maintaining ordered arrays.
    [Show full text]
  • Neurofilaments: Neurobiological Foundations for Biomarker Applications
    Neurofilaments: neurobiological foundations for biomarker applications Arie R. Gafson1, Nicolas R. Barthelmy2*, Pascale Bomont3*, Roxana O. Carare4*, Heather D. Durham5*, Jean-Pierre Julien6,7*, Jens Kuhle8*, David Leppert8*, Ralph A. Nixon9,10,11,12*, Roy Weller4*, Henrik Zetterberg13,14,15,16*, Paul M. Matthews1,17 1 Department of Brain Sciences, Imperial College, London, UK 2 Department of Neurology, Washington University School of Medicine, St Louis, MO, USA 3 a ATIP-Avenir team, INM, INSERM , Montpellier university , Montpellier , France. 4 Clinical Neurosciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom 5 Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Québec, Canada 6 Department of Psychiatry and Neuroscience, Laval University, Quebec, Canada. 7 CERVO Brain Research Center, 2601 Chemin de la Canardière, Québec, QC, G1J 2G3, Canada 8 Neurologic Clinic and Policlinic, Departments of Medicine, Biomedicine and Clinical Research, University Hospital Basel, University of Basel, Basel, Switzerland. 9 Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY, 10962, USA. 10Departments of Psychiatry, New York University School of Medicine, New York, NY, 10016, 11 Neuroscience Institute, New York University School of Medicine, New York, NY, 10016, USA. 12Department of Cell Biology, New York University School of Medicine, New York, NY, 10016, USA 13 University College London Queen Square Institute of Neurology, London, UK 14 UK Dementia Research Institute at University College London 15 Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden 16 Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden 17 UK Dementia Research Institute at Imperial College, London * Co-authors ordered alphabetically Address for correspondence: Prof.
    [Show full text]
  • Microtubule Nucleation by Γ-Tubulin Complexes
    REVIEWS Microtubule nucleation by γ‑tubulin complexes Justin M. Kollman*, Andreas Merdes‡, Lionel Mourey§ and David A. Agard* Abstract | Microtubule nucleation is regulated by the γ‑tubulin ring complex (γTuRC) and related γ‑tubulin complexes, providing spatial and temporal control over the initiation of microtubule growth. Recent structural work has shed light on the mechanism of γTuRC-based microtubule nucleation, confirming the long-standing hypothesis that the γTuRC functions as a microtubule template. The first crystallographic analysis of a non-γ‑tubulin γTuRC component (γ‑tubulin complex protein 4 (GCP4)) has resulted in a new appreciation of the relationships among all γTuRC proteins, leading to a refined model of their organization and function. The structures have also suggested an unexpected mechanism for regulating γTuRC activity via conformational modulation of the complex component GCP3. New experiments on γTuRC localization extend these insights, suggesting a direct link between its attachment at specific cellular sites and its activation. 4 Microtubule catastrophe The microtubule cytoskeleton is critically important in vitro from purified tubulin . In vivo, though, almost The rapid depolymerization of for the spatial and temporal organization of eukaryotic all microtubules have 13 protofilaments5–7, suggesting microtubules that occurs when cells, playing a central part in functions as diverse as that one level of cell­ular control involves defining unique GTP has been hydrolysed in all intra­cellular transport, organelle positioning, motil‑ microtubule geometry. The 13‑fold symmetry is probably tubulin subunits up to the growing tip. ity, signalling and cell division. The ability to play this preferred because it is the only geometry in which proto‑ range of parts requires microtubules to be arranged in filaments run straight along the microtubule length, as complex arrays that are capable of rapid reorganization.
    [Show full text]
  • Cytoskeleton Cytoskeleton
    CYTOSKELETON CYTOSKELETON The cytoskeleton is composed of three principal types of protein filaments: actin filaments, intermediate filaments, and microtubules, which are held together and linked to subcellular organelles and the plasma membrane by a variety of accessory proteins Muscle Contraction • Skeletal muscles are bundles of muscle fibers • Most of the cytoplasm consists of myofibrils, which are cylindrical bundles of two types of filaments: thick filaments of myosin (about 15 run in diameter) and thin filaments of actin (about 7 nm in diameter). • Each myofibril is organized as a chain of contractile units called sarcomeres, which are responsible for the striated appearance of skeletal and cardiac muscle. Structure of muscle cells Sarcomere • The ends of each sarcomere are defined by the Z disc. • Within each sarcomere, dark bands (called A bands because they are anisotropic when viewed with polarized light) alternate with light bands (called I bands for isotropic). • The I bands contain only thin (actin) filaments, whereas the A bands contain thick (myosin) filaments. • The myosin and actin filaments overlap in peripheral regions of the A band, whereas a middle region (called the H zone) contains only myosin. Muscle contraction • The basis for understanding muscle contraction is the sliding filament model, first proposed in 1954 both by Andrew Huxley and Ralph Niedergerke and by Hugh Huxley and Jean Hanson • During muscle contraction each sarcomere shortens, bringing the Z discs closer together. • There is no change in the width of the A band, but both the I bands and the H zone almost completely disappear. • These changes are explained by the actin and myosin filaments sliding past one another so that the actin filaments move into the A band and H zone.
    [Show full text]
  • The Relationship Between Intermediate Filaments and Microfilaments Before and During the Formation of Desmosomes and Adherens-Ty
    Published May 1, 1987 The Relationship between Intermediate Filaments and Microfilaments before and during the Formation of Desmosomes and Adherens-type Junctions in Mouse Epidermal Keratinocytes Kathleen J. Green, Benjamin Geiger,* Jonathan C. R. Jones, John C. Talian, and Robert D. Goldman Department of Cell Biology and Anatomy, Northwestern University Medical School, Chicago, Illinois 60611; and * Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel Abstract. Actin, keratin, vinculin and desmoplakin ermost of the concentric MFB. Individual IF often organization were studied in primary mouse keratino- splay out, becoming interwoven into these MFB in the cytes before and during Ca2+-induced cell contact forma- region of cell-substrate contact. In the first 30 min af- tion. Double-label fluorescence shows that in cells cul- ter the Ca 2+ switch, areas of submembranous dense Downloaded from tured in low Ca 2÷ medium, keratin-containing inter- material (identified as adherens junctions), which are mediate filament bundles (IFB) and desmoplakin- associated with the perpendicular MFB, can be seen at containing spots are both concentrated towards the cell newly formed cell-ceU contact sites. By 1-2 h, IFB- center in a region bounded by a series of concentric desmosomal component complexes are aligned with microfilament bundles (MFB). Within 5-30 min after the perpendicular MFB as the complexes become jcb.rupress.org raising Ca 2+ levels, a discontinuous actin/vinculin-rich, redistributed to cell-cell interfaces. Cytochalasin D submembranous zone of fluorescence appears at cell- treatment causes the redistribution of actin into numer- cell interfaces. This zone is usually associated with ous patches; keratin-containing Lr:B undergo a con- short, perpendicular MFB, which become wider and comitant redistribution, forming foci that coincide with longer with time.
    [Show full text]
  • Myosin-Driven Actin-Microtubule Networks Exhibit Self-Organized Contractile Dynamics Gloria Lee1, Michael J
    bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146662; this version posted June 12, 2020. 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-ND 4.0 International license. Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics Gloria Lee1, Michael J. Rust2, Moumita Das3, Ryan J. McGorty1, Jennifer L. Ross4, Rae M. Robertson-Anderson1* 1Department of Physics and Biophysics, University of San Diego, San Diego, CA 92110, USA 2Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA 3School of Physics and Astronomy, Rochester Institute of Technology, Rochester, NY 14623, USA 4Department of Physics, Syracuse University, Syracuse, NY 13244, USA Abstract The cytoskeleton is a dynamic network of proteins, including actin, microtubules, and myosin, that enables essential cellular processes such as motility, division, mechanosensing, and growth. While actomyosin networks are extensively studied, how interactions between actin and microtubules, ubiquitous in the cytoskeleton, influence actomyosin activity remains an open question. Here, we create a network of co-entangled actin and microtubules driven by myosin II. We combine dynamic differential microscopy, particle image velocimetry and particle-tracking to show that both actin and microtubules in the network undergo ballistic contraction with surprisingly indistinguishable characteristics. This controlled contractility is distinct from the faster turbulent motion and rupturing that active actin networks exhibit. Our results suggest that microtubules can enable self-organized myosin-driven contraction by providing flexural rigidity and enhanced connectivity to actin networks.
    [Show full text]
  • Signaling Function of Α-Catenin in Microtubule Regulation
    [Cell Cycle 7:15, 2377-2383; 1 August 2008]; ©2008 Landes Bioscience Report Signaling function of α-catenin in microtubule regulation Michael Shtutman,1,* Alexander Chausovsky,2 Masha Prager-Khoutorsky,2 Natalia Schiefermeier,2,† Shlomit Boguslavsky,2 Zvi Kam,2 Elaine Fuchs,3 Benjamin Geiger,2 Gary G. Borisy4 and Alexander D. Bershadsky2 1Cancer Center, Ordway Research Institute; Albany, New York USA; 2Department of Molecular and Cellular Biology; The Weizmann Institute of Science; Rehovot, Israel; 3Howard Hughes Medical Institute; Laboratory of Mammalian Cell Biology and Development; The Rockefeller University; New York, New York USA; 4Marine Biological Laboratory; Woods Hole; Massachusetts, USA †Current address: Innsbruck Medical University; Biocenter, Innsbruck, Austria Abbreviations: APC, adenomatous polyposis coli protein; AJ, adherens junction; CHO, chinese hamster ovary cells; DMEM, Dulbecco’s Modified Eagle’s Medium; EGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; IL2R, interleukin-2 receptor; MAPK, mitogen-activated protein kinase; mDia1, mouse diaphanous related formin 1; MT, microtubule; PBS, phosphate buffered saline; SD, stan- dard deviation; SEM, standard error of mean Key words: alpha-catenin, microtubules, beta-catenin, p120ctn, adherens junction, centrosome, cadherins, cytoplasts Centrosomes control microtubule dynamics in many cell stability depends on the minus end being anchored in the centrosome, types, and their removal from the cytoplasm leads to a shift from more precisely in the pericentriolar material surrounding the mother dynamic instability to treadmilling behavior and to a dramatic centriole.1,2 In contrast, epithelial and neuronal cells maintain decrease of microtubule mass (Rodionov et al., 1999; PNAS large populations of MTs that have no apparent connection to the 96:115).
    [Show full text]