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Biophysical Journal Jan 2018Moesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological...
Moesin, a protein of the ezrin, radixin, and moesin family, which links the plasma membrane to the cytoskeleton, is involved in multiple physiological and pathological processes, including viral budding and infection. Its interaction with the plasma membrane occurs via a key phosphoinositide, the phosphatidyl(4,5)inositol-bisphosphate (PIP), and phosphorylation of residue T558, which has been shown to contribute, in cellulo, to a conformationally open protein. We study the impact of a double phosphomimetic mutation of moesin (T235D, T558D), which mimics the phosphorylation state of the protein, on protein/PIP/microtubule interactions. Analytical ultracentrifugation in the micromolar range showed moesin in the monomer and dimer forms, with wild-type (WT) moesin containing a slightly larger fraction (∼30%) of dimers than DD moesin (10-20%). Only DD moesin was responsive to PIP in its micellar form. Quantitative cosedimentation assays using large unilamellar vesicles and quartz crystal microbalance on supported lipid bilayers containing PIP reveal a specific cooperative interaction for DD moesin with an ability to bind two PIP molecules simultaneously, whereas WT moesin was able to bind only one. In addition, DD moesin could subsequently interact with microtubules, whereas WT moesin was unable to do so. Altogether, our results point to an important role of these two phosphorylation sites in the opening of moesin: since DD moesin is intrinsically in a more open conformation than WT moesin, this intermolecular interaction is reinforced by its binding to PIP. We also highlight important differences between moesin and ezrin, which appear to be finely regulated and to exhibit distinct molecular behaviors.
Topics: Membranes, Artificial; Microfilament Proteins; Microtubules; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Protein Binding; Protein Multimerization
PubMed: 29320700
DOI: 10.1016/j.bpj.2017.10.041 -
Scientific Reports May 2024The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which...
The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell.
Topics: Microtubules; Dictyostelium; Mitosis; Microfilament Proteins; Actin-Related Protein 2-3 Complex; Protozoan Proteins; Protein Transport; Cytokinesis; Actins
PubMed: 38755233
DOI: 10.1038/s41598-024-61967-7 -
Molecular Biology of the Cell Aug 1998
Review
Topics: Actin Depolymerizing Factors; Actins; Amino Acid Sequence; Animals; Conserved Sequence; Evolution, Molecular; Fungi; Humans; Microfilament Proteins; Models, Molecular; Molecular Sequence Data; Neuropeptides; Phylogeny; Protein Structure, Secondary; Protein-Tyrosine Kinases; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Vertebrates
PubMed: 9693358
DOI: 10.1091/mbc.9.8.1951 -
The Journal of Biological Chemistry Jul 2005Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most...
Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop.
Topics: Actins; Amino Acid Sequence; Animals; Binding Sites; Cell Proliferation; Contractile Proteins; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Fungal; Immunoblotting; Immunoprecipitation; Microfilament Proteins; Molecular Sequence Data; Phosphorylation; Profilins; Protein Binding; Protein Structure, Tertiary; Rabbits; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Time Factors; Two-Hybrid System Techniques; Up-Regulation
PubMed: 15923184
DOI: 10.1074/jbc.M503094200 -
Molecular Biology of the Cell Jan 2016Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which...
Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility.
Topics: Actin Capping Proteins; Actin Cytoskeleton; Cell Movement; Cytoskeleton; Fetal Proteins; Formins; Kinetics; Microfilament Proteins; Models, Molecular; Nuclear Proteins; Profilins; Protein Binding
PubMed: 26715420
DOI: 10.1091/mbc.E15-06-0352 -
Proceedings. Biological Sciences Nov 1997Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites...
Cell motility is produced by changes in the dynamics and organization of actin filaments. The aim of the experiments described here was to test whether growing neurites contain two actin-binding proteins, gelsolin and profilin, that regulate polymerization of actin and affect non-neuronal cell motility. The distribution of gelsolin, profilin and the microfilaments was compared by immunocytochemistry of leech neurons growing in culture. We observed that microfilaments are enriched in the peripheral motile areas of the neurites. Both gelsolin and profilin are also concentrated in these regions. Gelsolin is abundant in filopodia and is associated with single identifiable microfilament bundles in lamellipodia. Profilin is not prominent in filopodia and shows a diffuse staining pattern in lamellipodia. The colocalization of gelsolin and profilin in motile, microfilament-rich areas supports the hypothesis that they synergistically regulate the actin dynamics that underlie neurite growth.
Topics: Actin Cytoskeleton; Animals; Cell Movement; Cells, Cultured; Contractile Proteins; Gelsolin; Leeches; Microfilament Proteins; Neurites; Profilins
PubMed: 9404032
DOI: 10.1098/rspb.1997.0236 -
The Journal of Cell Biology Feb 1986Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory...
Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.
Topics: Actin Cytoskeleton; Actins; Adrenal Medulla; Animals; Calcium-Binding Proteins; Carrier Proteins; Cattle; Egtazic Acid; Gelsolin; Microfilament Proteins; Molecular Weight; Solubility; Swine; Viscosity
PubMed: 3003118
DOI: 10.1083/jcb.102.2.636 -
BMC Cell Biology Nov 2007The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex...
BACKGROUND
The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1-/- mice.
RESULTS
The present results demonstrate that HS1 is not required for platelet activation, shape change or aggregation. Platelets from HS1-/- mice spread normally on a variety of adhesion proteins and have normal F-actin and Arp2/3 complex distributions. Clot retraction, an actin-dependent process, is also normal in these mice. Platelet aggregation and secretion is indistinguishable between knock out and littermates and there is no increase in bleeding using the tail bleeding assay.
CONCLUSION
This study concludes that HS1 does not play a major role in platelet function. It is possible that a role for HS1 is masked by the presence of cortactin.
Topics: Actins; Animals; Blood Platelets; Cell Shape; Genotype; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Platelet Activation; Thrombosis
PubMed: 17996076
DOI: 10.1186/1471-2121-8-46 -
Current Biology : CB Dec 2001Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when...
BACKGROUND
Assembly and organization of actin filaments are required for many cellular processes, including locomotion and division. In many cases, actin assembly is initiated when proteins of the WASP/Scar family respond to signals from Rho family G proteins and stimulate the actin-nucleating activity of the Arp2/3 complex. Two questions of fundamental importance raised in the study of actin dynamics concern the molecular mechanism of Arp2/3-dependent actin nucleation and how different signaling pathways that activate the same Arp2/3 complex produce actin networks with different three-dimensional architectures?
RESULTS
We directly compared the activity of the Arp2/3 complex in the presence of saturating concentrations of the minimal Arp2/3-activating domains of WASP, N-WASP, and Scar1 and found that each induces unique kinetics of actin assembly. In cell extracts, N-WASP induces rapid actin polymerization, while Scar1 fails to induce detectable polymerization. Using purified proteins, Scar1 induces the slowest rate of nucleation. WASP activity is 16-fold higher, and N-WASP activity is 70-fold higher. The data for all activators fit a mathematical model in which one activated Arp2/3 complex, one actin monomer, and an actin filament combine into a preactivation complex which then undergoes a first-order activation step to become a nucleus. The differences between Scar and N-WASP activity are explained by differences in the rate constants for the activation step. Changing the number of actin binding sites on a WASP family protein, either by removing a WH2 domain from N-WASP or by adding WH2 domains to Scar1, has no significant effect on nucleation activity. The addition of a three amino acid insertion found in the C-terminal acidic domains of WASP and N-WASP, however, increases the activity of Scar1 by more than 20-fold. Using chemical crosslinking assays, we determined that both N-WASP and Scar1 induce a conformational change in the Arp2/3 complex but crosslink with different efficiencies to the small molecular weight subunits p18 and p14.
CONCLUSION
The WA domains of N-WASP, WASP, and Scar1 bind actin and Arp2/3 with nearly identical affinities but stimulate rates of actin nucleation that vary by almost 100-fold. The differences in nucleation rate are caused by differences in the number of acidic amino acids at the C terminus, so each protein is tuned to produce a different rate of actin filament formation. Arp2/3, therefore, is not regulated by a simple on-off switch. Precise tuning of the filament formation rate may help determine the architecture of actin networks produced by different nucleation-promoting factors.
Topics: Actin-Related Protein 2; Actin-Related Protein 3; Actins; Animals; Biopolymers; Cytoskeletal Proteins; Humans; Microfilament Proteins; Nerve Tissue Proteins; Protein Conformation; Proteins; Wiskott-Aldrich Syndrome Protein; Wiskott-Aldrich Syndrome Protein Family; Wiskott-Aldrich Syndrome Protein, Neuronal
PubMed: 11747816
DOI: 10.1016/s0960-9822(01)00603-0 -
The Journal of Biological Chemistry Apr 2006Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with... (Comparative Study)
Comparative Study
Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.
Topics: Actin Depolymerizing Factors; Actins; Animals; Cattle; Cell Membrane; Golgi Apparatus; Microfilament Proteins; Neuropeptides; Protein Binding; Protein Structure, Tertiary; Rats; Subcellular Fractions
PubMed: 16452483
DOI: 10.1074/jbc.M510141200