scholarly journals Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells

2002 ◽  
Vol 156 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Gerhard Schratt ◽  
Ulrike Philippar ◽  
Jürgen Berger ◽  
Heinz Schwarz ◽  
Olaf Heidenreich ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Serum Response Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Spreading ◽  
Stress Fibers ◽  
Response Factor ◽  
Es Cell ◽  
And Migration ◽  
Serum Response

The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf(−/−) embryonic stem (ES) cells, we demonstrate that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), β1-integrin, talin, zyxin, and vinculin were downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf(−/−) ES cells were accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescued F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf(−/−) ES cells, on the other hand, strongly induced expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, we demonstrate for the first time the importance of SRF for the formation of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. Our findings suggest an involvement of SRF in cell migratory processes in multicellular organisms.

scholarly journals Serum Response Factor Is Required for Immediate-Early Gene Activation yet Is Dispensable for Proliferation of Embryonic Stem Cells

2001 ◽  
Vol 21 (8) ◽  
pp. 2933-2943 ◽  
Author(s):  
Gerhard Schratt ◽  
Birgit Weinhold ◽  
Ante S. Lundberg ◽  
Sebastian Schuck ◽  
Jürgen Berger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Serum Response Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Gene ◽  
Response Factor ◽  
Es Cell ◽  
Cycle Progression ◽  
Serum Response

ABSTRACT Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf −/− mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf −/− ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf−/− ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf −/− ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

scholarly journals Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1121-1133 ◽  
Author(s):  
Helen Priddle ◽  
Lance Hemmings ◽  
Susan Monkley ◽  
Alison Woods ◽  
Bipin Patel ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Es Cell ◽  
Wild Type

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

scholarly journals Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

1995 ◽  
Vol 128 (5) ◽  
pp. 979-988 ◽  
Author(s):  
R Fässler ◽  
M Pfaff ◽  
J Murphy ◽  
A A Noegel ◽  
S Johansson ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Junctions ◽  
Es Cell ◽  
Beta 1 Integrin ◽  
Integrin Gene ◽  
And Migration

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Selected Contribution: Skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent

2001 ◽  
Vol 90 (3) ◽  
pp. 1174-1183 ◽  
Author(s):  
Scott E. Gordon ◽  
Martin Flück ◽  
Frank W. Booth
Keyword(s):  
Skeletal Muscle ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Soleus Muscle ◽  
Skeletal Muscles ◽  
Serum Response Factor ◽  
Response Factor ◽  
Gastrocnemius Muscles ◽  
Fast Twitch ◽  
Serum Response

This investigation examined the effect of mechanical loading state on focal adhesion kinase (FAK), paxillin, and serum response factor (SRF) in rat skeletal muscle. We found that FAK concentration and tyrosine phosphorylation, paxillin concentration, and SRF concentration are all lower in the lesser load-bearing fast-twitch plantaris and gastrocnemius muscles compared with the greater load-bearing slow-twitch soleus muscle. Of these three muscles, 7 days of mechanical unloading via tail suspension elicited a decrease in FAK tyrosine phosphorylation only in the soleus muscle and decreases in FAK and paxillin concentrations only in the plantaris and gastrocnemius muscles. Unloading decreased SRF concentration in all three muscles. Mechanical overloading (via bilateral gastrocnemius ablation) for 1 or 8 days increased FAK and paxillin concentrations in the soleus and plantaris muscles. Additionally, whereas FAK tyrosine phosphorylation and SRF concentration were increased by ≤1 day of overloading in the soleus muscle, these increases did not occur until somewhere between 1 and 8 days of overloading in the plantaris muscle. These data indicate that, in the skeletal muscles of rats, the focal adhesion complex proteins FAK and paxillin and the transcription factor SRF are generally modulated in association with the mechanical loading state of the muscle. However, the somewhat different patterns of adaptation of these proteins to altered loading in slow- vs. fast-twitch skeletal muscles indicate that the mechanisms and time course of adaptation may partly depend on the prior loading state of the muscle.

scholarly journals Serum response factor orchestrates nascent sarcomerogenesis and silences the biomineralization gene program in the heart

2008 ◽  
Vol 105 (46) ◽  
pp. 17824-17829 ◽  
Author(s):  
Zhiyv Niu ◽  
Dinakar Iyer ◽  
Simon J. Conway ◽  
James F. Martin ◽  
Kathryn Ivey ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Serum Response Factor ◽  
Es Cells ◽  
Bone Morphogenetic Protein 4 ◽  
Response Factor ◽  
Down Regulation ◽  
Morphogenetic Protein ◽  
Calcium Activated Potassium Channel ◽  
Serum Response ◽  
Potassium Channel Gene

Our conditional serum response factor (SRF) knockout, Srf Cko, in the heart-forming region blocked the appearance of rhythmic beating myocytes, one of the earliest cardiac defects caused by the ablation of a cardiac-enriched transcription factor. The appearance of Hand1 and Smyd1, transcription and chromatin remodeling factors; Acta1, Acta2, Myl3, and Myom1, myofibril proteins; and calcium-activated potassium-channel gene activity (KCNMB1), the channel protein, were powerfully attenuated in the SrfCKO mutant hearts. A requisite role for combinatorial cofactor interactions with SRF, as a major determinant for regulating the appearance of organized sarcomeres, was shown by viral rescue of SRF-null ES cells with SRF point mutants that block cofactor interactions. In the absence of SRF genes associated with biomineralization, GATA-6, bone morphogenetic protein 4 (BMP4), and periostin were strongly up-regulated, coinciding with the down regulation of many SRF dependent microRNA, including miR1, which exerted robust silencer activity over the induction of GATA-6 leading to the down regulation of BMP4 and periostin.

scholarly journals Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation

2003 ◽  
Vol 23 (7) ◽  
pp. 2425-2437 ◽  
Author(s):  
Kevin L. Du ◽  
Hon S. Ip ◽  
Jian Li ◽  
Mary Chen ◽  
Frederic Dandre ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Molecular Mechanisms ◽  
Serum Response Factor ◽  
Es Cells ◽  
Regulatory Elements ◽  
Response Factor ◽  
Transcriptional Program ◽  
Serum Response

ABSTRACT The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF−/− ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22α promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22α, smooth muscle α-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.

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