scholarly journals The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P(2) influences cytoskeletal protein activity at the plasma membrane

2000 ◽  
Vol 113 (21) ◽  
pp. 3685-3695 ◽  
Author(s):  
A.S. Sechi ◽  
J. Wehland
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Adhesion Molecules ◽  
Focal Adhesion ◽  
Small Gtpases ◽  
Cytoskeletal Protein ◽  
Protein Activity ◽  
Associated Proteins ◽  
Tight Connection

The co-ordination of rearrangements of the actin cytoskeleton depends on its tight connection to the plasma membrane. Phosphatidylinositol 4,5-bisphosphate is thought to transmit signals originating at the plasma membrane to the underlying actin cytoskeleton. This lipid binds to, and influences the activity of, several actin-associated proteins in vitro that regulate the architecture of the actin cytoskeleton. Signalling intermediates in this process include focal adhesion molecules such as vinculin and members of two families of proteins, ERM and WASP. These proteins interact with phosphatidylinositol 4,5-bisphosphate and appear to be regulated by interplay between small GTPases and phosphatidylinositol 4,5-bisphosphate metabolism, and thus link the plasma membrane with cytoskeletal remodelling.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

scholarly journals Extracellular MIF, but not its homologue D-DT, promotes fibroblast motility independent of its receptor CD74/CD44

2020 ◽  
pp. jcs.217356
Author(s):  
Paweł Szczęśniak ◽  
Tamara Henke ◽  
Suada Fröhlich ◽  
Uwe Plessmann ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Biological Activities ◽  
Wide Spectrum ◽  
Oxidoreductase Activity ◽  
Dopachrome Tautomerase ◽  
Associated Proteins ◽  
Signalling Transduction ◽  
Thiol Protein

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are ubiquitous, pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities like migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. While the CD74/CD44 receptor complex is essential for signalling transduction in fibroblasts by extracellular MIF/D-DT, our interactome data rather suggested direct effects. We thus investigated whether MIF/D-DT can modulate cell migration independent of CD74/CD44. To differentiate between receptor- and non-receptor-mediated motility, we treated fibroblasts that are deficient in CD74 and CD44 or that express both proteins with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, likely, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro our findings establish a new intracellular role for MIF/D-DT in driving cell motility by modulating the actin cytoskeleton.

scholarly journals ARHGAP25, a novel Rac GTPase-activating protein, regulates phagocytosis in human neutrophilic granulocytes

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 573-582 ◽  
Author(s):  
Roland Csépányi-Kömi ◽  
Gábor Sirokmány ◽  
Miklós Geiszt ◽  
Erzsébet Ligeti
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpases ◽  
Active Form ◽  
Cell Types ◽  
Small Gtpases ◽  
Negative Regulator ◽  
Phagocytic Cells ◽  
Rac Gtpase

Members of the Rac/Rho family of small GTPases play an essential role in phagocytic cells in organization of the actin cytoskeleton and production of toxic oxygen compounds. GTPase-activating proteins (GAPs) decrease the amount of the GTP-bound active form of small GTPases, and contribute to the control of biologic signals. The number of potential Rac/RhoGAPs largely exceeds the number of Rac/Rho GTPases and the expression profile, and their specific role in different cell types is largely unknown. In this study, we report for the first time the properties of full-length ARHGAP25 protein, and show that it is specifically expressed in hematopoietic cells, and acts as a RacGAP both in vitro and in vivo. By silencing and overexpressing the protein in neutrophil model cell lines (PLB-985 and CosPhoxFcγR, respectively) and in primary macrophages, we demonstrate that ARHGAP25 is a negative regulator of phagocytosis acting probably via modulation of the actin cytoskeleton.

Change in distribution of cytoskeleton-associated proteins, lasp-1 and palladin, during uterine receptivity in the rat endometrium

2018 ◽  
Vol 30 (11) ◽  
pp. 1482 ◽  
Author(s):  
Leigh Nicholson ◽  
Laura Lindsay ◽  
Christopher R. Murphy
Keyword(s):  
Plasma Membrane ◽  
Focal Adhesion ◽  
Early Pregnancy ◽  
Morphological Changes ◽  
Cytoskeletal Proteins ◽  
Initial Contact ◽  
Luminal Epithelium ◽  
Uterine Receptivity ◽  
Uterine Lumen ◽  
Associated Proteins

The epithelium of the uterine lumen is the first point of contact with the blastocyst before implantation. To facilitate pregnancy, these uterine epithelial cells (UECs) undergo morphological changes specific to the receptive uterus. These changes include basal, lateral and apical alterations in the plasma membrane of UECs. This study looked at the cytoskeletal and focal adhesion-associated proteins, lasp-1 and palladin, in the uterus during early pregnancy in the rat. Two palladin isoforms, 140 kDa and 90 kDa, were analysed, with the migration-associated 140-kDa isoform increasing significantly at the time of implantation when compared with the time of fertilisation. Lasp-1 was similarly increased at this time, whilst also being located predominantly apically and laterally in the UECs, suggesting a role in the initial contact between the UECs and the blastocyst. This is the first study to investigate palladin and lasp-1 in the uterine luminal epithelium and suggests an importance for these cytoskeletal proteins in the morphological changes the UECs undergo for pregnancy to occur.

scholarly journals The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL)

2001 ◽  
Vol 154 (1) ◽  
pp. 161-176 ◽  
Author(s):  
Kip A. West ◽  
Huaye Zhang ◽  
Michael C. Brown ◽  
Sotiris N. Nikolopoulos ◽  
M.C. Riedy ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Deletion Mutant ◽  
Morphological Changes ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Random Motility ◽  
Phenotypic Changes ◽  
Rho Family ◽  
Mutant Cells

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

scholarly journals TBC1D10C is a cytoskeletal functional linker that modulates cell spreading and phagocytosis in macrophages

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fabian R. Villagomez ◽  
Juan D. Diaz-Valencia ◽  
Erasmo Ovalle-García ◽  
Armando Antillón ◽  
Iván Ortega-Blake ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ex Vivo ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Burkholderia Cenocepacia ◽  
In Vitro Assays ◽  
Inhibitory Protein ◽  
Intrinsically Disordered ◽  
Biological Potential

AbstractCell spreading and phagocytosis are notably regulated by small GTPases and GAP proteins. TBC1D10C is a dual inhibitory protein with GAP activity. In immune cells, TBC1D10C is one of the elements regulating lymphocyte activation. However, its specific role in macrophages remains unknown. Here, we show that TBC1D10C engages in functions dependent on the cytoskeleton and plasma membrane reorganization. Using ex vivo and in vitro assays, we found that elimination and overexpression of TBC1D10C modified the cytoskeletal architecture of macrophages by decreasing and increasing the spreading ability of these cells, respectively. In addition, TBC1D10C overexpression contributed to higher phagocytic activity against Burkholderia cenocepacia and to increased cell membrane tension. Furthermore, by performing in vitro and in silico analyses, we identified 27 TBC1D10C-interacting proteins, some of which were functionally classified as protein complexes involved in cytoskeletal dynamics. Interestingly, we identified one unreported TBC1D10C-intrinsically disordered region (IDR) with biological potential at the cytoskeleton level. Our results demonstrate that TBC1D10C shapes macrophage activity by inducing reorganization of the cytoskeleton-plasma membrane in cell spreading and phagocytosis. We anticipate our results will be the basis for further studies focused on TBC1D10C. For example, the specific molecular mechanism in Burkholderia cenocepacia phagocytosis and functional analysis of TBC1D10C-IDR are needed to further understand its role in health and disease.

scholarly journals The systemin signaling cascade as derived from phosphorylation time courses under stimulation by systemin and its inactive Thr17Ala (A17) analog

2019 ◽  
Author(s):  
Fatima Haj Ahmad ◽  
Xuna Wu ◽  
Annick Stintzi ◽  
Andreas Schaller ◽  
Waltraud X Schulze
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Map Kinases ◽  
Small Gtpases ◽  
Wound Response ◽  
Signaling Cascade ◽  
Transcriptional Responses ◽  
Data Set ◽  
Time Courses

AbstractSystemin is a small peptide with important functions in plant wound response signaling. Although transcriptional responses of systemin action are well described, the precise signaling cascades involved in its perception and signal transduction are poorly understood at the protein level. Here we use a phosphoproteomic profiling study involving stimulation time courses with systemin and its inactive analogon A17 to reconstruct a systemin-specific kinase/phosphatase signaling network. The time course analysis of systemin-induced phosphorylation patterns revealed early events at the plasma membrane, such dephosphorylation of H+-ATPase, rapid phosphorylation of NADPH-oxidase and Ca2+-ATPase. Later responses involved transient phosphorylation of small GTPases and vesicle trafficking proteins, as well as transcription factors. Based on a correlation analysis of systemin-specific phosphorylation profiles, we predict substrate candidates for 56 systemin specific kinases and 18 phosphatases. Among the kinases are several systemin-specific receptor kinases as well as kinases with downstream signaling functions, such as MAP-kinases. A regulatory circuit for plasma membrane H+-ATPase was predicted and confirmed by in-vitro activity assays. In this regulatory model we propose that upon systemin treatment, H+-ATPase LHA1 is rapidly de-phosphorylated at its C-terminal regulatory residue T955 by phosphatase PLL5, resulting in the alkalization of the growth medium within 2 minutes of systemin treatment. We further propose that the H+-ATPase LHA1 is re-activated by MAP-Kinase MPK2 later in the systemin response. MPK2 was identified with increased phosphorylation at its activating TEY-motif at 15 minutes of treatment and the predicted interaction with LHA1 was confirmed by in-vitro kinase assays. Our data set provides a valuable resource of proteomic events involved in the systemin signaling cascade with a focus on predictions of substrates to systemin-specific kinases and phosphatases.

The focal adhesions of chick retinal pigmented epithelial cells

1985 ◽  
Vol 63 (6) ◽  
pp. 553-563 ◽  
Author(s):  
Michał Opas
Keyword(s):  
Plasma Membrane ◽  
Incident Light ◽  
Focal Adhesions ◽  
Rpe Cells ◽  
Surface Reflection ◽  
Immersion Medium ◽  
Microfilament Bundles ◽  
Associated Proteins ◽  
Definition Of

Retinal pigmented epithelial (RPE) cells obtained from the eyes of chick embryos form colonies in vitro in which cells at the periphery of the colony express an undifferentiated, well-spread morphology and develop extremely large areas of cell–substratum adhesion. These adhesions can be classified as focal on the basis of the following: (a) their black surface reflection interference image, the contrast of which is not affected by changes in either the wavelength of the incident light or the refractive index of the immersion medium; (b) their association with the termini of actin-containing microfilament bundles; and (c) their ability to be labelled with antiserum against vinculin, a protein specific for adhesions of the focal type. The focal adhesions of RPE cells comprise laterally associated individual focal contacts, the mechanism by which this association is achieved and maintained is yet unknown. Because of the unusually large size and excellent microscopical definition of their focal adhesions, I used RPE cells to investigate the role of other actin-associated proteins in adhesion complexes. One of these, nonerythroid spectrin (fodrin), a protein suggested to play a role in anchoring actin filaments to the plasma membrane, was neither concentrated in nor excluded from the focal adhesions of RPE cells. Thus, at least in this cell type, spectrin seems unlikely to serve as a link between the major actin-containing microfilament bundles and the plasma membrane in the regions of cell-to-substratum contacts.

The effect of cytoskeletal protein mutations on cell motility and morphogenesis

1992 ◽  
Vol 50 (1) ◽  
pp. 594-595
Author(s):  
David A. Knecht
Keyword(s):  
Cell Motility ◽  
Actin Filaments ◽  
Cytoskeletal Proteins ◽  
Cross Linking ◽  
Cytoskeletal Protein ◽  
Homologous Gene ◽  
Complex Interactions ◽  
Associated Proteins ◽  
Protein Mutations

The cortical cytoskeleton of eukaryotic cells is composed of actin filaments and a variety of associated proteins. The polymerization, depolymerization, cross-linking and bundling of these filaments, are presumed to be intimately involved in such processes as cell motility, cell adhesion and cell shape. In developing systems, all of these processes are involved in the morphogenetic mechanisms that shape tissues, organs and organisms.We are investigating the complex interactions among cytoskeletal proteins using the simple eukaryotic amoebae, Dictyostelium discoideum. Our approach is to determine the function of the components of the cytoskeleton by creating mutants lacking particular proteins, or containing specific alterations in these proteins. Mutants lacking myosin heavy chain have been created using antisense RNA and homologous gene targetting. These cells have alterations in their shape and movement, and are incapable of accomplishing normal morphogenesis. Another cytoskeletal protein is ABP-120, which is capable of cross-linking actin filaments into orthogonal arrays, leading to the formation of an actin gel in vitro. ABP-120 is found in newly formed pseudopods extended during the chemotactic respose to extracellular cAMP. Mutants lacking this protein have been created by disruption of the chromosomal gene with a transformation vector. These cells are not as dramatically affected as the myosin mutants, but have clear alterations in their motility and in the pathway of responses in the cytoskeleton that correlate with the expected function of this protein. Mutations in several other cytoskeletal genes are currently being constructed.

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

1993 ◽  
Vol 105 (3) ◽  
pp. 765-775 ◽  
Author(s):  
E. Friederich ◽  
T.E. Kreis ◽  
D. Louvard
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Site ◽  
Actin Filaments ◽  
Living Cells ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Fast Growing ◽  
Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

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