scholarly journals Dimensionality changes actin network through lamin A and C and zyxin

2019 ◽  
Author(s):  
Jip Zonderland ◽  
Ivan Lorenzo Moldero ◽  
Carlos Mota ◽  
Lorenzo Moroni
Keyword(s):  
Actin Cytoskeleton ◽  
Stromal Cells ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Lamin A ◽  
Actin Network ◽  
Knock Down ◽  
Electrospun Scaffolds ◽  
Human Mesenchymal Stromal Cells ◽  
Stiff Material

AbstractThe actin cytoskeleton plays a key role in differentiation of human mesenchymal stromal cells (hMSCs), but its regulation in 3D tissue engineered scaffolds remains poorly studied. hMSCs cultured on 3D electrospun scaffolds made of a stiff material do not form actin stress fibers, contrary to hMSCs on 2D films of the same material. On 3D electrospun- and 3D additive manufactured scaffolds, hMSCs also displayed fewer focal adhesions, lower lamin A and C expression and less YAP1 nuclear localization. Together, this shows that dimensionality prevents the build-up of cellular tension, even on stiff materials. Knock down of either lamin A and C or zyxin resulted in fewer stress fibers in the cell center. Zyxin knock down reduced lamin A and C expression, but not vice versa, showing that this signal chain starts from the outside of the cell. Our study demonstrates that dimensionality changes the actin cytoskeleton through lamin A and C and zyxin, an important insight for future scaffold design, as the actin network, focal adhesions and nuclear stiffness are all critical for hMSC differentiation.

scholarly journals Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

2021 ◽  
Author(s):  
Erik S Linklater ◽  
Emily Duncan ◽  
Ke Jun Han ◽  
Algirdas Kaupinis ◽  
Mindaugas Valius ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Migration And Invasion ◽  
Matrix Remodeling ◽  
Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

scholarly journals Biological activity of human mesenchymal stromal cells on polymeric electrospun scaffolds

2019 ◽  
Vol 7 (3) ◽  
pp. 1088-1100 ◽  
Author(s):  
Febriyani F. R. Damanik ◽  
Gabriele Spadolini ◽  
Joris Rotmans ◽  
Silvia Farè ◽  
Lorenzo Moroni
Keyword(s):  
Biological Activity ◽  
Cell Fate ◽  
Structural Properties ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Electrospun Scaffolds ◽  
Human Mesenchymal Stromal Cells ◽  
And Migration

Controlling chemical and structural properties of electrospun scaffolds provide cues to regulate cell fate and migration.

Migration Potential of Human Mesenchymal Stromal Cells Is Determined by Origin of Tissue Rather Than Their Developmental Stage

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4743-4743
Author(s):  
Marijke W. Maijenburg ◽  
Willy A. Noort ◽  
Marion Kleijer ◽  
Charlotte J.A. Kompier ◽  
Kees Weijer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Synergistic Effect ◽  
Actin Cytoskeleton ◽  
Developmental Stage ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Focal Adhesions ◽  
Migration Potential ◽  
Migration Capacity

Abstract It is thought that adult mesenchymal stromal cells (MSC) are important for tissue repair and maintenance. Crucial in these processes is the presence of MSC at the site of injury, however the recruitment and migration of MSC towards their destiny is poorly understood. With respect to future cell therapy, we are studying the process of migration of various human mesenchymal stem cell sources, and hypothesize that only a subpopulation of ex vivo expanded mesenchymal stem cells is capable of specific homing. For this purpose, MSC from different sources i.e. fetal lung (FL), fetal bone marrow (FBM), adult bone marrow (ABM) and adult adipose tissue (AT) were derived by plastic adherence and subsequently expanded. All MSC sources were characterized as CD73+, CD90+, CD105+, CD34− and CD45−. MSC (P4-9) were allowed to migrate for 4h towards SDF-1a, PDGF-BB, HGF, bFGF or FCS over fibronectin-coated 12 mm pore size transwell plates. FL-MSC migrated significantly better towards SDF-1a as compared to ABM-MSC or AT-MSC. This enhanced migration capacity towards SDF-1a is specific for FL-MSC since AT-MSC migrated better towards FCS as compared to FL-MSC. Even ABM-MSC responded better to FCS than FL-MSC. This suggests that MSC originating from all sources are able to migrate but require different triggers to induce migration. In order to elucidate whether the observed differences in migration potential were due to developmental stage, cultured MSC derived from fetal bone marrow were tested as well. No significant differences in migration capacity were observed between adult and fetal BM- MSC for any of the (chemotactic) stimuli evaluated. Interestingly, FL-MSC had a significant increased migration capacity as compared to FBM-MSC towards SDF-1a, PDGF-BB and HGF, suggesting that the origin of tissue may determine migration capacity of ex vivo expanded MSC. Since it was observed that only a small percentage of the cultured MSC were able to migrate towards the various stimuli, checkerboard migration was performed to elucidate whether a synergistic effect could be observed. No synergistic effect was observed between SDF and PDGF, SDF and FCS or PDGF and FCS in FL-MSC, suggesting that there may be one subpopulation of MSC that possesses migratory capacities. When studying the SDF-1-induced migratory subpopulation in more detail, it was observed that, after migration, migratory MSC originating from all tissue sources maintain their proliferation and differentiation capacity and express CXCR4 at a higher level than MSC that did not migrate. To be able to migrate, cells have to rearrange their actin cytoskeleton and focal adhesions. These processes can be initiated by various chemokines and growth factors. In response to SDF or FCS, morphological changes were observed in ABM-MSC by confocal microscopy. Cells became smaller and membrane protrusions appeared, whereas this was absent in the control. Furthermore, upon stimulation with SDF, PDGF and FCS, tyrosine-phosphorylation of the adapter protein paxillin that links the actin cytoskeleton to focal adhesions was increased. In conclusion, our results suggest that migration potential of ex vivo expanded MSC derived from various adult and fetal tissues have different migratory capacity towards growth factor and chemokine stimuli and may involve paxillin phosphorylation. Our data indicate that further studies on the migratory subpopulation(s) within the heterogeneous population of culture expanded MSC will contribute to unravel how and which MSC will be of interest for future cellular therapies.

scholarly journals Septins promote stress fiber–mediated maturation of focal adhesions and renal epithelial motility

2014 ◽  
Vol 207 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Lee Dolat ◽  
John L. Hunyara ◽  
Jonathan R. Bowen ◽  
Eva Pauline Karasmanis ◽  
Maha Elgawly ◽  
...  
Keyword(s):  
G Proteins ◽  
Tumor Metastasis ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Stress Fibers ◽  
Actin Network ◽  
Septin 9 ◽  
The Stability ◽  
2D And 3D ◽  
Hepatocyte Growth

Organogenesis and tumor metastasis involve the transformation of epithelia to highly motile mesenchymal-like cells. Septins are filamentous G proteins, which are overexpressed in metastatic carcinomas, but their functions in epithelial motility are unknown. Here, we show that a novel network of septin filaments underlies the organization of the transverse arc and radial (dorsal) stress fibers at the leading lamella of migrating renal epithelia. Surprisingly, septin depletion resulted in smaller and more transient and peripheral focal adhesions. This phenotype was accompanied by a highly disorganized lamellar actin network and rescued by the actin bundling protein α-actinin-1. We show that preassembled actin filaments are cross-linked directly by Septin 9 (SEPT9), whose expression is increased after induction of renal epithelial motility with the hepatocyte growth factor. Significantly, SEPT9 overexpression enhanced renal cell migration in 2D and 3D matrices, whereas SEPT9 knockdown decreased migration. These results suggest that septins promote epithelial motility by reinforcing the cross-linking of lamellar stress fibers and the stability of nascent focal adhesions.

scholarly journals Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

2021 ◽  
pp. mbc.E21-03-0156
Author(s):  
Stefano Sala ◽  
Patrick W. Oakes
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Lim Domain ◽  
Lim Domains ◽  
Lim Domain Protein ◽  
The Family ◽  
Domain Protein ◽  
Cytoskeletal Elements ◽  
In Cells

The actin cytoskeleton is a key regulator of mechanical processes in cells. The family of LIM domain proteins have recently emerged as important mechanoresponsive cytoskeletal elements capable of sensing strain in the actin cytoskeleton. The mechanisms regulating this mechanosensitive behavior, however, remain poorly understood. Here we show that the LIM domain protein testin is peculiar in that despite the full-length protein primarily appearing diffuse in the cytoplasm, the C-terminal LIM domains alone recognize focal adhesions and strained actin while the N-terminal domains alone recognize stress fibers. Phosphorylation mutations in the dimerization regions of testin, however, reveal its mechanosensitivity and cause it to relocate to focal adhesions and sites of strain in the actin cytoskeleton. Finally, we demonstrate activated RhoA causes testin to adorn stress fibers and become mechanosensitive. Together, our data show that testin's mechanoresponse is regulated in cells and provide new insights into LIM domain protein recognition of the actin cytoskeleton mechanical state. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals Distinct Actions and Cooperative Roles of ROCK and mDia in Rho Small G Protein-induced Reorganization of the Actin Cytoskeleton in Madin–Darby Canine Kidney Cells

1999 ◽  
Vol 10 (8) ◽  
pp. 2481-2491 ◽  
Author(s):  
Katsutoshi Nakano ◽  
Kenji Takaishi ◽  
Atsuko Kodama ◽  
Akiko Mammoto ◽  
Hitoshi Shiozaki ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Kidney Cells ◽  
Madin Darby Canine Kidney ◽  
Downstream Target ◽  
Small G Protein ◽  
Target Molecules ◽  
Darby Canine Kidney ◽  
Canine Kidney

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin–Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate–induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.

scholarly journals Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration

2021 ◽  
Vol 220 (7) ◽  
Author(s):  
Erik S. Linklater ◽  
Emily D. Duncan ◽  
Ke-Jun Han ◽  
Algirdas Kaupinis ◽  
Mindaugas Valius ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Migration And Invasion ◽  
Matrix Remodeling ◽  
Cell Migration And Invasion

Rab40b is a SOCS box–containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b–Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b–Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

scholarly journals Lipid-directed cytoskeletal protein oligomerization at sites of cell adhesion

2014 ◽  
Vol 70 (a1) ◽  
pp. C1833-C1833
Author(s):  
Krishna Chinthalapudi ◽  
Erumbi Rangarajan ◽  
Dipak Patil ◽  
Tina Izard
Keyword(s):  
Actin Cytoskeleton ◽  
Lipid Membrane ◽  
Acyl Chain ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Head Group ◽  
Protein Oligomerization ◽  
Actin Network ◽  
Integrin Receptors ◽  
Dynamic Interactions

Vertebrate cell growth, division, migration, morphogenesis, and development, rely on the dynamic interactions of cells with components the extracellular matrix (ECM) via cell surface complexes. These focal adhesions (FAs) are comprised of integrin receptors, associated signaling molecules, and talin, which is required for "inside-out" signaling that stabilizes contacts of integrin receptors with the ECM by linking FAs to the actin cytoskeleton by binding to vinculin. The highly dynamic interactions with the actin cytoskeleton are also essential for the formation of membrane protrusions (lamellopodia and filopodia). Second messengers are found at the plasma cell membrane and include signaling lipids such as phosphoinositides, which play essential roles in signal transduction pathways and in directing the oligomerization of cytoskeletal proteins that function as essential links of FAs to the actin cytoskeleton. Notably, the most abundant phosphoinositide, phosphatidyl (4,5) bisphosphate (PIP2), directly binds to key cytoskeletal proteins, where it triggers homotypic and heterotypic interactions that amplify binding to the actin network. Binding of the inositol head group and the hydrophobic acyl chain pose difficulties in generating protein/PIP2 complex crystals and here we present the only second non-membrane protein structure of such a complex. Our crystal structure and biochemical approaches define the roles of PIP2 in controlling the oligomerization of cytoskeletal proteins and their binding to adhesion receptors and to the actin cytoskeleton. Importantly, we also determined the contribution of PIP2-directed oligomerization of cytoskeletal proteins to the formation and stabilization of adhesion complexes. These studies provide important new insights into how dynamic interactions of cytoskeletal proteins with the lipid membrane, adhesion complexes, and the actin network direct the mechanical behaviors of cells.

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