Evidence Against a Direct Role for Cortical Actin Arrays in Saltatory Organelle Motility in Hyphae of the Fungus Saprolegnia Ferax

1988 ◽  
Vol 91 (1) ◽  
pp. 41-47
Author(s):  
I. BRENT HEATH
Keyword(s):  
Actin Filaments ◽  
Normal Population ◽  
Tween 20 ◽  
Detectable Effect ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Direct Role ◽  
Saprolegnia Ferax ◽  
Organelle Motility ◽  
Actin Fibrils

Actin stained by rhodamine-labelled phalloidin in growing hyphae of the oomycete fungus Saprolegnia ferax is restricted to an approximately 0.25 μm deep layer of the cell periphery where it forms an apical cap of fine filaments and a subapical array of coarser longitudinal fibrils interspersed with plaques. The functions of this actin are unknown, but because actin-rich fibrils in other cells are involved in organelle motility I have sought evidence for a similar role in these hyphae. The most prominent motile structures are a population of spherical, predominantly sub-micrometre diameter, refractile vesicles of unknown function, which show typical saltatory movements along the hyphae. The motility of these vesicles is statistically identical in both the central cytoplasm, remote from the actin fibrils, and the peripheral cytoplasm adjacent to the fibrils. Treatment of hyphae with specific concentrations and durations of the detergents Tween 20 and Brij 58 causes extensive reorganization of the actin arrays with no effect on vesicle motility, whereas Triton X-100 severely inhibits motility with little detectable effect on the actin filaments. These observations show that normal vesicle motility does not depend on the proximity of a normal population of actin filaments and, therefore, suggest that the latter have some function other than a direct role in saltatory organelle motility.

scholarly journals Myo1c binding to submembrane actin mediates insulin-induced tethering of GLUT4 vesicles

2012 ◽  
Vol 23 (20) ◽  
pp. 4065-4078 ◽  
Author(s):  
Shlomit Boguslavsky ◽  
Tim Chiu ◽  
Kevin P. Foley ◽  
Cesar Osorio-Fuentealba ◽  
Costin N. Antonescu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Binding ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Vesicle Tethering ◽  
Extracellular Milieu ◽  
Intracellular Compartments ◽  
Insulin Dependent

GLUT4-containing vesicles cycle between the plasma membrane and intracellular compartments. Insulin promotes GLUT4 exocytosis by regulating GLUT4 vesicle arrival at the cell periphery and its subsequent tethering, docking, and fusion with the plasma membrane. The molecular machinery involved in GLUT4 vesicle tethering is unknown. We show here that Myo1c, an actin-based motor protein that associates with membranes and actin filaments, is required for insulin-induced vesicle tethering in muscle cells. Myo1c was found to associate with both mobile and tethered GLUT4 vesicles and to be required for vesicle capture in the total internal reflection fluorescence (TIRF) zone beneath the plasma membrane. Myo1c knockdown or overexpression of an actin binding–deficient Myo1c mutant abolished insulin-induced vesicle immobilization, increased GLUT4 vesicle velocity in the TIRF zone, and prevented their externalization. Conversely, Myo1c overexpression immobilized GLUT4 vesicles in the TIRF zone and promoted insulin-induced GLUT4 exposure to the extracellular milieu. Myo1c also contributed to insulin-dependent actin filament remodeling. Thus we propose that interaction of vesicular Myo1c with cortical actin filaments is required for insulin-mediated tethering of GLUT4 vesicles and for efficient GLUT4 surface delivery in muscle cells.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments.

1990 ◽  
Vol 111 (5) ◽  
pp. 1905-1911 ◽  
Author(s):  
L G Cao ◽  
Y L Wang
Keyword(s):  
Actin Filaments ◽  
Average Rate ◽  
Rat Kidney ◽  
Equatorial Region ◽  
Cell Cortex ◽  
Polar Regions ◽  
Cortical Actin ◽  
Animal Cells ◽  
Contractile Ring ◽  
Directional Movement

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

2005 ◽  
Vol 288 (1) ◽  
pp. C46-C56 ◽  
Author(s):  
Camille Ehre ◽  
Andrea H. Rossi ◽  
Lubna H. Abdullah ◽  
Kathleen De Pestel ◽  
Sandra Hill ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Yellow Fluorescent Protein ◽  
Goblet Cells ◽  
Actin Binding ◽  
Secretory Cells ◽  
Cortical Actin ◽  
Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

Actin filaments regulate epithelial Na+ channel activity

1991 ◽  
Vol 261 (5) ◽  
pp. C882-C888 ◽  
Author(s):  
H. F. Cantiello ◽  
J. L. Stow ◽  
A. G. Prat ◽  
D. A. Ausiello
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Functional Role ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Na Channel ◽  
Na Channels ◽  
Channel Activity ◽  
Cortical Actin ◽  
Excised Patches

The functional role of the cytoskeleton in the control of ion channel activity is unknown. In the present study, immunocolocalization of Na+ channels with specific antibodies and fluorescein isothiocyanate-phalloidin to stain the cortical cytoskeleton indicates that actin is always present in close proximity to apical Na+ channels in A6 cells. The patch-clamp technique was used to assess the effect of cortical actin networks on apical Na+ channels in these A6 epithelial cells. The actin filament disrupter, cytochalasin D (5 micrograms/ml), induced Na+ channel activity in cell-attached patches within 5 min of addition. Cytochalasin D also induced and/or increased Na+ channel activity in 90% of excised patches tested within 2 min. Addition of short actin filaments (greater than 5 microM) to excised patches also induced channel activity. This effect was enhanced by addition of ATP and/or cytochalasin D. The effect of actin on Na+ channel activity was reversed by addition of the G actin-binding protein DNase I or completely prevented by treatment of the excised patches with this enzyme. Addition of the actin-binding protein, filamin, reversibly inhibited both spontaneous and actin-induced Na+ channels. Thus actin filament networks, achieved by either depolymerizing endogenous actin filaments by treatment with cytochalasin D, the addition of exogenous short actin filaments plus ATP, or actin plus cytochalasin D, regulate apical Na+ channel activity. This conclusion was supported by the observation that the addition of short actin filaments in the form of actin-gelsolin complexes in molar ratios less than 8:1 was also effective in activating Na+ channels. We have thus demonstrated a functional role for the cortical actin network in the regulation of epithelial Na+ channels that may complement a structural role for membrane protein targetting and assembly.

scholarly journals Stress fibers are embedded in a contractile cortical network

2020 ◽  
Author(s):  
Timothée Vignaud ◽  
Calina Copos ◽  
Christophe Leterrier ◽  
Qingzong Tseng ◽  
Laurent Blanchoin ◽  
...  
Keyword(s):  
Cell Fate ◽  
Actin Filaments ◽  
Mechanical Response ◽  
Force Production ◽  
Traction Force ◽  
Cortical Network ◽  
Cortical Actin ◽  
Cell Fate Decisions ◽  
Production And Distribution ◽  
Intracellular Forces

ABSTRACTContractile actomyosin networks generate intracellular forces essential for the regulation of cell shape, migration, and cell-fate decisions, ultimately leading to the remodeling and patterning of tissues. Although actin filaments aligned in bundles represent the main source of traction-force production in adherent cells, there is increasing evidence that these bundles form interconnected and interconvertible structures with the rest of the intracellular actin network. In this study, we explored how these bundles are connected to the surrounding cortical network and the mechanical impact of these interconnected structures on the production and distribution of traction forces on the extracellular matrix and throughout the cell. By using a combination of hydrogel micropatterning, traction-force microscopy and laser photoablation, we measured the relaxation of the cellular traction field in response to local photoablations at various positions within the cell. Our experimental results and modeling of the mechanical response of the network revealed that bundles were fully embedded along their entire length in a continuous and contractile network of cortical filaments. Moreover, the propagation of the contraction of these bundles throughout the entire cell was dependent on this embedding. In addition, these bundles appeared to originate from the alignment and coalescence of thin and unattached cortical actin filaments from the surrounding mesh.

scholarly journals Coupled myosin VI motors facilitate unidirectional movement on an F-actin network

2009 ◽  
Vol 187 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Sivaraj Sivaramakrishnan ◽  
James A. Spudich
Keyword(s):  
Single Molecule ◽  
Membrane Trafficking ◽  
Cell Periphery ◽  
Myosin Vi ◽  
Actin Network ◽  
Cortical Actin ◽  
Actin Cortex ◽  
Unidirectional Movement ◽  
Transport Vesicle ◽  
Pointed End

Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 µm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.

The organization of tip-growth-related organelles and microtubules revealed by quantitative analysis of freeze-substituted oomycete hyphae

1989 ◽  
Vol 93 (1) ◽  
pp. 41-52
Author(s):  
I. BRENT HEATH ◽  
SUSAN G.W. KAMINSKYJ
Keyword(s):  
Golgi Body ◽  
Cell Periphery ◽  
Cross Sectional ◽  
Saprolegnia Ferax ◽  
Cytoskeleton Organization ◽  
Golgi Bodies ◽  
Section Electron ◽  
Serial Section Electron Microscopy ◽  
Body Production ◽  
Actin Cables

The distribution of organelles and microtubules in hyphal tips of the oomycete, Saprolegnia ferax, were quantitatively determined at high resolution from serial-section electron microscopy of freeze-substituted cells. All the organelles and the microtubules were non-uniformly distributed, each showing a characteristic longitudinal gradient starting at a different point behind the tip. In addition, when the cytoplasmic cross-sectional area was divided into radial regions, all organelles occurred preferentially in either the central (mitochondria and Golgi bodies) or the peripheral (microtubules, wall vesicles and spherical vesicles) region. The nuclei were so large as to span both regions but were always oriented with their centrioles facing the plasmalemma. Microtubules occurred in the extreme tips, became more abundant sub-apically, were predominantly short but increased in mean length with distance from the tip. The correlated patterns of organelle and cytoskeleton organization from this and previous work show that neither the microtubules nor the detected arrays of actin are sufficient to account for most organelle arrangements. However, on the basis of the distribution and orientation of the predominantly elongated wall vesicles, we suggest that the wall vesicles travel radially from their origin at the centrally located Golgi bodies to the cell periphery where they are transported longitudinally to the hyphal tip in conjunction with the plasmalemma-associated actin cables. Our data also suggest that the hyphae contain a cortical ectoplasm with which the nuclei interact, at least in part, via their centrioles and centriole-associated microtubules, and whose mechanical integrity is increased by both the peripheral actin cables and a high density of microtubules. We suggest that the endoplasm is less strong and has physiological properties that enhance the differentiation of endoplasmic reticulum and nuclear envelope into Golgi body production.

scholarly journals The ATP binding cassette transporter, ABCG1, localizes to cortical actin filaments

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Elvis Pandzic ◽  
Ingrid C. Gelissen ◽  
Renee Whan ◽  
Philip J. Barter ◽  
Dmitri Sviridov ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Atp Binding ◽  
Cortical Actin ◽  
Atp Binding Cassette Transporter ◽  
Atp Binding Cassette
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