scholarly journals Class II formin targeting to the cell cortex by binding PI(3,5)P2 is essential for polarized growth

2012 ◽  
Vol 198 (2) ◽  
pp. 235-250 ◽  
Author(s):  
Peter A.C. van Gisbergen ◽  
Ming Li ◽  
Shu-Zon Wu ◽  
Magdalena Bezanilla
Keyword(s):  
Cell Growth ◽  
Actin Filaments ◽  
De Novo ◽  
Class Ii ◽  
Lipid Binding ◽  
Cell Cortex ◽  
Polarized Growth ◽  
Cortical Actin ◽  
Cortical Localization

Class II formins are key regulators of actin and are essential for polarized plant cell growth. Here, we show that the class II formin N-terminal phosphatase and tensin (PTEN) domain binds phosphoinositide-3,5-bisphosphate (PI(3,5)P2). Replacing the PTEN domain with polypeptides of known lipid-binding specificity, we show that PI(3,5)P2 binding was required for formin-mediated polarized growth. Via PTEN, formin also localized to the cell apex, phragmoplast, and to the cell cortex as dynamic cortical spots. We show that the cortical localization driven by binding to PI(3,5)P2 was required for function. Silencing the kinases that produce PI(3,5)P2 reduced cortical targeting of formin and inhibited polarized growth. We show a subset of cortical formin spots moved in actin-dependent linear trajectories. We observed that the linearly moving subpopulation of cortical formin generated new actin filaments de novo and along preexisting filaments, providing evidence for formin-mediated actin bundling in vivo. Taken together, our data directly link PI(3,5)P2 to generation and remodeling of the cortical actin array.

scholarly journals Mechanism of the formation of contractile ring in dividing cultured animal cells. II. Cortical movement of microinjected actin filaments.

1990 ◽  
Vol 111 (5) ◽  
pp. 1905-1911 ◽  
Author(s):  
L G Cao ◽  
Y L Wang
Keyword(s):  
Actin Filaments ◽  
Average Rate ◽  
Rat Kidney ◽  
Equatorial Region ◽  
Cell Cortex ◽  
Polar Regions ◽  
Cortical Actin ◽  
Animal Cells ◽  
Contractile Ring ◽  
Directional Movement

The contractile ring in dividing animal cells is formed primarily through the reorganization of existing actin filaments (Cao, L.-G., and Y.-L. Wang. 1990. J. Cell Biol. 110:1089-1096), but it is not clear whether the process involves a random recruitment of diffusible actin filaments from the cytoplasm, or a directional movement of cortically associated filaments toward the equator. We have studied this question by observing the distribution of actin filaments that have been labeled with fluorescent phalloidin and microinjected into dividing normal rat kidney (NRK) cells. The labeled filaments are present primarily in the cytoplasm during prometaphase and early metaphase, but become associated extensively with the cell cortex 10-15 min before the onset of anaphase. This process is manifested both as an increase in cortical fluorescence intensity and as movements of discrete aggregates of actin filaments toward the cortex. The concentration of actin fluorescence in the equatorial region, accompanied by a decrease of fluorescence in polar regions, is detected 2-3 min after the onset of anaphase. By directly tracing the distribution of aggregates of labeled actin filaments, we are able to detect, during anaphase and telophase, movements of cortical actin filaments toward the equator at an average rate of 1.0 micron/min. Our results, combined with previous observations, suggest that the organization of actin filaments during cytokinesis probably involves an association of cytoplasmic filaments with the cortex, a movement of cortical filaments toward the cleavage furrow, and a dissociation of filaments from the equatorial cortex.

scholarly journals Filopodia formation and endosome clustering induced by mutant plus-end–directed myosin VI

2017 ◽  
Vol 114 (7) ◽  
pp. 1595-1600 ◽  
Author(s):  
Thomas A. Masters ◽  
Folma Buss
Keyword(s):  
Actin Filaments ◽  
Leucine Zipper ◽  
Adaptor Protein ◽  
Cell Cortex ◽  
Myosin Vi ◽  
Actin Network ◽  
Ph Domain ◽  
Cortical Actin ◽  
Directed Movement ◽  
Interacting Protein

Myosin VI (MYO6) is the only myosin known to move toward the minus end of actin filaments. It has roles in numerous cellular processes, including maintenance of stereocilia structure, endocytosis, and autophagosome maturation. However, the functional necessity of minus-end–directed movement along actin is unclear as the underlying architecture of the local actin network is often unknown. To address this question, we engineered a mutant of MYO6, MYO6+, which undergoes plus-end–directed movement while retaining physiological cargo interactions in the tail. Expression of this mutant motor in HeLa cells led to a dramatic reorganization of cortical actin filaments and the formation of actin-rich filopodia. MYO6 is present on peripheral adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) signaling endosomes and MYO6+ expression causes a dramatic relocalization and clustering of this endocytic compartment in the cell cortex. MYO6+ and its adaptor GAIP interacting protein, C terminus (GIPC) accumulate at the tips of these filopodia, while APPL1 endosomes accumulate at the base. A combination of MYO6+ mutagenesis and siRNA-mediated depletion of MYO6 binding partners demonstrates that motor activity and binding to endosomal membranes mediated by GIPC and PI(4,5)P2 are crucial for filopodia formation. A similar reorganization of actin is induced by a constitutive dimer of MYO6+, indicating that multimerization of MYO6 on endosomes through binding to GIPC is required for this cellular activity and regulation of actin network structure. This unique engineered MYO6+ offers insights into both filopodia formation and MYO6 motor function at endosomes and at the plasma membrane.

scholarly journals EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

1997 ◽  
Vol 17 (8) ◽  
pp. 4294-4304 ◽  
Author(s):  
H Y Tang ◽  
A Munn ◽  
M Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Growth Factor Receptor ◽  
Cell Cortex ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cortical Actin ◽  
Kinase Substrate ◽  
Physiological Relevance ◽  
Eh Domain

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

scholarly journals Interaction of Sla2p's ANTH Domain with PtdIns(4,5)P2 Is Important for Actin-dependent Endocytic Internalization

2005 ◽  
Vol 16 (2) ◽  
pp. 717-730 ◽  
Author(s):  
Yidi Sun ◽  
Marko Kaksonen ◽  
David T. Madden ◽  
Randy Schekman ◽  
David G. Drubin
Keyword(s):  
Cell Growth ◽  
Fluorescence Microscopy ◽  
Particle Tracking ◽  
Binding Assay ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Organization ◽  
Lysine Residues ◽  
Endocytic Machinery

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

scholarly journals Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

1993 ◽  
Vol 120 (2) ◽  
pp. 503-512 ◽  
Author(s):  
F Widmer ◽  
P Caroni
Keyword(s):  
Phosphorylation Site ◽  
Growth Cones ◽  
Cell Cortex ◽  
Membrane Association ◽  
Major Protein ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

scholarly journals Daip1, a Dictyostelium Homologue of the Yeast Actin-Interacting Protein 1, Is Involved in Endocytosis, Cytokinesis, and Motility

1999 ◽  
Vol 146 (2) ◽  
pp. 453-464 ◽  
Author(s):  
Angelika Konzok ◽  
Igor Weber ◽  
Evelyn Simmeth ◽  
Ulrike Hacker ◽  
Markus Maniak ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Gene Replacement ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Interacting Protein ◽  
Phagocytic Cups ◽  
Green Fluorescent

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.

scholarly journals Bee1, a Yeast Protein with Homology to Wiscott-Aldrich Syndrome Protein, Is Critical for the Assembly of Cortical Actin Cytoskeleton

1997 ◽  
Vol 136 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Rong Li
Keyword(s):  
Actin Cytoskeleton ◽  
Protein Function ◽  
Actin Filaments ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Yeast Protein ◽  
Wiskott Aldrich Syndrome ◽  
Striking Change ◽  
And Function ◽  
Syndrome Protein

Yeast protein, Bee1, exhibits sequence homology to Wiskott-Aldrich syndrome protein (WASP), a human protein that may link signaling pathways to the actin cytoskeleton. Mutations in WASP are the primary cause of Wiskott-Aldrich syndrome, characterized by immuno-deficiencies and defects in blood cell morphogenesis. This report describes the characterization of Bee1 protein function in budding yeast. Disruption of BEE1 causes a striking change in the organization of actin filaments, resulting in defects in budding and cytokinesis. Rather than assemble into cortically associated patches, actin filaments in the buds of Δbee1 cells form aberrant bundles that do not contain most of the cortical cytoskeletal components. It is significant that Δbee1 is the only mutation reported so far that abolishes cortical actin patches in the bud. Bee1 protein is localized to actin patches and interacts with Sla1p, a Src homology 3 domain–containing protein previously implicated in actin assembly and function. Thus, Bee1 protein may be a crucial component of a cytoskeletal complex that controls the assembly and organization of actin filaments at the cell cortex.

scholarly journals Yeast actin-binding proteins: evidence for a role in morphogenesis.

1988 ◽  
Vol 107 (6) ◽  
pp. 2551-2561 ◽  
Author(s):  
D G Drubin ◽  
K G Miller ◽  
D Botstein
Keyword(s):  
Yeast Cell ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Binding Proteins ◽  
Binding Protein ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Binding Protein

Three yeast actin-binding proteins were identified using yeast actin filaments as an affinity matrix. One protein appears to be a yeast myosin heavy chain; it is dissociated from actin filaments by ATP, it is similar in size (200 kD) to other myosins, and antibodies directed against Dictyostelium myosin heavy chain bind to it. Immunofluorescence experiments show that a second actin-binding protein (67 kD) colocalizes in vivo with both cytoplasmic actin cables and cortical actin patches, the only identifiable actin structures in yeast. The cortical actin patches are concentrated at growing surfaces of the yeast cell where they might play a role in membrane and cell wall insertion, and the third actin-binding protein (85 kD) is only detected in association with these structures. This 85-kD protein is therefore a candidate for a determinant of growth sites. The in vivo role of this protein was tested by overproduction; this overproduction causes a reorganization of the actin cytoskeleton which in turn dramatically affects the budding pattern and spatial growth organization of the yeast cell.

scholarly journals Computational Approaches to Investigate and Design Lipid-binding Domains for Membrane Biosensing

2021 ◽  
Vol 75 (12) ◽  
pp. 1031-1036
Author(s):  
Sriraksha Srinivasan ◽  
Stefano Vanni
Keyword(s):  
Membrane Trafficking ◽  
De Novo ◽  
Md Simulations ◽  
Lipid Binding ◽  
Broad Perspective ◽  
Computational Approaches ◽  
Binding Domains ◽  
Specific Lipid

Association of proteins with cellular membranes is critical for signaling and membrane trafficking processes. Many peripheral lipid-binding domains have been identified in the last few decades and have been investigated for their specific lipid-sensing properties using traditional in vivo and in vitro studies. However, several knowledge gaps remain owing to intrinsic limitations of these methodologies. Thus, novel approaches are necessary to further our understanding in lipid–protein biology. This review briefly discusses lipid-binding domains that act as specific lipid biosensors and provides a broad perspective on the computational approaches such as molecular dynamics (MD) simulations and machine learning (ML)-based techniques that can be used to study protein–membrane interactions. We also highlight the need for de novo design of proteins that elicit specific lipid-binding properties.

WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement

2001 ◽  
Vol 114 (10) ◽  
pp. 1801-1809 ◽  
Author(s):  
T. Takenawa ◽  
H. Miki
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Cell Movement ◽  
Activation Mechanism ◽  
Cortical Actin ◽  
C Terminus ◽  
Inactive Form ◽  
Extracellular Stimuli ◽  
Proline Rich Region

Reorganization of cortical actin filaments plays critical roles in cell movement and pattern formation. Recently, the WASP and WAVE family proteins WASP and N-WASP, and WAVE1, WAVE2 and WAVE3 have been shown to regulate cortical actin filament reorganization in response to extracellular stimuli. These proteins each have a verprolin-homology (V) domain, cofilin-homology (C) domain and an acidic (A) region at the C-terminus, through which they activate the Arp2/3 complex, leading to rapid actin polymerization. N-WASP is usually present as an inactive form in which the VCA region is masked. Cooperative binding of Cdc42 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) exposes the VCA region, activating N-WASP. In addition to this activation mechanism, WISH also activates N-WASP independently of Cdc42 and PtdIns(4,5)P(2), by binding to the proline-rich region of N-WASP. N-WASP activation induces formation of filopodia in vivo. In contrast, the ubiquitously expressed form of WAVE2 is activated downstream of Rac, leading to formation of lamellipodia. In this case, IRSp53 transmits a signal from Rac to WAVE2 through formation of a ternary Rac-IRSp53-WAVE2 complex. Thus, N-WASP, which is activated downstream of Cdc42 or independently by WISH, induces formation of filopodia and WAVE2, which is activated via IRSp53 downstream of Rac, induces formation of lamellipodia.

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