Disruption of the actin cytoskeleton of mammalian cells by the capping complex actin-fragmin is inhibited by actin phosphorylation and regulated by Ca2+ ions

1998 ◽  
Vol 111 (12) ◽  
pp. 1695-1706 ◽  
Author(s):  
B. Constantin ◽  
K. Meerschaert ◽  
J. Vandekerckhove ◽  
J. Gettemans
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Morphology ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Actin Binding ◽  
General Mechanism ◽  
Resting Cells ◽  
Dependent Manner

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding protein and interferes with the growth of actin filaments in vitro by severing actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragmin dimer is phosphorylated in vivo and in vitro on the actin subunit by the actin-fragmin kinase. We have studied the properties of these capping proteins and their regulation by actin phosphorylation and Ca2+ ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection or by expression in conjunction with immunostaining and fluorescence microscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect could be blocked by phosphorylation of the actin subunit by the actin-fragmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin. This suggests that in Physarum microplasmodia, actin polymerization can be controlled in a Ca2+-dependent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytoskeleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containing buffer. The cells were able to revert to their normal phenotype which indicates that endogenous regulatory mechanisms counteracted fragmin activity, probably by uncapping fragmin from the barbed ends of filaments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2+ concentration and this provides a basis for a more general mechanism in higher organisms to regulate microfilament organization.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

scholarly journals Mechanosensing through direct binding of tensed F-actin by LIM domains

2020 ◽  
Author(s):  
Xiaoyu Sun ◽  
Donovan Y. Z. Phua ◽  
Lucas Axiotakis ◽  
Mark A. Smith ◽  
Elizabeth Blankman ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Point Mutations ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
General Mechanism ◽  
Protein Protein Interaction ◽  
Cytoplasmic Actin ◽  
Lim Domains ◽  
Lim Proteins

SummaryMechanical signals transmitted through the cytoplasmic actin cytoskeleton must be relayed to the nucleus to control gene expression. LIM domains are protein-protein interaction modules found in cytoskeletal proteins and transcriptional regulators; however, it is unclear if there is a direct link between these two functions. Here we identify three LIM protein families (zyxin, paxillin, and FHL) whose members preferentially localize to the actin cytoskeleton in mechanically-stimulated cells through their tandem LIM domains. A minimal actin-myosin reconstitution system reveals that representatives of all three families directly bind F-actin only in the presence of mechanical force. Point mutations at a site conserved in each LIM domain of these proteins selectively disrupt tensed F-actin binding in vitro and cytoskeletal localization in cells, demonstrating a common, avidity-based mechanism. Finally, we find that binding to tensed F-actin in the cytoplasm excludes the cancer-associated transcriptional co-activator FHL2 from the nucleus in stiff microenvironments. This establishes direct force-activated F-actin binding by FHL2 as a mechanosensing mechanism. Our studies suggest that force-dependent sequestration of LIM proteins on the actin cytoskeleton could be a general mechanism for controlling nuclear localization to effect mechanical signaling.

Understanding Biological Structures Through Exploring the Mechanical Response of Cell-Like Systems

2008 ◽  
Author(s):  
Ying Zhang ◽  
Philip R. LeDuc
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Living Cell ◽  
Cellular Response ◽  
Cancer Metastasis ◽  
Cell Structure ◽  
Polymer Physics ◽  
Wide Range

The actin cytoskeleton provides mechanical support for the cell and influences activities such as cancer metastasis and chemotaxis. While their mechanical responses have been studied in vivo and in vitro, understanding the link between these two forms remains challenging. To explore this gap and further understand cell structure, we reconstructed the cell cytoskeleton in a membrane-like spherical liposome to mimic the cellular environment; this enables an artificial “cell like” system. Through this approach, we are pursuing a path to compare in vitro mechanics from a polymer physics perspective of individual actin filaments with the in vivo mechanics of a living cell [1]. A living cell contains many organelles, which are in a highly packed environment and require significant organization to function. The actin cytoskeleton provides both structural and organizational regulation that is essential for cellular response. Here, we first encapsulated G-actin into giant unilamellar vesicles through an electroformation technique and then polymerized them into actin filaments (F-actin) within individual vesicles. To probe their conformation, we visualized these vesicles with fluorescence and laser scanning confocal microscopy. We then used a tapping mode atomic force microscopy to determine the mechanical properties of these cell-like systems. These results provide insight into a wide range of fields and studies including polymer physics, cell biology, and biotechnology.

scholarly journals Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

2020 ◽  
Vol 117 (41) ◽  
pp. 25532-25542 ◽  
Author(s):  
Jonathan D. Winkelman ◽  
Caitlin A. Anderson ◽  
Cristian Suarez ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Mammalian Cells ◽  
Strain Sensing ◽  
Lim Domain ◽  
Lim Domains ◽  
Mechanical Homeostasis ◽  
Functionally Diverse

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

scholarly journals SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

scholarly journals The toxofilin–actin–PP2C complex of Toxoplasma: identification of interacting domains

2007 ◽  
Vol 401 (3) ◽  
pp. 711-719 ◽  
Author(s):  
Gaelle Jan ◽  
Violaine Delorme ◽  
Violaine David ◽  
Celine Revenu ◽  
Angelita Rebollo ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Actin Filaments ◽  
Function Analysis ◽  
Protozoan Parasite ◽  
Actin Binding ◽  
Protein Protein Interactions ◽  
The Third ◽  
Parasite Motility

Toxofilin is a 27 kDa protein isolated from the human protozoan parasite Toxoplasma gondii, which causes toxoplasmosis. Toxofilin binds to G-actin, and in vitro studies have shown that it controls elongation of actin filaments by sequestering actin monomers. Toxofilin affinity for G-actin is controlled by the phosphorylation status of its Ser53, which depends on the activities of a casein kinase II and a type 2C serine/threonine phosphatase (PP2C). To get insights into the functional properties of toxofilin, we undertook a structure–function analysis of the protein using a combination of biochemical techniques. We identified a domain that was sufficient to sequester G-actin and that contains three peptide sequences selectively binding to G-actin. Two of these sequences are similar to sequences present in several G- and F-actin-binding proteins, while the third appears to be specific to toxofilin. Additionally, we identified two toxofilin domains that interact with PP2C, one of which contains the Ser53 substrate. In addition to characterizing the interacting domains of toxofilin with its partners, the present study also provides information on an in vivo-based approach to selectively and competitively disrupt the protein–protein interactions that are important to parasite motility.

scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

scholarly journals Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

2012 ◽  
Vol 109 (38) ◽  
pp. E2533-E2542 ◽  
Author(s):  
Michal Skruzny ◽  
Thorsten Brach ◽  
Rodolfo Ciuffa ◽  
Sofia Rybina ◽  
Malte Wachsmuth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Lipid Binding ◽  
Actin Binding ◽  
Dependent Manner ◽  
Vesicle Budding ◽  
Cooperative Action ◽  
Binding Domains ◽  
Membrane Invagination

Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane–actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2’s ANTH and Ent1’s ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.

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