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.

Live dynamics of Dictyostelium cofilin suggests a role in remodeling actin latticework into bundles

1997 ◽  
Vol 110 (19) ◽  
pp. 2333-2344 ◽  
Author(s):  
H. Aizawa ◽  
Y. Fukui ◽  
I. Yahara
Keyword(s):  
Fluorescent Protein ◽  
Dorsal Surface ◽  
Actin Binding ◽  
Cortical Actin ◽  
Actin Bundles ◽  
Phagocytic Cups ◽  
Green Fluorescent ◽  
High Level

Cofilin, an indispensable, actin-regulating protein represents the ‘cofilin family’ of actin-binding proteins existing in a wide variety of organisms. Our previous and other in vitro studies have implied that cofilin can accelerate transformation of filamentous (F)-actin and (alpha)-actinin latticework into bundles, and overexpression of cofilin induces formation of F-actin bundles in Dictyostelium. Here we expressed an Aequorea green fluorescent protein (GFP)-Dictyostelium cofilin fusion protein in Dictyostelium, and observed the live dynamics to examine the physiological function of cofilin. We show that purified GFP-cofilin binds to actin filaments and decreases the apparent viscosity of actin solution in a similar manner to authentic Dictyostelium cofilin. Expressed GFP-cofilin exhibits normal actin-binding activities in the cytoplasm as represented by incorporation into the actin rods induced with dimethyl sulfoxide. Free moving cells form a crown-like cortical structure on the dorsal surface, and GFP-cofilin exhibits dynamic assembly into actin bundles being formed beneath the cortex. During phagocytosis, GFP-cofilin accumulates into actin bundles formed in the region underlying the phagocytic cups. In cells chemotactically activated with cyclic AMP, GFP-cofilin exhibits a high level of accumulation in projecting leading edges. When the chemo-attraction is experimentally changed, the redistribution of GFP-cofilin towards the new pseudopod occurs in a matter of 30–60 seconds. These results demonstrate that cofilin plays a crucial role in vivo in rapid remodeling of the cortical actin meshwork into bundles.

scholarly journals Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding Yeast

2002 ◽  
Vol 13 (3) ◽  
pp. 854-865 ◽  
Author(s):  
K. L. Fehrenbacher ◽  
D. Davis ◽  
M. Wu ◽  
I. Boldogh ◽  
Liza A. Pon
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Cell Cortex ◽  
Filamentous Actin ◽  
Protein Fusion ◽  
Directed Movement ◽  
Apical Bud ◽  
M Phase ◽  
Green Fluorescent

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement.

scholarly journals Mal3, the Fission Yeast Homologue of the Human APC-interacting Protein EB-1 Is Required for Microtubule Integrity and the Maintenance of Cell Form

1997 ◽  
Vol 139 (3) ◽  
pp. 717-728 ◽  
Author(s):  
Jens D. Beinhauer ◽  
Iain M. Hagan ◽  
Johannes H. Hegemann ◽  
Ursula Fleig
Keyword(s):  
Fission Yeast ◽  
Gene Product ◽  
Fluorescent Protein ◽  
Interacting Protein ◽  
Tumour Suppressor Protein ◽  
Cytoplasmic Microtubules ◽  
Green Fluorescent ◽  
Yeast Genes ◽  
And Function

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

scholarly journals Temporally Distinct and Ligand-Specific Recruitment of Nuclear Receptor-Interacting Peptides and Cofactors to Subnuclear Domains Containing the Estrogen Receptor

2000 ◽  
Vol 14 (12) ◽  
pp. 2024-2039 ◽  
Author(s):  
Fred Schaufele ◽  
Ching-yi Chang ◽  
Weiqun Liu ◽  
John D. Baxter ◽  
Steven K. Nordeen ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Fluorescent Protein ◽  
Interacting Protein ◽  
Ici 182,780 ◽  
Blue Fluorescent Protein ◽  
Time Courses ◽  
Dissociation Mechanisms ◽  
Green Fluorescent ◽  
Temporal Sequences

Abstract Ligand binding to estrogen receptor (ER) is presumed to regulate the type and timing of ER interactions with different cofactors. Using fluorescence microscopy in living cells, we characterized the recruitment of five different green fluorescent protein (GFP)-labeled ER-interacting peptides to the distinct subnuclear compartment occupied by blue fluorescent protein (BFP)-labeled ERα. Different ligands promoted the recruitment of different peptides. One peptide was recruited in response to estradiol (E2), tamoxifen, raloxifene, or ICI 182,780 incubation whereas other peptides were recruited specifically by E2 or tamoxifen. Peptides containing different sequences surrounding the ER-interacting motif LXXLL were recruited with different time courses after E2 addition. Complex temporal kinetics also were observed for recruitment of the full-length, ER cofactor glucocorticoid receptor-interacting protein 1 (GRIP1); rapid, E2-dependent recruitment of GRIP1 was blocked by mutation of the GRIP1 LXXLL motifs to LXXAA whereas slower E2 recruitment persisted for the GRIP1 LXXAA mutant. This suggested the presence of multiple, temporally distinct GRIP 1 recruitment mechanisms. E2 recruitment of GRIP1 and LXXLL peptides was blocked by coincubation with excess ICI 182,780. In contrast, preformed E2/ER/GRIP1 and E2/ER/LXXLL complexes were resistant to subsequent ICI 182,780 addition whereas ICI 182,780 dispersed preformed complexes containing the GRIP1 LXXAA mutant. This suggested that E2-induced LXXLL binding altered subsequent ligand/ER interactions. Thus, alternative, ligand-selective recruitment and dissociation mechanisms with distinct temporal sequences are available for ERα action in vivo.

Fluid-phase uptake by macropinocytosis in Dictyostelium

1997 ◽  
Vol 110 (2) ◽  
pp. 105-112 ◽  
Author(s):  
U. Hacker ◽  
R. Albrecht ◽  
M. Maniak
Keyword(s):  
Fluorescent Protein ◽  
Fluid Phase ◽  
Average Diameter ◽  
Cell Cortex ◽  
Dictyostelium Cell ◽  
Scanning Electron Micrographs ◽  
Extracellular Medium ◽  
Green Fluorescent ◽  
Cytoskeletal Rearrangements ◽  
Shape Changes

To study fluid-phase endocytosis in living cells and its relationship to changes in the cell cortex, we have used a green fluorescent protein (GFP)-tagged version of coronin, an actin-associated protein that localises to dynamic regions of the Dictyostelium cell cortex. In the confocal microscope, internalisation of fluorescently labelled dextran as a fluid-phase marker can be recorded simultaneously with the recruitment of the coronin-GFP fusion-protein from the cytoplasm of the phagocyte. At crown-shaped surface protrusions, extracellular medium is taken up into vesicles with an average diameter of 1.6 microns, which is significantly larger than the 0.1 microns diameter of clathrin-coated pinosomes. The observed frequency of macropinosome formation can account for a large portion, if not all, of the fluid-phase uptake. The redistribution of coronin-GFP strongly resembles cytoskeletal rearrangements during phagocytosis. Scanning-electron micrographs indicate that crown-shaped cell-surface extensions can undergo shape changes, without a particle bound, that are similar to shape changes that occur during phagocytosis. In quantitative assays, the uptake of particles and fluid are about equally dependent on F-actin and coronin.

A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis

2000 ◽  
Vol 113 (18) ◽  
pp. 3309-3319 ◽  
Author(s):  
B. Gagny ◽  
A. Wiederkehr ◽  
P. Dumoulin ◽  
B. Winsor ◽  
H. Riezman ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Fluid Phase ◽  
Cortical Actin ◽  
Entire Genome ◽  
Functional Link ◽  
Cytoskeleton Organization ◽  
Eh Domain ◽  
Actin Cytoskeleton Organization ◽  
Green Fluorescent

Sequencing of the entire genome of S. cerevisiae has revealed the existence of five proteins containing EH domains. These are protein-protein interaction modules first described in mammalian Eps15, a protein that is involved in clathrin-dependent endocytosis. Two of the yeast proteins, End3p and Pan1p, are required for the internalization step of endocytosis. We report characterization of the nonessential ORF YBL047c which, like Eps15, encodes a protein with three N-terminal EH domains. Deletion of YBL047c leads to a defective fluid-phase endocytosis and to defective internalization of the pheromone (alpha)-factor and uracil permease. We therefore named YBL047c EDE1, for EH Domains and Endocytosis. Ede1p expressed as a chromosomally encoded fusion to the green fluorescent protein is localized in punctate cortical spots that only partially colocalize with actin patches. This localization is maintained when actin is depolymerized. Deletion of EDE1 impairs the diploid budding pattern, but has only a small impact on actin cytoskeleton organization, in contrast to the effects observed in pan1 cells and many end mutants impaired in proteins colocalizing with cortical actin patches. Genetic interaction was observed between EDE1 and RSP5, which encodes the ubiquitin ligase Rsp5p essential for ubiquitin-dependent endocytosis of many plasma membrane proteins, thus further emphasizing the functional link between Rsp5p and the EH domain proteins. We also observed genetic interaction between EDE1, and END3 or PAN1, suggesting that Ede1p might be part of a yeast EH network implicated in endocytosis.

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 Cellular Distribution and Functions of Wild-Type and Constitutively ActivatedDictyosteliumPakB

2005 ◽  
Vol 16 (1) ◽  
pp. 238-247 ◽  
Author(s):  
Marc de la Roche ◽  
Amjad Mahasneh ◽  
Sheu-Fen Lee ◽  
Francisco Rivero ◽  
Graham P. Côté
Keyword(s):  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Leading Edge ◽  
Cell Cortex ◽  
Wild Type ◽  
Cortical Actin ◽  
Myosin I ◽  
N Terminus ◽  
Green Fluorescent ◽  
Migrating Cells

Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis.

scholarly journals Targeted gene disruption reveals a role for vacuolin B in the late endocytic pathway and exocytosis

1998 ◽  
Vol 111 (1) ◽  
pp. 61-70 ◽  
Author(s):  
N. Jenne ◽  
R. Rauchenberger ◽  
U. Hacker ◽  
T. Kast ◽  
M. Maniak
Keyword(s):  
Gene Disruption ◽  
Fluorescent Protein ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Knock Out ◽  
Targeted Gene Disruption ◽  
Green Fluorescent ◽  
Cytoplasmic Side ◽  
Dependent Mechanism

Cells of Dictyostelium discoideum take up fluid by macropinocytosis. The contents of macropinosomes are acidified and digested by lysosomal enzymes. Thereafter, an endocytic marker progresses in an F-actin dependent mechanism from the acidic lysosomal phase to a neutral post-lysosomal phase. From the post-lysosomal compartment indigestible remnants are released by exocytosis. This compartment is characterised by two isoforms of vacuolin, A and B, which are encoded by different genes. Fusions of the vacuolin isoforms to the green fluorescent protein associate with the cytoplasmic side of post-lysosomal vacuoles in vivo. Vacuolin isoforms also localise to patches at the plasma membrane. Since vacuolins have no homologies to known proteins and do not contain domains of obvious function, we investigated their role by knocking out the genes separately. Although the sequences of vacuolins A and B are about 80% identical, only deletion of the vacuolin B gene results in a defect in the endocytic pathway; the vacuolin A knock-out appeared to be phenotypically normal. In vacuolin B- mutants endocytosis is normal, but the progression of fluid-phase marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker, suggesting a role for vacuolin B in targeting the vacuole for exocytosis.

scholarly journals Sequential Assembly of Myosin II, an IQGAP-like Protein, and Filamentous Actin to a Ring Structure Involved in Budding Yeast Cytokinesis

1998 ◽  
Vol 140 (2) ◽  
pp. 355-366 ◽  
Author(s):  
John Lippincott ◽  
Rong Li
Keyword(s):  
Cell Cycle ◽  
Fluorescent Protein ◽  
Budding Yeast ◽  
Sequence Similarity ◽  
Myosin Ii ◽  
Ring Structure ◽  
Binding Activity ◽  
Filamentous Actin ◽  
Green Fluorescent

We have identified a Saccharomyces cerevisiae protein, Cyk1p, that exhibits sequence similarity to the mammalian IQGAPs. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother–bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homologue. In vivo observation with green fluorescent protein–tagged Myo1p showed that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring was not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs.

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