The Dictyostelium RasS protein is required for macropinocytosis, phagocytosis and the control of cell movement

2000 ◽  
Vol 113 (4) ◽  
pp. 709-719 ◽  
Author(s):  
J.R. Chubb ◽  
A. Wilkins ◽  
G.M. Thomas ◽  
R.H. Insall
Keyword(s):  
Cell Migration ◽  
Significant Proportion ◽  
Cell Movement ◽  
Fluid Phase ◽  
Small Gtpases ◽  
Cell Cortex ◽  
Fluid Phase Endocytosis ◽  
Ras Family ◽  
Actin Protrusions ◽  
Mutant Cells

Endocytosis and cell migration both require transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. We have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in endocytosis and cell migration. Mutant amoebae are defective in phagocytosis and fluid-phase endocytosis and are impaired in growth. Conversely, the rasS(-)cells show an enhanced rate of cell migration, moving three times faster than wild-type controls. The mutant cells display an aberrant morphology, are highly polarised, carry many elongated actin protrusions and show a concomitant decrease in formation of pinocytic crowns on the cell surface. These morphological aberrations are paralleled by changes in the actin cytoskeleton, with a significant proportion of the cortical F-actin relocalised to prominent pseudopodia. Rapid migration and endocytosis appear to be mutually incompatible and it is likely that RasS protein is required to maintain the normal balance between these two actin-dependent processes.

scholarly journals Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

2004 ◽  
Vol 15 (2) ◽  
pp. 861-869 ◽  
Author(s):  
Yaya Lefkir ◽  
Marilyne Malbouyres ◽  
Daniel Gotthardt ◽  
Adrian Ozinsky ◽  
Sophie Cornillon ◽  
...  
Keyword(s):  
Early Stage ◽  
Fluid Phase ◽  
Small Particles ◽  
Medium Chain ◽  
Murine Macrophages ◽  
Fluid Phase Endocytosis ◽  
Phagocytic Cups ◽  
Golgi Network ◽  
Mutant Cells

The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1-cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1-cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1-cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation.

Assessing the role of the ASP56/CAP homologue of Dictyostelium discoideum and the requirements for subcellular localization

1999 ◽  
Vol 112 (19) ◽  
pp. 3195-3203 ◽  
Author(s):  
A.A. Noegel ◽  
F. Rivero ◽  
R. Albrecht ◽  
K.P. Janssen ◽  
J. Kohler ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Fluid Phase ◽  
Axenic Medium ◽  
Terminal Domain ◽  
Defect Growth ◽  
Fluid Phase Endocytosis ◽  
Altered Cell ◽  
Mutant Cells ◽  
Proline Rich Region

The CAP (cyclase-associated protein) homologue of Dictyostelium discoideum is a phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulated G-actin sequestering protein which is present in the cytosol and shows enrichment at plasma membrane regions. It is composed of two domains separated by a proline rich stretch. The sequestering activity has been localized to the C-terminal domain of the protein, whereas the presence of the N-terminal domain seems to be required for PIP(2)-regulation of the sequestering activity. Here we have constructed GFP-fusions of N- and C-domain and found that the N-terminal domain showed CAP-specific enrichment at the anterior and posterior ends of cells like endogenous CAP irrespective of the presence of the proline rich region. Mutant cells expressing strongly reduced levels of CAP were generated by homologous recombination. They had an altered cell morphology with very heterogeneous cell sizes and exhibited a cytokinesis defect. Growth on bacteria was normal both in suspension and on agar plates as was phagocytosis of yeast and bacteria. In suspension in axenic medium mutant cells grew more slowly and did not reach saturation densities observed for wild-type cells. This was paralleled by a reduction in fluid phase endocytosis. Development was delayed by several hours under all conditions assayed, furthermore, motile behaviour was affected.

scholarly journals Cortical dynamics during cell motility are regulated by CRL3KLHL21 E3 ubiquitin ligase

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Thibault Courtheoux ◽  
Radoslav I. Enchev ◽  
Fabienne Lampert ◽  
Juan Gerez ◽  
Jochen Beck ◽  
...  
Keyword(s):  
Cell Migration ◽  
Ubiquitin Ligase ◽  
Cortical Plasticity ◽  
Cortical Microtubule ◽  
E3 Ubiquitin Ligase ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Cortex ◽  
Cortical Dynamics

Abstract Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

scholarly journals The conserved Pkh–Ypk kinase cascade is required for endocytosis in yeast

2002 ◽  
Vol 156 (2) ◽  
pp. 241-248 ◽  
Author(s):  
Amy K.A. deHart ◽  
Joshua D. Schnell ◽  
Damian A. Allen ◽  
Linda Hicke
Keyword(s):  
Fluid Phase ◽  
Receptor Internalization ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Extracellular Signals ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery ◽  
Kinase Cascade ◽  
Signaling Receptors ◽  
Mutant Cells

Internalization of activated signaling receptors by endocytosis is one way cells downregulate extracellular signals. Like many signaling receptors, the yeast α-factor pheromone receptor is downregulated by hyperphosphorylation, ubiquitination, and subsequent internalization and degradation in the lysosome-like vacuole. In a screen to detect proteins involved in ubiquitin-dependent receptor internalization, we identified the sphingoid base–regulated serine–threonine kinase Ypk1. Ypk1 is a homologue of the mammalian serum– and glucocorticoid-induced kinase, SGK, which can substitute for Ypk1 function in yeast. The kinase activity of Ypk1 is required for receptor endocytosis because mutations in two residues important for its catalytic activity cause a severe defect in α-factor internalization. Ypk1 is required for both receptor-mediated and fluid-phase endocytosis, and is not necessary for receptor phosphorylation or ubiquitination. Ypk1 itself is phosphorylated by Pkh kinases, homologues of mammalian PDK1. The threonine in Ypk1 that is phosphorylated by Pkh1 is required for efficient endocytosis, and pkh mutant cells are defective in α-factor internalization and fluid-phase endocytosis. These observations demonstrate that Ypk1 acts downstream of the Pkh kinases to control endocytosis by phosphorylating components of the endocytic machinery.

scholarly journals Scd5p and Clathrin Function Are Important for Cortical Actin Organization, Endocytosis, and Localization of Sla2p in Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2607-2625 ◽  
Author(s):  
Kenneth R. Henry ◽  
Kathleen D'Hondt ◽  
JiSuk Chang ◽  
Thomas Newpher ◽  
Kristen Huang ◽  
...  
Keyword(s):  
Fluid Phase ◽  
Cell Cortex ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Animal Cells ◽  
Interacting Protein ◽  
Actin Organization ◽  
Multicopy Suppressor ◽  
Fluid Phase Endocytosis ◽  
Huntingtin Interacting Protein

SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but anscd5-Δ338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Δ338affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Δ338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars.scd5-Δ338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression ofSCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Δ338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells.

scholarly journals The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

1998 ◽  
Vol 142 (5) ◽  
pp. 1289-1299 ◽  
Author(s):  
Lisa D. Belmont ◽  
David G. Drubin
Keyword(s):  
Fluid Phase ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Synthetically Lethal ◽  
Actin Patch ◽  
Actin Cables ◽  
Mutant Cells

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.

scholarly journals Novel Proteins Linking the Actin Cytoskeleton to the Endocytic Machinery in Saccharomyces cerevisiae

2002 ◽  
Vol 13 (10) ◽  
pp. 3646-3661 ◽  
Author(s):  
H. Dewar ◽  
D. T. Warren ◽  
F. C. Gardiner ◽  
C. G. Gourlay ◽  
N. Satish ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Elevated Temperatures ◽  
Adaptor Protein ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Fluid Phase Endocytosis ◽  
Endocytic Machinery

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

scholarly journals Nonmuscle Myosin IIB Is Involved in the Guidance of Fibroblast Migration

2004 ◽  
Vol 15 (3) ◽  
pp. 982-989 ◽  
Author(s):  
Chun-Min Lo ◽  
Denis B. Buxton ◽  
Gregory C.H. Chua ◽  
Micah Dembo ◽  
Robert S. Adelstein ◽  
...  
Keyword(s):  
Cell Migration ◽  
Large Fraction ◽  
Myosin Ii ◽  
Cell Movement ◽  
Traction Forces ◽  
Migration Patterns ◽  
Fibroblast Migration ◽  
Substrate Rigidity ◽  
Migration Analysis ◽  
Mutant Cells

Although myosin II is known to play an important role in cell migration, little is known about its specific functions. We have addressed the function of one of the isoforms of myosin II, myosin IIB, by analyzing the movement and mechanical characteristics of fibroblasts where this protein has been ablated by gene disruption. Myosin IIB null cells displayed multiple unstable and disorganized protrusions, although they were still able to generate a large fraction of traction forces when cultured on flexible polyacrylamide substrates. However, the traction forces were highly disorganized relative to the direction of cell migration. Analysis of cell migration patterns indicated an increase in speed and decrease in persistence, which were likely responsible for the defects in directional movements as demonstrated with Boyden chambers. In addition, unlike control cells, mutant cells failed to respond to mechanical signals such as compressing forces and changes in substrate rigidity. Immunofluorescence staining indicated that myosin IIB was localized preferentially along stress fibers in the interior region of the cell. Our results suggest that myosin IIB is involved not in propelling but in directing the cell movement, by coordinating protrusive activities and stabilizing the cell polarity.

scholarly journals RLIP76 (RalBP1) is an R-Ras effector that mediates adhesion-dependent Rac activation and cell migration

2006 ◽  
Vol 174 (6) ◽  
pp. 877-888 ◽  
Author(s):  
Lawrence E. Goldfinger ◽  
Celeste Ptak ◽  
Erin D. Jeffery ◽  
Jeffrey Shabanowitz ◽  
Donald F. Hunt ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
Malignant Transformation ◽  
Cell Spreading ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Ras Family ◽  
Ras Effectors ◽  
And Migration

The Ras family of small GTPases regulates cell proliferation, spreading, migration and apoptosis, and malignant transformation by binding to several protein effectors. One such GTPase, R-Ras, plays distinct roles in each of these processes, but to date, identified R-Ras effectors were shared with other Ras family members (e.g., H-Ras). We utilized a new database of Ras-interacting proteins to identify RLIP76 (RalBP1) as a novel R-Ras effector. RLIP76 binds directly to R-Ras in a GTP-dependent manner, but does not physically associate with the closely related paralogues H-Ras and Rap1A. RLIP76 is required for adhesion-induced Rac activation and the resulting cell spreading and migration, as well as for the ability of R-Ras to enhance these functions. RLIP76 regulates Rac activity through the adhesion-induced activation of Arf6 GTPase and activation of Arf6 bypasses the requirement for RLIP76 in Rac activation and cell spreading. Thus, we identify a novel R-Ras effector, RLIP76, which links R-Ras to adhesion-induced Rac activation through a GTPase cascade that mediates cell spreading and migration.

Sign in / Sign up

Export Citation Format

Share Document