Actin-interacting Protein Research Articles

Cooperation of two actin-binding proteins, cofilin and Aip1, in Saccharomyces cerevisiae.

Cofilin is a low-molecular weight actin-modulating protein, and is structurally and functionally conserved among eukaryotes. Cofilin is encoded by COF1 in Saccharomyces cerevisiae, and is essential for cell viability. Cofilin binds to and severs actin filaments in vitro, and also enhances their depolymerization. A partner protein that cooperates with cofilin in vivo has not been identified. When COF1 was over-expressed in yeast cells under the GAL1 promoter in a medium containing galactose as a sole carbon source, the cells did not survive. These results indicate that cells can grow only when the expression of cofilin is appropriately regulated. Several temperature sensitive (ts-) mutants were independently created by the random mutagenesis of COF1 with hydroxylamine. Mutated amino acids in ts-mutants were mapped in the sequences that were presumed to be involved in actin binding. A gene on a multicopy plasmid which suppresses the ts-phenotype of cof1-101, a typical ts-cofilin mutant, was isolated. The suppressor gene, SCF1, was found to be identical to AIP1, a gene encoding an actin-interacting protein. Although SCF1/AIP1 is not essential for cell viability, a combination of cof1-101 and Deltascf1/aip1 is synthetic lethal. Immunofluorescence staining of a wild-type strain using anti-Aip1 antibodies revealed that Aip1 was distributed in cortical actin patches where cofilin was also co-localized. Thick and long fibres stained with anti-cofilin antibody were detected in Deltascf1/aip1 cells, but not in SCF1/AIP1 cells. These results suggest the cooperative modulation of the actin cytoskeleton by cofilin and Aip1.

A novel 66-kDa stress protein, p66, associated with the process of cyst formation of Physarum polycephalum is a Physarum homologue of a yeast actin-interacting protein, AIP1.

When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p66 to homogeneity and isolated a p66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P. polycephalum.

High Rates of Actin Filament Turnover in Budding Yeast and Roles for Actin in Establishment and Maintenance of Cell Polarity Revealed Using the Actin Inhibitor Latrunculin-A

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites.

A search for Saccharomyces cerevisiae proteins that interact with actin in the two-hybrid system and a screen for mutants that affect the bipolar budding pattern identified the same gene, AIP3/BUD6. This gene is not essential for mitotic growth but is necessary for normal morphogenesis. MATa/alpha daughter cells lacking Aip3p place their first buds normally at their distal poles but choose random sites for budding in subsequent cell cycles. This suggests that actin and associated proteins are involved in placing the bipolar positional marker at the division site but not at the distal tip of the daughter cell. In addition, although aip3 mutant cells are not obviously defective in the initial polarization of the cytoskeleton at the time of bud emergence, they appear to lose cytoskeletal polarity as the bud enlarges, resulting in the formation of cells that are larger and rounder than normal. aip3 mutant cells also show inefficient nuclear migration and nuclear division, defects in the organization of the secretory system, and abnormal septation, all defects that presumably reflect the involvement of Aip3p in the organization and/or function of the actin cytoskeleton. The sequence of Aip3p is novel but contains a predicted coiled-coil domain near its C terminus that may mediate the observed homo-oligomerization of the protein. Aip3p shows a distinctive localization pattern that correlates well with its likely sites of action: it appears at the presumptive bud site prior to bud emergence, remains near the tips of small bund, and forms a ring (or pair of rings) in the mother-bud neck that is detectable early in the cell cycle but becomes more prominent prior to cytokinesis. Surprisingly, the localization of Aip3p does not appear to require either polarized actin or the septin proteins of the neck filaments.

Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites.

Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites.

Caldesmon, a calmodulin-binding, F actin-interacting protein, is present in aorta, uterus and platelets

Caldesmon, a protein originally found in chicken gizzard, was concluded also to be present in bovine aorta, uterus, and human platelets by demonstration of a protein with the following properties: (a) Ca 2+-dependent calmodulin-binding; (b) binding to F actin in such way that the binding was broken on Ca 2+-dependent binding of calmodulin; (c) cross-reactivity in immune blotting procedures with affinity-purified antibody against gizzard caldesmon; (d) similar subunit M r-values on SDS-gel to those of gizzard caldesmon. Like gizzard caldesmon, platelet caldesmon was composed of two polypeptide bands of M r 150000 and 147000, but caldesmon in aorta and uterus gave a single band of M r 150000. A polypeptide of M r 165000 that was immunologically distinct from caldesmon but, like caldesmon, bound to calmodulin and F actin in a flip-flop fashion, was also demonstrated in aorta and uterus.