scholarly journals Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hydrated collagen gels.

1984 ◽  
Vol 99 (2) ◽  
pp. 536-549 ◽  
Author(s):  
J J Tomasek ◽  
E D Hay
Keyword(s):  
Actin Cytoskeleton ◽  
Shape Change ◽  
Cell Cortex ◽  
Collagen Gels ◽  
Actin Cortex ◽  
Microtubule Disruption ◽  
Corneal Fibroblasts ◽  
Fourth Step ◽  
Collagenous Stroma

Fibroblasts in situ reside within a collagenous stroma and are elongate and bipolar in shape. If isolated and grown on glass, they change from elongate to flat shape, lose filopodia, and acquire ruffles. This shape change can be reversed to resemble that in situ by suspending the cells in hydrated collagen gels. In this study of embryonic avian corneal fibroblasts grown in collagen gels, we describe for the first time the steps in the acquisition of the elongate shape and analyze the effect of cytoskeleton-disrupting drugs on filopodial activity, assumption of bipolarity, and cell elongation within extracellular matrix. We have previously shown by immunofluorescence that filopodia contain actin but not myosin and are free of organelles. The cell cortex is rich in actin and the cytosol, in myosin. By using antitubulin, we show in the present study that microtubules are aligned along the long axis of the bipolar cell body. The first step in assumption of the elongate shape is extension of filopodia by the round cells suspended in collagen, and this is not significantly affected by the drugs we used: taxol to stabilize microtubules; nocodazole to disassemble microtubules; and cytochalasin D to disrupt microfilaments. The second step, movement of filopodia to opposite ends of the cell, is disrupted by cytochalasin, but not by taxol or nocodazole. The third step, extension of pseudopodia and acquisition of bipolarity similarly requires intact actin, but not microtubules. If fibroblasts are allowed to become bipolar before drug treatment, moreover, they remain so in the presence of the drugs. To complete the fourth step, extensive elongation of the cell, both intact actin and microtubules are required. Retraction of the already elongated cell occurs on microtubule disruption, but retraction requires an intact actin cytoskeleton. We suggest that the cell interacts with surrounding collagen fibrils via its actin cytoskeleton to become bipolar in shape, and that microtubules interact with the actin cortex to bring about the final elongation of the fibroblast.

scholarly journals Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules.

1981 ◽  
Vol 91 (1) ◽  
pp. 45-54 ◽  
Author(s):  
S P Sugrue ◽  
E D Hay
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Basal Cell ◽  
Actin Filaments ◽  
Cell Cortex ◽  
Collagen Gels ◽  
Basal Surface ◽  
Extracellular Matrix Components ◽  
Cytoplasmic Processes

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.

scholarly journals A Role for the Actin Cytoskeleton of Saccharomyces cerevisiae in Bipolar Bud-Site Selection

1997 ◽  
Vol 136 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Shirley Yang ◽  
Kathryn R. Ayscough ◽  
David G. Drubin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Site Selection ◽  
Mitotic Checkpoint ◽  
Cell Cortex ◽  
Spatial Cues ◽  
Genes Encoding ◽  
Mother And Daughter ◽  
Mother Cells ◽  
Bud Site

Saccharomyces cerevisiae cells select bud sites according to one of two predetermined patterns. MATa and MATα cells bud in an axial pattern, and MATa/α cells bud in a bipolar pattern. These budding patterns are thought to depend on the placement of spatial cues at specific sites in the cell cortex. Because cytoskeletal elements play a role in organizing the cytoplasm and establishing distinct plasma membrane domains, they are well suited for positioning bud-site selection cues. Indeed, the septin-containing neck filaments are crucial for establishing the axial budding pattern characteristic of MATa and MATα cells. In this study, we determined the budding patterns of cells carrying mutations in the actin gene or in genes encoding actin-associated proteins: MATa/α cells were defective in the bipolar budding pattern, but MATa and MATα cells still exhibit a normal axial budding pattern. We also observed that MATa/α actin cytoskeleton mutant daughter cells correctly position their first bud at the distal pole of the cell, but mother cells position their buds randomly. The actin cytoskeleton therefore functions in generation of the bipolar budding pattern and is required specifically for proper selection of bud sites in mother MATa/α cells. These observations and the results of double mutant studies support the conclusion that different rules govern bud-site selection in mother and daughter MATa/α cells. A defective bipolar budding pattern did not preclude an sla2-6 mutant from undergoing pseudohyphal growth, highlighting the central role of daughter cell bud-site selection cues in the formation of pseudohyphae. Finally, by examining the budding patterns of mad2-1 mitotic checkpoint mutants treated with benomyl to depolymerize their microtubules, we confirmed and extended previous evidence indicating that microtubules do not function in axial or bipolar bud-site selection.

scholarly journals Pan1p, End3p, and Sla1p, Three Yeast Proteins Required for Normal Cortical Actin Cytoskeleton Organization, Associate with Each Other and Play Essential Roles in Cell Wall Morphogenesis

2000 ◽  
Vol 20 (1) ◽  
pp. 12-25 ◽  
Author(s):  
Hsin-Yao Tang ◽  
Jing Xu ◽  
Mingjie Cai
Keyword(s):  
Cell Wall ◽  
Actin Cytoskeleton ◽  
Normal Cell ◽  
Cell Cortex ◽  
Repeat Region ◽  
Cortical Actin ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Cell Wall Morphogenesis

ABSTRACT The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Δ, andend3Δ mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.

scholarly journals Golgi Body Motility in the Plant Cell Cortex Correlates with Actin Cytoskeleton Organization

2011 ◽  
Vol 52 (10) ◽  
pp. 1844-1855 ◽  
Author(s):  
Miriam Akkerman ◽  
Elysa J. R. Overdijk ◽  
Jan H. N. Schel ◽  
Anne Mie C. Emons ◽  
Tijs Ketelaar
Keyword(s):  
Actin Cytoskeleton ◽  
Plant Cell ◽  
Golgi Body ◽  
Cell Cortex ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization ◽  
Body Motility

scholarly journals Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sofia Duarte ◽  
Álvaro Viedma-Poyatos ◽  
Elena Navarro-Carrasco ◽  
Alma E. Martínez ◽  
María A. Pajares ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Types ◽  
Hiv Protease ◽  
Cell Cortex ◽  
Tail Domain ◽  
Cellular Functions ◽  
Cortical Actin ◽  
Intrinsically Disordered ◽  
Actin Cortex ◽  
Vimentin Filaments

Abstract The vimentin network displays remarkable plasticity to support basic cellular functions and reorganizes during cell division. Here, we show that in several cell types vimentin filaments redistribute to the cell cortex during mitosis, forming a robust framework interwoven with cortical actin and affecting its organization. Importantly, the intrinsically disordered tail domain of vimentin is essential for this redistribution, which allows normal mitotic progression. A tailless vimentin mutant forms curly bundles, which remain entangled with dividing chromosomes leading to mitotic catastrophes or asymmetric partitions. Serial deletions of vimentin tail domain gradually impair cortical association and mitosis progression. Disruption of f-actin, but not of microtubules, causes vimentin bundling near the chromosomes. Pathophysiological stimuli, including HIV-protease and lipoxidation, induce similar alterations. Interestingly, full filament formation is dispensable for cortical association, which also occurs in vimentin particles. These results unveil implications of vimentin dynamics in cell division through its interplay with the actin cortex.

scholarly journals Disruption of Astral Microtubule Contact with the Cell Cortex Activates a Bub1, Bub3, and Mad3-dependent Checkpoint in Fission Yeast

2004 ◽  
Vol 15 (7) ◽  
pp. 3345-3356 ◽  
Author(s):  
Sylvie Tournier ◽  
Yannick Gachet ◽  
Vicky Buck ◽  
Jeremy S. Hyams ◽  
Jonathan B.A. Millar
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Checkpoint Proteins ◽  
Sister Chromatids ◽  
Astral Microtubule ◽  
Spindle Rotation

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.

Swelling and Curling Behaviors of Articular Cartilage

1998 ◽  
Vol 120 (3) ◽  
pp. 355-361 ◽  
Author(s):  
L. A. Setton ◽  
H. Tohyama ◽  
V. C. Mow
Keyword(s):  
Articular Cartilage ◽  
Residual Strain ◽  
Shape Change ◽  
Articular Surface ◽  
Ion Concentration ◽  
Ex Situ ◽  
Surface Zone ◽  
Sample Orientation ◽  
Residual Strains

A new experimental method was developed to quantify parameters of swelling-induced shape change in articular cartilage. Full-thickness strips of cartilage were studied in free-swelling tests and the swelling-induced stretch, curvature, and areal change were measured. In general, swelling-induced stretch and curvature were found to increase in cartilage with decreasing ion concentration, reflecting an increasing tendency to swell and “curl” at higher swelling pressures. An exception was observed at the articular surface, which was inextensible for all ionic conditions. The swelling-induced residual strain at physiological ionic conditions was estimated from the swelling-induced stretch and found to be tensile and from 3–15 percent. Parameters of swelling were found to vary with sample orientation, reflecting a role for matrix anisotropy in controlling the swelling-induced residual strains. In addition, the surface zone was found to be a structurally important element, which greatly limits swelling of the entire cartilage layer. The findings of this study provide the first quantitative measures of swelling-induced residual strain in cartilage ex situ, and may be readily adapted to studies of cartilage swelling in situ.

scholarly journals Gap43, Marcks, and Cap23 Modulate Pi(4,5)p2 at Plasmalemmal Rafts, and Regulate Cell Cortex Actin Dynamics through a Common Mechanism

2000 ◽  
Vol 149 (7) ◽  
pp. 1455-1472 ◽  
Author(s):  
Thorsten Laux ◽  
Kiyoko Fukami ◽  
Marcus Thelen ◽  
Tamara Golub ◽  
Dunja Frey ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Dynamic Properties ◽  
Peripheral Nerve Regeneration ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Common Mechanism ◽  
Cell Cortex ◽  
Kinase Substrate ◽  
Nerve Sprouting ◽  
Process Formation

The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P2, and promote its retention and clustering. Binding and modulation of PI(4,5)P2 depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P2 modulation. In the neuronlike cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by ΔED mutants. Agents that globally mask PI(4,5)P2 mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(ΔED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P2 modulating proteins, upstream of actin and cell cortex dynamics regulation.

Interactions between Sla1p, Lsb5p and Arf3p in yeast endocytosis

2005 ◽  
Vol 33 (6) ◽  
pp. 1273-1275 ◽  
Author(s):  
R. Costa ◽  
K.R. Ayscough
Keyword(s):  
Actin Cytoskeleton ◽  
Mammalian Cells ◽  
Cell Signalling ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Pheromone Receptor ◽  
Cargo Proteins ◽  
Endocytic Machinery ◽  
Adp Ribosylation Factor ◽  
Uptake Of Nutrients

Endocytosis is critical for controlling the protein–lipid composition of the plasma membrane, uptake of nutrients as well as pathogens, and also plays an important role in regulation of cell signalling. While a number of pathways for endocytosis have been characterized in different organisms, all of these require remodelling of the cell cortex. The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognized for many years in budding yeast, and is increasingly supported by studies in mammalian cells. Our studies have focused on proteins that we have shown to act at the interface between the actin cytoskeleton and the endocytic machinery. In particular, we have studied interactions of Sla1p, which binds to both activators of actin dynamics, i.e. Abp1p, Las17p and Pan1p, and to cargo proteins such as the pheromone receptor Ste2p. More recently we have mapped the interaction of Sla1p with Lsb5p, a protein that has a similar structure to the GGA [Golgi-localizing, γ-adaptin ear homology domain, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS (Vps27p/Hrs/STAM)-domain and a GAT (GGAs and TOM1) domain. We show that Lsb5p can interact with yeast Arf3p (orthologous with mammalian Arf6) and we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the cell cortex.

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