scholarly journals Novel protein Callipygian defines the back of migrating cells

2015 ◽  
Vol 112 (29) ◽  
pp. E3845-E3854 ◽  
Author(s):  
Kristen F. Swaney ◽  
Jane Borleis ◽  
Pablo A. Iglesias ◽  
Peter N. Devreotes
Keyword(s):  
Actin Polymerization ◽  
Protein Localization ◽  
Leading Edge ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Opposite Edge ◽  
Cell Axis ◽  
Mutual Antagonism ◽  
And Migration ◽  
Novel Protein

Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or “clustered,” to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.

scholarly journals Role of Phosphatidylinositol 3′ Kinase and a Downstream Pleckstrin Homology Domain–Containing Protein in Controlling Chemotaxis inDictyostelium

2001 ◽  
Vol 153 (4) ◽  
pp. 795-810 ◽  
Author(s):  
Satoru Funamoto ◽  
Kristina Milan ◽  
Ruedi Meili ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Adaptor Protein ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Wild Type ◽  
Pi3k Inhibitor Ly294002 ◽  
Effector Pathways

We show that cells lacking two Dictyostelium class I phosphatidylinositol (PI) 3′ kinases (PI3K and pi3k1/2-null cells) or wild-type cells treated with the PI3K inhibitor LY294002 are unable to properly polarize, are very defective in the temporal, spatial, and quantitative regulation of chemoattractant-mediated filamentous (F)-actin polymerization, and chemotax very slowly. PI3K is thought to produce membrane lipid-binding sites for localization of PH domain–containing proteins. We demonstrate that in response to chemoattractants three PH domain–containing proteins do not localize to the leading edge in pi3k1/2-null cells, and the translocation is blocked in wild-type cells by LY294002. Cells lacking one of these proteins, phdA-null cells, exhibit defects in the level and kinetics of actin polymerization at the leading edge and have chemotaxis phenotypes that are distinct from those described previously for protein kinase B (PKB) (pkbA)-null cells. Phenotypes of PhdA-dominant interfering mutations suggest that PhdA is an adaptor protein that regulates F-actin localization in response to chemoattractants and links PI3K to the control of F-actin polymerization at the leading edge during pseudopod formation. We suggest that PKB and PhdA lie downstream from PI3K and control different downstream effector pathways that are essential for proper chemotaxis.

scholarly journals Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

2001 ◽  
Vol 21 (16) ◽  
pp. 5437-5446 ◽  
Author(s):  
Tetsuya Hosooka ◽  
Tetsuya Noguchi ◽  
Hiroshi Nagai ◽  
Tatsuya Horikawa ◽  
Takashi Matozaki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Tyrosine Kinases ◽  
Cell Spreading ◽  
Melanoma Cells ◽  
Dominant Negative ◽  
Pleckstrin Homology Domain ◽  
Pleckstrin Homology ◽  
Cell Growth And Migration ◽  
Mouse Melanoma ◽  
And Migration

ABSTRACT Dok-1 (p62Dok) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOH-terminal truncation mutants of this protein: one (DokPH+PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH+PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH+PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.

scholarly journals Local Myo9b RhoGAP activity regulates cell motility

2020 ◽  
pp. jbc.RA120.013623
Author(s):  
Sandra Angela Hemkemeyer ◽  
Veith Vollmer ◽  
Vera Schwarz ◽  
Birgit Lohmann ◽  
Ulrike Honnert ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Cell Morphology ◽  
Actin Polymerization ◽  
Rho Gtpase ◽  
Leading Edge ◽  
Cell Extension ◽  
Local Inhibition ◽  
And Migration ◽  
Directional Cell Migration

To migrate, cells assume a polarized morphology, extending forward with a leading edge with their trailing edge retracting back toward the cell body. Both cell extension and retraction critically depend on the organization and dynamics of the actin cytoskeleton, and the small, monomeric GTPases Rac and Rho are important regulators of actin. Activation of Rac induces actin polymerization and cell extension whereas activation of Rho enhances acto-myosin II contractility and cell retraction. To coordinate migration, these processes must be carefully regulated. The myosin Myo9b, a Rho GTPase activating protein (GAP), negatively regulates Rho activity and deletion of Myo9b in leukocytes impairs cell migration through increased Rho activity. However, it is not known whether cell motility is regulated by global or local inhibition of Rho activity by Myo9b. Here, we addressed this question by using Myo9b-deficient macrophage-like cells that expressed different recombinant Myo9b constructs. We found that Myo9b accumulates in lamellipodial extensions generated by Rac-induced actin polymerization as a function of its motor activity. Deletion of Myo9b in HL-60 derived macrophages altered cell morphology and impaired cell migration. Reintroduction of Myo9b or Myo9b motor and GAP mutants revealed that local GAP activity rescues cell morphology and migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration. In summary, Rac activation leads to actin polymerization and recruitment of Myo9b, which locally inhibits Rho activity to enhance directional cell migration.

scholarly journals Lamellipodin tunes cell migration by stabilizing protrusions and promoting adhesion formation

2019 ◽  
Author(s):  
Georgi Dimchev ◽  
Behnam Amiri ◽  
Ashley C. Humphries ◽  
Matthias Schaks ◽  
Vanessa Dimchev ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Binding Protein ◽  
Critical Role ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Actin Binding ◽  
Membrane Ruffling ◽  
Genetic Ablation ◽  
And Migration

ABSTRACTEfficient migration on adhesive surfaces involves the protrusion of lamellipodial actin networks and their subsequent stabilization by nascent adhesions. The actin binding protein lamellipodin (Lpd) is thought to play a critical role in lamellipodium protrusion, by delivering Ena/VASP proteins onto the growing plus ends of actin filaments and by interacting with the WAVE regulatory complex (WRC), an activator of the Arp2/3 complex, at the leading edge. Using B16-F1 melanoma cell lines, we demonstrate that genetic ablation of Lpd compromises protrusion efficiency and coincident cell migration without altering essential parameters of lamellipodia, including their maximal rate of forward advancement and actin polymerization. We also confirmed lamellipodia and migration phenotypes with CRISPR/Cas9-mediated Lpd knockout Rat2 fibroblasts, excluding cell type-specific effects. Moreover, computer-aided analysis of cell edge morphodynamics on B16-F1 cell lamellipodia revealed that loss of Lpd correlates with reduced temporal protrusion maintenance as a prerequisite of nascent adhesion formation. We conclude that Lpd optimizes protrusion and nascent adhesion formation by counteracting frequent, chaotic retraction and membrane ruffling.Summary statementWe describe how genetic ablation of the prominent actin- and VASP-binding protein lamellipodin combined with software-aided protrusion analysis uncovers mechanistic insights into its cellular function during cell migration.

scholarly journals The actin regulators Enabled and Diaphanous direct distinct protrusive behaviors in different tissues during Drosophila development

2014 ◽  
Vol 25 (20) ◽  
pp. 3147-3165 ◽  
Author(s):  
Stephanie H. Nowotarski ◽  
Natalie McKeon ◽  
Rachel J. Moser ◽  
Mark Peifer
Keyword(s):  
Actin Polymerization ◽  
Cultured Cells ◽  
Leading Edge ◽  
Dorsal Closure ◽  
Primary Role ◽  
Loss Of Function ◽  
Timely Completion ◽  
And Migration ◽  
Different Tissues

Actin-based protrusions are important for signaling and migration during development and homeostasis. Defining how different tissues in vivo craft diverse protrusive behaviors using the same genomic toolkit of actin regulators is a current challenge. The actin elongation factors Diaphanous and Enabled both promote barbed-end actin polymerization and can stimulate filopodia in cultured cells. However, redundancy in mammals and Diaphanous’ role in cytokinesis limited analysis of whether and how they regulate protrusions during development. We used two tissues driving Drosophila dorsal closure—migratory leading-edge (LE) and nonmigratory amnioserosal (AS) cells—as models to define how cells shape distinct protrusions during morphogenesis. We found that nonmigratory AS cells produce filopodia that are morphologically and dynamically distinct from those of LE cells. We hypothesized that differing Enabled and/or Diaphanous activity drives these differences. Combining gain- and loss-of-function with quantitative approaches revealed that Diaphanous and Enabled each regulate filopodial behavior in vivo and defined a quantitative “fingerprint”—the protrusive profile—which our data suggest is characteristic of each actin regulator. Our data suggest that LE protrusiveness is primarily Enabled driven, whereas Diaphanous plays the primary role in the AS, and reveal each has roles in dorsal closure, but its robustness ensures timely completion in their absence.

scholarly journals The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis

2011 ◽  
Vol 22 (18) ◽  
pp. 3442-3455 ◽  
Author(s):  
Michiko Nakamura-Kubo ◽  
Aiko Hirata ◽  
Chikashi Shimoda ◽  
Taro Nakamura
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Pleckstrin Homology ◽  
Phosphatidylinositol 3 Kinase ◽  
Pole Body ◽  
Phosphatidylinositol 3

Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM.

scholarly journals HGF-induced migration depends on the PI(3,4,5)P3-binding microexon-spliced variant of the Arf6 exchange factor cytohesin-1

2018 ◽  
Vol 218 (1) ◽  
pp. 285-298 ◽  
Author(s):  
Colin D.H. Ratcliffe ◽  
Nadeem Siddiqui ◽  
Paula P. Coelho ◽  
Nancy Laterreur ◽  
Tumini N. Cookey ◽  
...  
Keyword(s):  
Cell Migration ◽  
Leading Edge ◽  
Pleckstrin Homology Domain ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Dependent Cell ◽  
And Migration ◽  
Phosphoinositide Recognition

Differential inclusion or skipping of microexons is an increasingly recognized class of alternative splicing events. However, the functional significance of microexons and their contribution to signaling diversity is poorly understood. The Met receptor tyrosine kinase (RTK) modulates invasive growth and migration in development and cancer. Here, we show that microexon switching in the Arf6 guanine nucleotide exchange factor cytohesin-1 controls Met-dependent cell migration. Cytohesin-1 isoforms, differing by the inclusion of an evolutionarily conserved three-nucleotide microexon in the pleckstrin homology domain, display differential affinity for PI(4,5)P2 (triglycine) and PI(3,4,5)P3 (diglycine). We show that selective phosphoinositide recognition by cytohesin-1 isoforms promotes distinct subcellular localizations, whereby the triglycine isoform localizes to the plasma membrane and the diglycine to the leading edge. These data highlight microexon skipping as a mechanism to spatially restrict signaling and provide a mechanistic link between RTK-initiated phosphoinositide microdomains and Arf6 during signal transduction and cancer cell migration.

scholarly journals WASP family proteins and formins compete in pseudopod- and bleb-based migration

2017 ◽  
Vol 217 (2) ◽  
pp. 701-714 ◽  
Author(s):  
Andrew J. Davidson ◽  
Clelia Amato ◽  
Peter A. Thomason ◽  
Robert H. Insall
Keyword(s):  
Hydrostatic Pressure ◽  
Dictyostelium Discoideum ◽  
Actin Polymerization ◽  
Leading Edge ◽  
Eukaryotic Cells ◽  
Double Knockout ◽  
Wave Drive ◽  
Bleb Formation ◽  
And Migration ◽  
Wasp Family Proteins

Actin pseudopods induced by SCAR/WAVE drive normal migration and chemotaxis in eukaryotic cells. Cells can also migrate using blebs, in which the edge is driven forward by hydrostatic pressure instead of actin. In Dictyostelium discoideum, loss of SCAR is compensated by WASP moving to the leading edge to generate morphologically normal pseudopods. Here we use an inducible double knockout to show that cells lacking both SCAR and WASP are unable to grow, make pseudopods or, unexpectedly, migrate using blebs. Remarkably, amounts and dynamics of actin polymerization are normal. Pseudopods are replaced in double SCAR/WASP mutants by aberrant filopods, induced by the formin dDia2. Further disruption of the gene for dDia2 restores cells’ ability to initiate blebs and thus migrate, though pseudopods are still lost. Triple knockout cells still contain near-normal F-actin levels. This work shows that SCAR, WASP, and dDia2 compete for actin. Loss of SCAR and WASP causes excessive dDia2 activity, maintaining F-actin levels but blocking pseudopod and bleb formation and migration.

Sign in / Sign up

Export Citation Format

Share Document