scholarly journals Actin-independent association of vinculin with the cytoplasmic aspect of the plasma membrane in cell-contact areas.

1983 ◽  
Vol 96 (6) ◽  
pp. 1622-1630 ◽  
Author(s):  
Z Avnur ◽  
J V Small ◽  
B Geiger
Keyword(s):  
Actin Filaments ◽  
Molecular Mechanisms ◽  
Cell Contact ◽  
Membrane Association ◽  
Focal Contact ◽  
Permeabilized Cells ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
Associated Proteins ◽  
Independent Association

We investigated the mode of association of vinculin with areas of contact between the termini of microfilament bundles and the cell membrane in sites of focal contact with the substrate by selective removal of actin from these areas. Opened-up substrate-attached membranes of chick fibroblasts as well as detergent-permeabilized cells were treated with fragmin from Physarum in the presence of Ca+2. This treatment removed actin filaments from the cytoplasmic faces of the membranes, along with several actin-associated proteins (alpha-actinin, tropomyosin, myosin, and filamin). Vinculin distribution was not affected by treatment. Moreover, rhodamine- or fluorescein-conjugated vinculin, when added to these preparations, became specifically associated with the focal contacts regardless of whether the latter were pretreated with fragmin or not. We conclude that the association of vinculin with focal contacts is largely actin-independent. We discuss the implications of these findings in the molecular mechanisms of microfilament membrane association in areas of cell contact.

The sequence of alignment of microtubules, focal contacts and actin filaments in fibroblasts spreading on smooth and grooved titanium substrata

1993 ◽  
Vol 106 (1) ◽  
pp. 343-354 ◽  
Author(s):  
C. Oakley ◽  
D.M. Brunette
Keyword(s):  
Actin Filaments ◽  
Human Gingival Fibroblasts ◽  
Contact Guidance ◽  
Gingival Fibroblasts ◽  
Cell Orientation ◽  
Cell Height ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
Titanium Surfaces ◽  
Oriented Parallel

Contact guidance refers to the reactions of cells with the topography of their substratum. Current hypotheses on the mechanism of contact guidance focus on the dynamic behaviour of the cytoskeletal components, but most observations have been made on cells that have already become oriented with topographic features of the substratum. The purpose of this study was to examine the sequence in which microtubules, focal contacts and microfilament bundles become aligned to the substratum topography as fibroblasts spread on grooved substrata. Human gingival fibroblasts were trypsinized and seeded onto grooved titanium surfaces produced by micromachining, as well as onto control smooth surfaces. After observation and photography of the spreading cells at times up to 6 hours, the cells were fixed and exposed to one or more of the following antibodies or fluorescent stains: phallacidin to stain actin filaments, monoclonal anti-tubulin, monoclonal anti-vinculin, anti-mouse IgG labelled with Texas-Red or FITC, and/or an aldehyde-reactive stain to identify the cell outline. The cells were photographed and cell area, shape and orientation were calculated. Cells were also examined with confocal microscopy to obtain optical sections so that cell height as well as the precise locations of the cytoskeletal components with respect to the vertical dimension of the grooved substrata could be determined. Microtubules were the first element to become oriented parallel to the direction of the grooves and were first aligned at the bottom of the grooves. This alignment of microtubules was evident as early as 20 minutes after plating and preceded the orientation of the cell as a whole. Aligned actin microfilament bundles were not observed until 40–60 minutes and were observed first at the wall-ridge edges. At early times, focal contacts were distributed radially, but only after 3 hours did the majority of cells demonstrate aligned focal contacts. If the first cytoskeletal component to become aligned is the prime determinant of cell orientation, then these data suggest that microtubules in human gingival fibroblasts may determine cell orientation on grooved titanium surfaces. By analogy with microtubule behaviour in other systems, we suggest that microtubule orientation on grooved substrata may occur as a result of the substratum establishing shear-free planes.

scholarly journals Microfilament-organizing centers in areas of cell contact: cytoskeletal interactions during cell attachment and locomotion.

1984 ◽  
Vol 99 (1) ◽  
pp. 83s-91s ◽  
Author(s):  
B Geiger ◽  
Z Avnur ◽  
G Rinnerthaler ◽  
H Hinssen ◽  
V J Small
Keyword(s):  
Dynamic Equilibrium ◽  
Cell Attachment ◽  
Dynamic Properties ◽  
Leading Edge ◽  
Stress Fibers ◽  
Cell Contact ◽  
Molecular Architecture ◽  
Contact Areas ◽  
Focal Contacts ◽  
Associated Proteins

In this article we discuss three aspects of cell contact formation: (a) the molecular architecture of the cytomatrix in cell-to-substrate focal contacts, (b) the dynamic properties of membrane- and microfilament-associated proteins in the contact areas, and (c) the involvement of microtubules in the coordinated and directed formation of new substrate contacts during cell locomotion. We show that different microfilament-associated proteins exhibit distinct patterns of association with focal contacts: some proteins are specifically associated with focal contacts (vinculin and talin); alpha-actinin is enriched in the contact areas but also is present along the stress fibers and in the lamellipodium; actin and filamin are detected throughout the contact areas but in apparently reduced amounts compared with the associated stress fibers; and tropomyosin, myosin, and spectrin are either absent from the endofacial surfaces of contact areas or are present in only very small amounts. Fluorescence photobleaching recovery analyses performed with living cells microinjected with fluorescently labeled actin, vinculin, and alpha-actinin indicate that each of these proteins maintains a dynamic equilibrium between a soluble cytoplasmic pool and a membrane-bound fraction. Correlation of the distribution of vinculin and tubulin in motile fibroblasts to local movements of the leading edge of the same cells indicates that free-end microtubules extend into actively ruffling areas along the lamellipodium and that new vinculin-containing contacts are preferentially formed in these protruding regions.

scholarly journals Arginylation Regulates Intracellular Actin Polymer Level by Modulating Actin Properties and Binding of Capping and Severing Proteins

2010 ◽  
Vol 21 (8) ◽  
pp. 1350-1361 ◽  
Author(s):  
Sougata Saha ◽  
Maureen M. Mundia ◽  
Fangliang Zhang ◽  
Ryan W. Demers ◽  
Farida Korobova ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Biochemical Properties ◽  
Cytoskeletal Organization ◽  
Actin Networks ◽  
Severe Impairment ◽  
Associated Proteins

Actin arginylation regulates lamella formation in motile fibroblasts, but the underlying molecular mechanisms are unknown. To understand how arginylation affects the actin cytoskeleton, we investigated the biochemical properties and the structural organization of actin filaments in wild-type and arginyltransferase (Ate1) knockout cells. We found that Ate1 knockout results in a dramatic reduction of the actin polymer levels in vivo accompanied by a corresponding increase in the monomer level. Purified nonarginylated actin has altered polymerization properties, and actin filaments from Ate1 knockout cells show altered interactions with several associated proteins. Ate1 knockout cells have severe impairment of cytoskeletal organization throughout the cell. Thus, arginylation regulates the ability of actin to form filaments in the whole cell rather than preventing the collapse of preformed actin networks at the cell leading edge as proposed in our previous model. This regulation is achieved through interconnected mechanisms that involve actin polymerization per se and through binding of actin-associated proteins.

scholarly journals Human fetal liver MSCs are more effective than adult bone marrow MSCs for their immunosuppressive, immunomodulatory, and Foxp3+ T reg induction capacity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yi Yu ◽  
Alejandra Vargas Valderrama ◽  
Zhongchao Han ◽  
Georges Uzan ◽  
Sina Naserian ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Immune Responses ◽  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Cytotoxic T Cells ◽  
Cell Contact ◽  
Adult Bone Marrow ◽  
Adult Bone

Abstract Background Mesenchymal stem cells (MSCs) exhibit active abilities to suppress or modulate deleterious immune responses by various molecular mechanisms. These cells are the subject of major translational efforts as cellular therapies for immune-related diseases and transplantations. Plenty of preclinical studies and clinical trials employing MSCs have shown promising safety and efficacy outcomes and also shed light on the modifications in the frequency and function of regulatory T cells (T regs). Nevertheless, the mechanisms underlying these observations are not well known. Direct cell contact, soluble factor production, and turning antigen-presenting cells into tolerogenic phenotypes, have been proposed to be among possible mechanisms by which MSCs produce an immunomodulatory environment for T reg expansion and activity. We and others demonstrated that adult bone marrow (BM)-MSCs suppress adaptive immune responses directly by inhibiting the proliferation of CD4+ helper and CD8+ cytotoxic T cells but also indirectly through the induction of T regs. In parallel, we demonstrated that fetal liver (FL)-MSCs demonstrates much longer-lasting immunomodulatory properties compared to BM-MSCs, by inhibiting directly the proliferation and activation of CD4+ and CD8+ T cells. Therefore, we investigated if FL-MSCs exert their strong immunosuppressive effect also indirectly through induction of T regs. Methods MSCs were obtained from FL and adult BM and characterized according to their surface antigen expression, their multilineage differentiation, and their proliferation potential. Using different in vitro combinations, we performed co-cultures of FL- or BM-MSCs and murine CD3+CD25−T cells to investigate immunosuppressive effects of MSCs on T cells and to quantify their capacity to induce functional T regs. Results We demonstrated that although both types of MSC display similar cell surface phenotypic profile and differentiation capacity, FL-MSCs have significantly higher proliferative capacity and ability to suppress both CD4+ and CD8+ murine T cell proliferation and to modulate them towards less active phenotypes than adult BM-MSCs. Moreover, their substantial suppressive effect was associated with an outstanding increase of functional CD4+CD25+Foxp3+ T regs compared to BM-MSCs. Conclusions These results highlight the immunosuppressive activity of FL-MSCs on T cells and show for the first time that one of the main immunoregulatory mechanisms of FL-MSCs passes through active and functional T reg induction.

scholarly journals Actin-membrane interaction in fibroblasts: what proteins are involved in this association?

1984 ◽  
Vol 99 (1) ◽  
pp. 95s-103s ◽  
Author(s):  
P Mangeat ◽  
K Burridge
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Stress Fiber ◽  
Membrane Interaction ◽  
Microfilament Bundles ◽  
The Family ◽  
Form Part ◽  
Membrane Attachment ◽  
A Chain ◽  
And Function

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.

scholarly journals The polymerization of actin: II. how nonfilamentous actin becomes nonrandomly distributed in sperm: evidence for the association of this actin with membranes

1976 ◽  
Vol 69 (1) ◽  
pp. 51-72 ◽  
Author(s):  
LG Tilney
Keyword(s):  
Plasma Membrane ◽  
Nuclear Envelope ◽  
Thin Section ◽  
Actin Filaments ◽  
Early Stage ◽  
Mature Sperm ◽  
Vacuole Membrane ◽  
Other Substances ◽  
Associated Proteins ◽  
Basal Half

At an early stage in spermiogenesis the acrosomal vacuole and other organelles including ribosomes are located at the basal end of the cell. From here actin must be transported to its future location at the anterior end of the cell. At no stage in the accumulation of actin in the periacrosomal region is the actin sequestered in a membrane-bounded compartment such as a vacuole or vesicle. Since filaments are not present in the periacrosomal region during the accumulation of the actin even though the fixation of these cells is sufficiently good to distinguish actin filaments in thin section, the actin must accumulate in the nonfilamentous state. The membranes in the periacrosomal region, specifically a portion of the nuclear envelope and the basal half of the acrosomal vacuole membrane, become specialized morphologically in advance of the accumulation of actin in this region. My working hypothesis is that the actin in combination with other substances binds to these specialized membranes and to itself and thus can accumulate in the periacrosmoal region by being trapped on these specialized membranes. Diffusion would then be sufficient to move these substances to this region. In support of this hypothesis are experiments in which I treated mature sperm with detergents, glycols, and hypotonic media, which solubilize or lift away the plasma membrane. The actin and its associated proteins remain attached to these specialized membranes. Thus actin can be nonrandomly distributed in cells in a nonfilamentous state presumably by its association with specialized membranes.

scholarly journals Novel Roles for α3β1 Integrin as a Regulator of Cytoskeletal Assembly and as a Trans-dominant Inhibitor of Integrin Receptor Function in Mouse Keratinocytes

1998 ◽  
Vol 142 (5) ◽  
pp. 1357-1369 ◽  
Author(s):  
Kairbaan M. Hodivala-Dilke ◽  
C. Michael DiPersio ◽  
Jordan A. Kreidberg ◽  
Richard O. Hynes
Keyword(s):  
Stress Fiber ◽  
Fiber Formation ◽  
The Other ◽  
Collagen Type Iv ◽  
Focal Contact ◽  
Actin Organization ◽  
Α3β1 Integrin ◽  
Stress Fiber Formation ◽  
Associated Proteins ◽  
Mouse Keratinocytes

Previously we found that α3β1 integrin–deficient neonatal mice develop micro-blisters at the epidermal–dermal junction. These micro-blisters were associated with poor basement membrane organization. In the present study we have investigated the effect of α3β1-deficiency on other keratinocyte integrins, actin-associated proteins and F-actin organization. We show that the absence of α3β1 results in an increase in stress fiber formation in keratinocytes grown in culture and at the basal face of the basal keratinocytes of α3-null epidermis. Moreover, we see a higher concentration of actin-associated proteins such as vinculin, talin, and α-actinin at focal contact sites in the α3-deficient keratinocytes. These changes in focal contact composition were not due to a change in steady-state levels of these proteins, but rather to reorganization due to α3β1 deficiency. Apart from the loss of α3β1 there is no change in expression of the other integrins expressed by the α3-null keratinocytes. However, in functional assays, α3β1 deficiency allows an increase in fibronectin and collagen type IV receptor activities. Thus, our findings provide evidence for a role of α3β1 in regulating stress fiber formation and as a trans-dominant inhibitor of the functions of the other integrins in mouse keratinocytes. These results have potential implications for the regulation of keratinocyte adhesion and migration during wound healing.

scholarly journals Structures linking microfilament bundles to the membrane at focal contacts

1993 ◽  
Vol 122 (2) ◽  
pp. 485-496 ◽  
Author(s):  
SJ Samuelsson ◽  
PW Luther ◽  
DW Pumplin ◽  
RJ Bloch
Keyword(s):  
Single Bundle ◽  
Actin Microfilaments ◽  
Lateral Interactions ◽  
Membrane Interactions ◽  
Membrane Particles ◽  
Focal Contacts ◽  
Microfilament Bundles ◽  
Immunogold Cytochemistry ◽  
Beta 1 Integrin

We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including beta 1-integrin, vinculin, and talin, but they appear to contain less alpha-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained beta 1-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and beta 1-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.

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