scholarly journals Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides.

1996 ◽  
Vol 135 (1) ◽  
pp. 169-179 ◽  
Author(s):  
D A Schafer ◽  
P B Jennings ◽  
J A Cooper
Keyword(s):  
Protein Binding ◽  
Rate Constant ◽  
Half Life ◽  
Actin Polymerization ◽  
Beta Subunit ◽  
Actin Binding ◽  
Capping Protein ◽  
Subunit Isoforms

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

scholarly journals Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila.

1996 ◽  
Vol 133 (6) ◽  
pp. 1293-1305 ◽  
Author(s):  
R Hopmann ◽  
J A Cooper ◽  
K G Miller
Keyword(s):  
Binding Proteins ◽  
Beta Subunit ◽  
Actin Binding ◽  
Filament Length ◽  
Actin Binding Proteins ◽  
Actin Organization ◽  
Capping Protein ◽  
Actin Capping Protein

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

2007 ◽  
Vol 179 (6) ◽  
pp. 1247-1259 ◽  
Author(s):  
Jacco van Rheenen ◽  
Xiaoyan Song ◽  
Wies van Roosmalen ◽  
Michael Cammer ◽  
Xiaoming Chen ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Carcinoma Cells ◽  
Actin Binding ◽  
Temporal Regulation ◽  
Vitro Binding ◽  
Epidermal Growth ◽  
Membrane Bound ◽  
Regulation Of Actin Cytoskeleton

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

scholarly journals Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

2008 ◽  
Vol 295 (5) ◽  
pp. C1113-C1122 ◽  
Author(s):  
Anne E. Kruchten ◽  
Eugene W. Krueger ◽  
Yu Wang ◽  
Mark A. McNiven
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Actin Polymerization ◽  
Control Cell ◽  
Focal Adhesions ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

scholarly journals The Putative Pocket Protein Binding Site of Autographa californica Nucleopolyhedrovirus BV/ODV-C42 Is Required for Virus-Induced Nuclear Actin Polymerization

2010 ◽  
Vol 84 (15) ◽  
pp. 7857-7868 ◽  
Author(s):  
Kun Li ◽  
Yun Wang ◽  
Huimin Bai ◽  
Qian Wang ◽  
Jianhua Song ◽  
...  
Keyword(s):  
Protein Binding ◽  
Actin Polymerization ◽  
Autographa Californica ◽  
Polymerization Process ◽  
Nuclear Actin ◽  
Filamentous Actin ◽  
Specific Staining ◽  
Pocket Protein

ABSTRACT Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAcp78/83nls-gfp, with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAcp78/83nls-gfp-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

scholarly journals Thymosin β4 is essential for adherens junction stability and epidermal planar cell polarity

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev193425
Author(s):  
Krishnanand Padmanabhan ◽  
Hanna Grobe ◽  
Jonathan Cohen ◽  
Arad Soffer ◽  
Adnan Mahly ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Polarity ◽  
Actin Polymerization ◽  
Planar Cell Polarity ◽  
Adherens Junction ◽  
Actin Binding ◽  
Thymosin Β4 ◽  
Eyelid Closure

ABSTRACTPlanar cell polarity (PCP) is essential for tissue morphogenesis and homeostasis; however, the mechanisms that orchestrate the cell shape and packing dynamics required to establish PCP are poorly understood. Here, we identified a major role for the globular (G)-actin-binding protein thymosin-β4 (TMSB4X) in PCP establishment and cell adhesion in the developing epidermis. Depletion of Tmsb4x in mouse embryos hindered eyelid closure and hair-follicle angling owing to PCP defects. Tmsb4x depletion did not preclude epidermal cell adhesion in vivo or in vitro; however, it resulted in abnormal structural organization and stability of adherens junction (AJ) due to defects in filamentous (F)-actin and G-actin distribution. In cultured keratinocytes, TMSB4X depletion increased the perijunctional G/F-actin ratio and decreased G-actin incorporation into junctional actin networks, but it did not change the overall actin expression level or cellular F-actin content. A pharmacological treatment that increased the G/F-actin ratio and decreased actin polymerization mimicked the effects of Tmsb4x depletion on both AJs and PCP. Our results provide insights into the regulation of the actin pool and its involvement in AJ function and PCP establishment.

scholarly journals Distribution to the Brain and Protein Binding of 3′and 5-Substituted 2′,3′-Dideoxyuridine Derivatives, Studied by Microdialysis

1997 ◽  
Vol 8 (1) ◽  
pp. 47-53 ◽  
Author(s):  
N Borg ◽  
X-X Zhou ◽  
N-G Johansson ◽  
B Öberg ◽  
L Ståhle
Keyword(s):  
Protein Binding ◽  
Half Life ◽  
Partition Coefficients ◽  
Transport Mechanism ◽  
Chemical Properties ◽  
Peripheral Muscle ◽  
Pharmacokinetic Properties ◽  
The Brain

The aim of this study was to investigate a series of 3′ and 5-substituted 2′,3′-dideoxyuridine derivatives (ddUD) with respect to plasma protein binding, half-life and distribution across the blood-brain barrier in the rat. The microdialysis technique was used to study protein binding in human plasma ( in vitro), and to sample the extracellular space of rats with microdialysis probes implanted into the striatum of the brain and the gastrocnemic muscle ( in vivo). The compounds were analysed by HPLC with UV-detection. The octanol/water partition coefficients of the ddUD varied from 0.08-0.84. The protein binding of the ddUDs was approximately 80%. After s.c. administration (25 or 50 mg kg−1), the brain and muscle extracellular levels differed; brain levels were 0.18-0.36 of peripheral (muscle) concentrations. A multivariate analysis, which included data on zidovudine, alovudine and thymidine, demonstrated a relationship between the physicochemical and some of the pharmacokinetic properties of uridine analogues. The analysis shows that half-life and protein binding increases with decreasing p Ka. However, penetration to the brain is not correlated with the partition into octanol. It is concluded that the transport to the brain is not primarily dependent upon passive diffusion over a lipophilic barrier but, rather, to other chemical properties of the ddUDs. This is suggestive of a specific transport mechanism, e.g. the thymidine carrier.

scholarly journals Identification and characterization of an actin-binding site of CapZ.

1992 ◽  
Vol 116 (4) ◽  
pp. 923-931 ◽  
Author(s):  
C Hug ◽  
T M Miller ◽  
M A Torres ◽  
J F Casella ◽  
J A Cooper
Keyword(s):  
Binding Site ◽  
Primary Structure ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Terminal Region ◽  
Beta Subunit ◽  
In Vitro Translation ◽  
Actin Binding ◽  
Actin Binding Site

A mAb (1E5) that binds the COOH-terminal region of the beta subunit of chicken CapZ inhibits the ability of CapZ to bind the barbed ends of actin filaments and nucleate actin polymerization. CapZ prepared as fusion proteins in bacteria or nonfusion proteins by in vitro translation has activity similar to that of CapZ purified from muscle. Deletion of the COOH-terminus of the beta subunit of CapZ leads to a loss of CapZ's ability to bind the barbed ends of actin filaments. A peptide corresponding to the COOH-terminal region of CapZ beta, expressed as a fusion protein, binds actin monomers. The mAb 1E5 also inhibits the binding of this peptide to actin. These results suggest that the COOH-terminal region of the beta subunit of CapZ is an actin-binding site. The primary structure of this region is not similar to that of potential actin-binding sites identified in other proteins. In addition, the primary structure of this region is not conserved across species.

scholarly journals Comparative pharmacokinetics, distributions in tissue, and interactions with blood proteins of conventional and sterically stabilized liposomes containing 2',3'-dideoxyinosine.

1996 ◽  
Vol 40 (1) ◽  
pp. 225-229 ◽  
Author(s):  
P Harvie ◽  
A Désormeaux ◽  
M C Bergeron ◽  
M Tremblay ◽  
D Beauchamp ◽  
...  
Keyword(s):  
Protein Binding ◽  
Intravenous Injection ◽  
Half Life ◽  
Peak Level ◽  
Free Drug ◽  
Systemic Clearance ◽  
Blood Proteins ◽  
Sterically Stabilized Liposomes

The pharmacokinetics and distribution in tissue of 2',3'-dideoxyinosine (ddI) encapsulated in sterically stabilized liposomes have been evaluated in rats. Most of the sterically stabilized liposomes concentrated in the spleen with a peak level at 24 h after their intravenous injection. An extended half-life in plasma was observed for sterically stabilized liposomes (14.5 h) compared with that of conventional liposomes (3.9 h). The systemic clearance of ddI incorporated in sterically stabilized liposomes was 180 times lower than that of the free drug. The levels of in vitro and in vivo protein binding on both conventional and sterically stabilized liposomes were also evaluated. Results suggest that the amount of proteins associated with liposomes might not be the only factor involved in the in vivo clearance of liposomes, as this process may also be influenced by the nature of the bound blood proteins.

scholarly journals Actin filaments in yeast are unstable in the absence of capping protein or fimbrin.

1995 ◽  
Vol 131 (6) ◽  
pp. 1483-1493 ◽  
Author(s):  
T S Karpova ◽  
K Tatchell ◽  
J A Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Filament ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Capping Protein ◽  
Filament Assembly

Many actin-binding proteins affect filament assembly in vitro and localize with actin in vivo, but how their molecular actions contribute to filament assembly in vivo is not understood well. We report here that capping protein (CP) and fimbrin are both important for actin filament assembly in vivo in Saccharomyces cerevisiae, based on finding decreased actin filament assembly in CP and fimbrin mutants. We have also identified mutations in actin that enhance the CP phenotype and find that those mutants also have decreased actin filament assembly in vivo. In vitro, actin purified from some of these mutants is defective in polymerization or binding fimbrin. These findings support the conclusion that CP acts to stabilize actin filaments in vivo. This conclusion is particularly remarkable because it is the opposite of the conclusion drawn from recent studies in Dictyostelium (Hug, C., P.Y. Jay, I. Reddy, J.G. McNally, P.C. Bridgman, E.L. Elson, and J.A. Cooper. 1995. Cell. 81:591-600). In addition, we find that the unpolymerized pool of actin in yeast is very small relative to that found in higher cells, which suggests that actin filament assembly is less dynamic in yeast than higher cells.

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