Function of spindle microtubules in directing cortical movement and actin filament organization in dividing cultured cells

1996 ◽  
Vol 109 (8) ◽  
pp. 2041-2051 ◽  
Author(s):  
D.J. Fishkind ◽  
J.D. Silverman ◽  
Y.L. Wang
Keyword(s):  
Cell Division ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Linear Dichroism ◽  
Single Particle Tracking ◽  
Contractile Apparatus ◽  
Cortical Actin ◽  
Surface Receptors ◽  
Spindle Microtubules

The mitotic spindle has long been recognized to play an essential role in determining the position of the cleavage furrow during cell division, however little is known about the mechanisms involved in this process. One attractive hypothesis is that signals from the spindle may function to induce reorganization of cortical structures and transport of actin filaments to the equator during cytokinesis. While an important idea, few experiments have directly tested this model. In the present study, we have used a variety of experimental approaches to identify microtubule-dependent effects on key cortical events during normal cell cleavage, including cortical flow, reorientation of actin filaments, and formation of the contractile apparatus. Single-particle tracking experiments showed that the microtubule disrupting drug nocodazole induces an inhibition of the movements of cell surface receptors following anaphase onset, while the microtubule stabilizing drug taxol causes profound changes in the overall pattern of receptor movements. These effects were accompanied by a related set of changes in the organization of the actin cytoskeleton. In nocodazole-treated cells, the three-dimensional organization of cortical actin filaments appeared less ordered than in controls. Measurements with fluorescence-detected linear dichroism indicated a decrease in the alignment of filaments along the spindle axis. In contrast, actin filaments in taxol-treated cells showed an increased alignment along the equator on both the ventral and dorsal cortical surfaces, mirroring the redistribution pattern of surface receptors. Together, these experiments show that spindle microtubules are involved in directing bipolar flow of surface receptors and reorganization of actin filaments during cell division, thus acting as a stimulus for positioning cortical cytoskeletal components and organizing the contractile apparatus of dividing tissue culture cells.

scholarly journals Clathrin plaques and associated actin anchor intermediate filaments in skeletal muscle

2019 ◽  
Vol 30 (5) ◽  
pp. 579-590 ◽  
Author(s):  
Agathe Franck ◽  
Jeanne Lainé ◽  
Gilles Moulay ◽  
Eline Lemerle ◽  
Michaël Trichet ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cell Biology ◽  
Striated Muscle ◽  
Three Dimensional ◽  
Cell Types ◽  
Contractile Apparatus ◽  
Cortical Actin ◽  
Muscle Formation ◽  
And Function ◽  
Dynamin 2

Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.

scholarly journals Three-dimensional reconstruction of the membrane skeleton at the plasma membrane interface by electron tomography

2006 ◽  
Vol 174 (6) ◽  
pp. 851-862 ◽  
Author(s):  
Nobuhiro Morone ◽  
Takahiro Fujiwara ◽  
Kotono Murase ◽  
Rinshi S. Kasai ◽  
Hiroshi Ike ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
High Speed ◽  
Plasma Membranes ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Membrane Skeleton ◽  
Single Particle Tracking ◽  
Coated Pits ◽  
Cytoplasmic Surface

Three-dimensional images of the undercoat structure on the cytoplasmic surface of the upper cell membrane of normal rat kidney fibroblast (NRK) cells and fetal rat skin keratinocytes were reconstructed by electron tomography, with 0.85-nm–thick consecutive sections made ∼100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeleton (MSK) primarily consists of actin filaments and associated proteins. The MSK covers the entire cytoplasmic surface and is closely linked to clathrin-coated pits and caveolae. The actin filaments that are closely apposed to the cytoplasmic surface of the plasma membrane (within 10.2 nm) are likely to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules, thus partitioning the plasma membrane with regard to their lateral diffusion. The distribution of the MSK mesh size as determined by electron tomography and that of the compartment size as determined from high speed single-particle tracking of phospholipid diffusion agree well in both cell types, supporting the MSK fence and MSK-anchored protein picket models.

scholarly journals Orientation and three-dimensional organization of actin filaments in dividing cultured cells.

1993 ◽  
Vol 123 (4) ◽  
pp. 837-848 ◽  
Author(s):  
D J Fishkind ◽  
Y L Wang
Keyword(s):  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Dorsal Surface ◽  
Ventral Surface ◽  
Equatorial Region ◽  
Optical Sectioning ◽  
Rhodamine Phalloidin ◽  
Anaphase Onset ◽  
Spindle Axis

The current hypothesis of cytokinesis suggests that contractile forces in the cleavage furrow are generated by a circumferential band of actin filaments. However, relatively little is known about the global organization of actin filaments in dividing cells. To approach this problem we have used fluorescence-detected linear dichroism (FDLD) microscopy to measure filament orientation, and digital optical sectioning microscopy to perform three-dimensional reconstructions of dividing NRK cells stained with rhodamine-phalloidin. During metaphase, actin filaments in the equatorial region show a slight orientation along the spindle axis, while those in adjacent regions appear to be randomly distributed. Upon anaphase onset and through cytokinesis, the filaments become oriented along the equator in the furrow region, and along the spindle axis in adjacent regions. The degree of orientation appears to be dependent on cell-cell and cell-substrate adhesions. By performing digital optical sectioning microscopy on a highly spread NRK subclone, we show that actin filaments organize as a largely isotropic cortical meshwork in metaphase cells and convert into an anisotropic network shortly after anaphase onset, becoming more organized as cytokinesis proceeds. The conversion is most dramatic on the adhering ventral surface which shows little or no cleavage activity, and results in the formation of large bundles along the equator. On the dorsal surface, where cleavage occurs actively, actin filaments remain isotropic, showing only subtle alignment late in cytokinesis. In addition, stereo imaging has led to the discovery of a novel set of filaments that are associated with the cortex and traverse through the cytoplasm. Together, these studies provide important insights into the process of actin remodeling during cell division and point to possible additional mechanisms for force generation.

Super-Resolution Three-Dimensional Imaging of Actin Filaments in Cultured Cells and the Brain via Expansion Microscopy

ACS Nano ◽  
2020 ◽  
Vol 14 (11) ◽  
pp. 14999-15010
Author(s):  
Chan E Park ◽  
Youngbin Cho ◽  
In Cho ◽  
Hyunsu Jung ◽  
Byeongyeon Kim ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Super Resolution ◽  
Three Dimensional Imaging ◽  
The Brain

scholarly journals The Small GTP-binding Protein Rho Regulates Cortical Activities in Cultured Cells during Division

1999 ◽  
Vol 144 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Christopher B. O'Connell ◽  
Sally P. Wheatley ◽  
Sohail Ahmed ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Binding Protein ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Metaphase Plate ◽  
Cortical Actin ◽  
Gtp Binding Protein ◽  
Gtp Binding

We have investigated the role of the small GTP-binding protein Rho in cytokinesis by microinjecting an inhibitor, C3 ribosyltransferase, into cultured cells. Microinjection of C3 into prometaphase or metaphase normal rat kidney epithelial cells induced immediate and global cortical movement of actin toward the metaphase plate, without an apparent effect on the mitotic spindle. During anaphase, concentrated cortical actin filaments migrated with separating chromosomes, leaving no apparent concentration of actin filaments along the equator. Myosin II in injected epithelial cells showed a diffuse distribution throughout cell division. All treated, well-adherent cells underwent cleavage-like activities and most of them divided successfully. However, cytokinesis became abnormal, generating irregular ingressions and ectopic cleavage sites even when mitosis was blocked with nocodazole. The effects of C3 appeared to be dependent on cell adhesion; less adherent 3T3 fibroblasts exhibited irregular cortical ingression only when cells started to increase attachment during respreading, but managed to complete cytokinesis. Poorly adherent HeLa cells showed neither ectopic cleavage nor completion of cytokinesis. Our results indicate that Rho does not simply activate actin–myosin II interactions during cytokinesis, but regulates the spatial pattern of cortical activities and completion of cytokinesis possibly through modulating the mechanical strength of the cortex.

scholarly journals Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells

2001 ◽  
Vol 155 (4) ◽  
pp. 511-518 ◽  
Author(s):  
Lisa A. Flanagan ◽  
Janet Chou ◽  
Hervé Falet ◽  
Ralph Neujahr ◽  
John H. Hartwig ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Mechanical Stability ◽  
Three Dimensional ◽  
Leading Edge ◽  
Human Melanoma ◽  
Actin Binding ◽  
Filamin A ◽  
Cortical Actin ◽  
Specific Antibodies ◽  
Actin Organization

The Arp2/3 complex and filamin A (FLNa) branch actin filaments. To define the role of these actin-binding proteins in cellular actin architecture, we compared the morphology of FLNa-deficient human melanoma (M2) cells and three stable derivatives of these cells expressing normal FLNa concentrations. All the cell lines contain similar amounts of the Arp2/3 complex. Serum addition causes serum-starved M2 cells to extend flat protrusions transiently; thereafter, the protrusions turn into spherical blebs and the cells do not crawl. The short-lived lamellae of M2 cells contain a dense mat of long actin filaments in contrast to a more three-dimensional orthogonal network of shorter actin filaments in lamellae of identically treated FLNa-expressing cells capable of translational locomotion. FLNa-specific antibodies localize throughout the leading lamellae of these cells at junctions between orthogonally intersecting actin filaments. Arp2/3 complex–specific antibodies stain diffusely and label a few, although not the same, actin filament overlap sites as FLNa antibody. We conclude that FLNa is essential in cells that express it for stabilizing orthogonal actin networks suitable for locomotion. Contrary to some proposals, Arp2/3 complex–mediated branching of actin alone is insufficient for establishing an orthogonal actin organization or maintaining mechanical stability at the leading edge.

3-D imaging of freeze-dried actin filaments in fibroblast using high-resolution cryo-SEM

1993 ◽  
Vol 51 ◽  
pp. 120-121
Author(s):  
Ya Chen ◽  
Alexander B. Verkhovsky ◽  
Gary G. Borisy
Keyword(s):  
Grain Size ◽  
High Resolution ◽  
Low Temperature ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Metal Layer ◽  
Three Dimensional ◽  
Fine Grain ◽  
Freeze Dried ◽  
Temperature Scanning

A new approach to image the macromolecular structure of actin filaments from detergent-extracted cultured cells using high resolution low-temperature scanning electron microscopy (cryo-SEM) is described. An advantage of studying the cytoskeleton by SEM is its large depth of focus which makes it easy to reveal the three-dimensional relationship between the cytoskeleton filaments. Recently, procedures have been investigated for high resolution visualization of biological samples by SEM. These studies have demonstrated the importance of cryo preparation techniques for preserving the morphology of the specimen and advanced metal coating techniques for revealing its topography.Coating for HRSEM requires a metal layer that will not obscure fine details while still yielding adequate SE signal. Magnetron-sputter coating of the samples with an ultrathin layer of metal at low temperature provides finer grain size than coating at ambient temperature. Chromium has many properties that make it suitable as coating metal for cytoskeleton: it has fine grain size, low BSE yield due to its low Z number, and it provides adequate SE signal.

scholarly journals Single particle tracking of surface receptor movement during cell division.

1994 ◽  
Vol 127 (4) ◽  
pp. 963-971 ◽  
Author(s):  
Y L Wang ◽  
J D Silverman ◽  
L G Cao
Keyword(s):  
Cell Division ◽  
Imaging Techniques ◽  
Equatorial Region ◽  
Random Motion ◽  
Single Particle Tracking ◽  
Membrane Receptors ◽  
Cortical Reorganization ◽  
Cortical Actin ◽  
Anaphase Onset ◽  
Surface Receptor

We have used fluorescent latex beads to label membrane receptors on cultured NRK cells. Movement of individual beads during cell division was recorded with digital imaging techniques. Surface-bound beads showed no organized movement during metaphase but started to migrate toward the equator approximately 1 min after anaphase onset, when chromosomes moved out of the equatorial region to create the interzone. The movement was most active in the central region of the cell near separating chromosomes, while beads located near the poles of the cell underwent primarily random motion. Most beads showed a surge in speed upon the passage of chromosomes, suggesting a possible link between chromosome separation and cortical reorganization. Furthermore, treatment of anaphase cells with cytochalasin D induced a rapid, simultaneous collapse of beads and cortical actin filaments into aggregates, indicating that the movement of beads was closely related to the reorganization of the actin cortex. In contrast to normal directional movement, cytochalasin-induced movement occurred in random directions and caused some beads in the equatorial region to move toward poles. Our results indicate that cytokinesis involves contractile activities, not only along the equator, but over a wide area of the actin-containing cortex. In addition, organized cortical activities appear to be temporally activated at anaphase onset, and spatially modulated by the spindle interzone or separating chromosomes.

scholarly journals Direct Involvement of Ezrin/Radixin/Moesin (ERM)-binding Membrane Proteins in the Organization of Microvilli in Collaboration with Activated ERM Proteins

1999 ◽  
Vol 145 (7) ◽  
pp. 1497-1509 ◽  
Author(s):  
Shigenobu Yonemura ◽  
Sachiko Tsukita ◽  
Shoichiro Tsukita
Keyword(s):  
Membrane Proteins ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Site Directed Mutagenesis ◽  
Cortical Actin ◽  
A431 Cells ◽  
Cultured Fibroblasts ◽  
Erm Proteins ◽  
Binding Domains ◽  
Cultured Epithelial Cells

Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

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