Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins

2002 ◽  
Vol 4 (4) ◽  
pp. 286-293 ◽  
Author(s):  
Zenon Rajfur ◽  
Partha Roy ◽  
Carol Otey ◽  
Lewis Romer ◽  
Ken Jacobson
Keyword(s):  
Fusion Proteins ◽  
Focal Adhesions

scholarly journals Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

1995 ◽  
Vol 182 (4) ◽  
pp. 1089-1099 ◽  
Author(s):  
K Tachibana ◽  
T Sato ◽  
N D'Avirro ◽  
C Morimoto
Keyword(s):  
Amino Acids ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fusion Proteins ◽  
Focal Adhesions ◽  
Immunohistochemical Analysis ◽  
Binding Activity ◽  
Glutathione S Transferase ◽  
Cytoskeleton Protein ◽  
Direct Association

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

scholarly journals Conditional Expression of a Truncated Fragment of Nonmuscle Myosin II-A Alters Cell Shape but Not Cytokinesis in HeLa Cells

2000 ◽  
Vol 11 (10) ◽  
pp. 3617-3627 ◽  
Author(s):  
Qize Wei ◽  
Robert S. Adelstein
Keyword(s):  
Hela Cells ◽  
Fusion Proteins ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Full Length ◽  
Stress Fibers ◽  
Amino Terminus ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Conditional Expression

A truncated fragment of the nonmuscle myosin II-A heavy chain (NMHC II-A) lacking amino acids 1–591, ΔN592, was used to examine the cellular functions of this protein. Green fluorescent protein (GFP) was fused to the amino terminus of full-length human NMHC II-A, NMHC II-B, and ΔN592 and the fusion proteins were stably expressed in HeLa cells by using a conditional expression system requiring absence of doxycycline. The HeLa cell line studied normally expressed only NMHC II-A and not NMHC II-B protein. Confocal microscopy indicated that the GFP fusion proteins of full-length NMHC II-A, II-B, and ΔN592 were localized to stress fibers. However, in vitro assays showed that baculovirus-expressed ΔN592 did not bind to actin, suggesting that ΔN592 was localized to actin stress fibers through incorporation into endogenous myosin filaments. There was no evidence for the formation of heterodimers between the full-length endogenous nonmuscle myosin and truncated nonmuscle MHCs. Expression of ΔN592, but not full-length NMHC II-A or NMHC II-B, induced cell rounding with rearrangement of actin filaments and disappearance of focal adhesions. These cells returned to their normal morphology when expression of ΔN592 was repressed by addition of doxycycline. We also show that GFP-tagged full-length NMHC II-A or II-B, but not ΔN592, were localized to the cytokinetic ring during mitosis, indicating that, in vertebrates, the amino-terminus part of mammalian nonmuscle myosin II may be necessary for localization to the cytokinetic ring.

scholarly journals p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

2013 ◽  
Vol 91 (6) ◽  
pp. 404-418 ◽  
Author(s):  
Margaret D. George ◽  
Robert N. Wine ◽  
Brad Lackford ◽  
Grace E. Kissling ◽  
Steven K. Akiyama ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Cell Adhesion ◽  
Fatty Acid ◽  
Protein Kinase ◽  
Actin Filaments ◽  
Fusion Proteins ◽  
Active Form ◽  
Focal Adhesions ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein

Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia, whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading, and that this association can be regulated by factors in the tumor microenvironment.

scholarly journals Talin contains three actin-binding sites each of which is adjacent to a vinculin-binding site

1996 ◽  
Vol 109 (11) ◽  
pp. 2715-2726 ◽  
Author(s):  
L. Hemmings ◽  
D.J. Rees ◽  
V. Ohanian ◽  
S.J. Bolton ◽  
A.P. Gilmore ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Binding Sites ◽  
Fusion Proteins ◽  
Focal Adhesions ◽  
Actin Binding ◽  
Mouse Protein ◽  
Protein Alignments ◽  
Terminal Site ◽  
Actin Binding Site

We have determined the sequence of chicken talin (2,541 amino acids, M(r) 271,881) which is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, we have identified at least three regions of the protein which can bind F-actin: residues 102–497, 951–1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from a binding site for vinculin, a protein which also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localised weakly to stress fibres, whereas one containing the C-terminal site initially localised predominantly to focal adhesions. The former was readily solubilised, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupted actin stress fibres whereas the C-terminal polypeptide was without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site did disrupt stress fibres and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct.

Identification of common and distinct epigenetic re-programming properties of Core-Binding Factor (CBF) Fusion Proteins

2016 ◽  
Vol 228 (03) ◽  
Author(s):  
J Loke ◽  
A Ptasinska ◽  
MR Imperato ◽  
SA Assi ◽  
P Cauchy ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Core Binding Factor ◽  
Binding Factor

Fusion proteins CDGM and 4CDGM enhance B cell proliferation

2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Shiqiang Lu ◽  
Xiaoyi Lu ◽  
Zehua Sun
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Fusion Proteins

Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain

2018 ◽  
Author(s):  
Sarah Klass ◽  
Matthew J. Smith ◽  
Tahoe Fiala ◽  
Jessica Lee ◽  
Anthony Omole ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fusion Proteins ◽  
Organic Molecules ◽  
Intrinsically Disordered Protein ◽  
Protein Domain ◽  
Enzymatic Cleavage ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Self Assembling ◽  
Protein Tag

Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values below 1 µM being observed in some cases. The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications.

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