In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

2016 ◽  
pp. 151-179 ◽  
Author(s):  
Dennis Zimmermann ◽  
Alisha N. Morganthaler ◽  
David R. Kovar ◽  
Cristian Suarez
Keyword(s):  
Binding Proteins ◽  
Biochemical Characterization ◽  
Actin Binding ◽  
Actin Binding Proteins

Purification and characterization of Ca2+ /calmodulin-dependent actin-binding proteins from squid retina

FEBS Letters ◽  
1989 ◽  
Vol 247 (2) ◽  
pp. 377-380 ◽  
Author(s):  
Haruo Asai ◽  
Takao Arai ◽  
Toshihiro Fujii ◽  
Gen Matsumoto
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Purification And Characterization ◽  
Actin Binding Proteins

scholarly journals STIP1/HOP Regulates the Actin Cytoskeleton through Interactions with Actin and Changes in Actin-Binding Proteins Cofilin and Profilin

2020 ◽  
Vol 21 (9) ◽  
pp. 3152 ◽  
Author(s):  
Samantha Joy Beckley ◽  
Morgan Campbell Hunter ◽  
Sarah Naulikha Kituyi ◽  
Ianthe Wingate ◽  
Abantika Chakraborty ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Binding Proteins ◽  
Major Effect ◽  
Vital Role ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nuclear Accumulation ◽  
Actin Binding Proteins ◽  
Hsp90 Chaperone

Cell migration plays a vital role in both health and disease. It is driven by reorganization of the actin cytoskeleton, which is regulated by actin-binding proteins cofilin and profilin. Stress-inducible phosphoprotein 1 (STIP1) is a well-described co-chaperone of the Hsp90 chaperone system, and our findings identify a potential regulatory role of STIP1 in actin dynamics. We show that STIP1 can be isolated in complex with actin and Hsp90 from HEK293T cells and directly interacts with actin in vitro via the C-terminal TPR2AB-DP2 domain of STIP1, potentially due to a region spanning two putative actin-binding motifs. We found that STIP1 could stimulate the in vitro ATPase activity of actin, suggesting a potential role in the modulation of F-actin formation. Interestingly, while STIP1 depletion in HEK293T cells had no major effect on total actin levels, it led to increased nuclear accumulation of actin, disorganization of F-actin structures, and an increase and decrease in cofilin and profilin levels, respectively. This study suggests that STIP1 regulates the cytoskeleton by interacting with actin, or via regulating the ratio of proteins known to affect actin dynamics.

Cloning and Characterization of Mouse ACF7, a Novel Member of the Dystonin Subfamily of Actin Binding Proteins

Genomics ◽  
1996 ◽  
Vol 38 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Gilbert Bernier ◽  
Martine Mathieu ◽  
Yves De Repentigny ◽  
Silvia M. Vidal ◽  
Rashmi Kothary
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

scholarly journals A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

1990 ◽  
Vol 111 (4) ◽  
pp. 1477-1489 ◽  
Author(s):  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A A Noegel ◽  
J E Segall ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Mutant Cell ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Binding Proteins ◽  
Cell Extracts ◽  
Nonmuscle Cells

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

scholarly journals Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells

1997 ◽  
Vol 327 (3) ◽  
pp. 909-915 ◽  
Author(s):  
C. Bryan GIBBON ◽  
Haiyun REN ◽  
J. Christopher STAIGER
Keyword(s):  
Binding Proteins ◽  
Dissociation Constants ◽  
Critical Concentration ◽  
Tryptophan Fluorescence ◽  
Actin Binding ◽  
Live Cells ◽  
Post Translational Modification ◽  
Actin Binding Proteins ◽  
Maize Pollen

Profilin is a small, 12-15 kDa, actin-binding protein that interacts with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd = 1.0-1.5 μM). The ability of maize profilin isoforms to bind poly-L-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.

Characterization of actin-binding proteins in slow axonal transport

1991 ◽  
Vol 16 ◽  
pp. 48
Author(s):  
Kiyoshi Tanaka ◽  
Tomoko Tashiro ◽  
Sumito Sekimoto ◽  
Yoshiaki Komiya
Keyword(s):  
Axonal Transport ◽  
Binding Proteins ◽  
Actin Binding ◽  
Slow Axonal Transport ◽  
Actin Binding Proteins

scholarly journals Novel stromal biomarker screening in pancreatic cancer patients using the in vitro cancer-stromal interaction model

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Yasunori Nishida ◽  
Akiko Kawano Nagatsuma ◽  
Motohiro Kojima ◽  
Naoto Gotohda ◽  
Atsushi Ochiai
Keyword(s):  
Pancreatic Cancer ◽  
Prognostic Marker ◽  
Microarray Data ◽  
Binding Proteins ◽  
Interaction Model ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Stromal Fibroblasts ◽  
Data Set

Abstract Background Stromal fibroblasts associated with pancreatic ductal adenocarcinoma (PDAC) play an important role in tumor progression through interactions with cancer cells. Our proposed combination strategies of in vitro and in silico biomarker screening through a cancer-stromal interaction model were previously identified several actin-binding proteins in human colon cancer stroma. The main aim of the present study was to identify novel prognostic markers in human PDAC stroma using our strategies. Methods Five primary cultivated fibroblasts from human pancreas were stimulated by two types of pancreatic cancer-cell-conditioned medium (Capan-1 and MIA PaCa-2) followed by gene expression analysis to identify up-regulated genes. Publicly available microarray data set concomitant with overall survival was collected and prognostic marker candidates were selected among the genes that were found to be up-regulated. The mRNA expression levels of the selected genes were evaluated in 5 human fresh PDAC tissues. Finally, survival analysis was performed based on immunohistochemical results on tissue microarrays consisting of 216 surgically resected PDAC tissues. Results The microarray data of the cancer-stromal interaction model revealed that 188 probes were significantly regulated in pancreatic fibroblasts. Further, six genes were selected using publicly available microarray data set, and a single Diaphanous-related formin-3 (DIAPH3), actin-binding protein, was identified as a stromal biomarker in PDAC fibroblasts by RNA validation analysis. DIAPH3 exhibited strong immunohistochemical expression in stromal fibroblasts. The high stromal expression of DIAPH3 was associated with shorter survival times of PDAC patients. Conclusions DIAPH3 was identified as a prognostic marker in PDAC fibroblasts using our biomarker screening strategies through the cancer-stromal interaction model, indicating that stromal actin-binding proteins might have an important biological role in cancer progression. These strategies were also available in PDAC, and can be used for stromal biomarker screening in various cancers.

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