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.

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

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.

scholarly journals Mapping the binding site of thymosin β4 on actin by competition with G-actin binding proteins indicates negative co-operativity between binding sites located on opposite subdomains of actin

1997 ◽  
Vol 327 (3) ◽  
pp. 787-793 ◽  
Author(s):  
Edda BALLWEBER ◽  
Ewald HANNAPPEL ◽  
Thomas HUFF ◽  
Hans Georg MANNHERZ
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Ternary Complex ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Critical Concentration ◽  
Dnase I ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Actin Binding Proteins

The β-thymosins are small monomeric (G-)actin-binding proteins of 5 kDa that are supposed to act intracellularly as actin-sequestering factors stabilizing the cytoplasmic monomeric pool of actin. The binding region of thymosin β4 was determined by analysing the binding of thymosin β4 to actin complexed with DNase I, gelsolin or gelsolin segment 1. Binding was analysed by determining the increase in the critical concentration of actin polymerization by native gel electrophoresis or chemical cross-linking. The formation of a ternary complex including thymosin β4 should indicate that the actin-binding proteins attach to different sites on actin. Competition would be indicative of binding to identical or overlapping sites on actin or of a negative co-operative linkage between the two binding sites. Competition of thymosin β4 for actin binding was observed in the presence of intact gelsolin or the N-terminal gelsolin fragment, segment 1, indicating that thymosin β4 binds to a site close to or identical with the gelsolin segment 1-binding site. The ternary complex of actin-DNase I-thymosin β4 was obtained only when using the chemically cross-linked actin-thymosin β4 complex, indicating that thymosin β4 is dissociated by the binding of DNase I to actin. It is suggested that the dissociation of thymosin β4 by DNase I binding to actin is caused by negative co-operativity between their spatially separated binding sites on actin. A similar negative co-operativity was observed between DNase I and gelsolin segment 1 binding to actin. The results therefore indicate that the respective binding sites for DNase I and segment 1 on subdomains 1 and 2 of actin are linked in a negative co-operative manner.

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 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.

scholarly journals Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

2007 ◽  
Vol 18 (8) ◽  
pp. 3002-3014 ◽  
Author(s):  
Faisal Chaudhry ◽  
Christophe Guérin ◽  
Matthias von Witsch ◽  
Laurent Blanchoin ◽  
Christopher J. Staiger
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filament ◽  
Binding Proteins ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Nucleotide Exchange ◽  
Suspension Cells ◽  
Actin Binding Proteins ◽  
Cell Shapes

The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP–actin.

scholarly journals Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells

1994 ◽  
Vol 124 (6) ◽  
pp. 971-983 ◽  
Author(s):  
KA Giuliano ◽  
DL Taylor
Keyword(s):  
Binding Proteins ◽  
Imaging Techniques ◽  
Leading Edge ◽  
Living Cells ◽  
Actin Binding ◽  
Actin Assembly ◽  
Membrane Interface ◽  
Actin Binding Proteins ◽  
High Affinity

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non-cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum-deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.

scholarly journals Actin-Binding Proteins from Burkholderia mallei and Burkholderia thailandensis Can Functionally Compensate for the Actin-Based Motility Defect of a Burkholderia pseudomallei bimA Mutant

2005 ◽  
Vol 187 (22) ◽  
pp. 7857-7862 ◽  
Author(s):  
Joanne M. Stevens ◽  
Ricky L. Ulrich ◽  
Lowrie A. Taylor ◽  
Michael W. Wood ◽  
David DeShazer ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Binding Proteins ◽  
Actin Binding ◽  
Burkholderia Mallei ◽  
Terminal Sequence ◽  
Actin Binding Proteins ◽  
Burkholderia Thailandensis ◽  
Amino Terminal ◽  
Amino Terminal Sequence

ABSTRACT Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.

scholarly journals N-terminal modification of actin by acetylation and arginylation determines the architecture and assembly rate of linear and branched actin networks

2020 ◽  
Author(s):  
Samantha M. Chin ◽  
Tomoyuki Hatano ◽  
Lavanya Sivashanmugam ◽  
Andrejus Suchenko ◽  
Anna S. Kashina ◽  
...  
Keyword(s):  
Cell Migration ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Network ◽  
Post Translational Modification ◽  
Actin Binding Proteins ◽  
N Terminus

AbstractThe great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, recent work implicates post-translational modification of the actin N-terminus by either acetylation or arginylation itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing, and modification of the N-terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in Cofilin-mediated severing. Taken together, these results suggest that cells can employ the differently modified actins to precisely regulate actin dynamics. In addition, unprocessed, or non-acetylated actin show very different effects on formin-mediated-elongation, Arp2/3-mediated nucleation, and severing by Cofilin. Altogether, this study shows that the nature of the N-terminus of actin can induce distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge.

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