Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essential<i>in vivo</i>function(s)

Liu, Xiong; Osherov, Nir; Yamashita, Roxanne A.; Brzeska, Hanna; Korn, Edward D.; May, Gregory S.

doi:10.1073/pnas.161285698

Cited by 35 publications

(30 citation statements)

References 45 publications

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“…7A). Three observations indicated that this Arp3-GFP fusion protein is associated with cortical actin patches, as in S. cerevisiae (40). First, the Arp3-GFP signal was localized in patches at the hyphal tips of the wildtype strain (Fig.…”

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confidence: 73%

“…In Aspergillus, replacement of the TEDS-rule site with alanine dramatically affected the Mg 2ϩ -ATPase activity of myosin I, whereas replacement with the phosphorylation mimic glutamic acid reduced but did not abolish this activity, suggesting that ATPase activity in MyoA is subject to modulation by phosphorylation of the TEDS-rule site. However, the significance of this phosphorylation is not clear because both of the myoA substitution mutants are equally functional in restoring the viability of the null mutant (40).…”

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confidence: 99%

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Functional Characterization of Myosin I Tail Regions in Candida albicans

et al. 2004

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The molecular motor myosin I is required for hyphal growth in the pathogenic yeast Candida albicans. Specific myosin I functions were investigated by a deletion analysis of five neck and tail regions. Hyphal formation requires both the TH1 region and the IQ motifs. The TH2 region is important for optimal hyphal growth. All of the regions, except for the SH3 and acidic (A) regions that were examined individually, were required for the localization of myosin I at the hyphal tip. Similarly, all of the domains were required for the association of myosin I with pelletable actin-bound complexes. Moreover, the hyphal tip localization of cortical actin patches, identified by both rhodamine-phalloidin staining and Arp3-green fluorescent protein signals, was dependent on myosin I. Double deletion of the A and SH3 domains depolarized the distribution of the cortical actin patches without affecting the ability of the mutant to form hyphae, suggesting that myosin I has distinct functions in these processes. Among the six myosin I tail domain mutants, the ability to form hyphae was strictly correlated with endocytosis. We propose that the uptake of cell wall remodeling enzymes and excess plasma membrane is critical for hyphal formation.

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confidence: 73%

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Functional Characterization of Myosin I Tail Regions in Candida albicans

et al. 2004

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“…It is possible that some contractile activity below the limit of our assays is present in ELC-myosin, and this is sufficient to allow myosin II to integrate the cortex. It has been determined that Aspergillus nidulans myosin I mutants with less than 1% of wild-type actin-activated MgATPase activity retain essential in vivo functions (Liu et al, 2001). However, we have shown that mlcE -cells cannot undergo contraction of detergent extracted cortices, which would be a direct test of contractile activity of myosin in situ (Xu et al, 2001).…”

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confidence: 99%

Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments

Laevsky

Knecht

2003

Journal of Cell Science

105

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Cells are frequently required to move in a local environment that physically restricts locomotion, such as during extravasation or metastatic invasion. In order to model these events, we have developed an assay in which vegetative Dictyostelium amoebae undergo chemotaxis under a layer of agarose toward a source of folic acid [Laevsky, G. and Knecht, D. A. (2001). Biotechniques 31, 1140-1149]. As the concentration of agarose is increased from 0.5% to 3% the cells are increasingly inhibited in their ability to move under the agarose. The contribution of myosin II and actin cross-linking proteins to the movement of cells in this restrictive environment has now been examined. Cells lacking myosin II heavy chain (mhcA -) are unable to migrate under agarose overlays of greater than 0.5%, and even at this concentration they move only a short distance from the trough. While attempting to move, the cells become stretched and fragmented due to their inability to retract their uropods. At higher agarose concentrations, the mhcA -cells protrude pseudopods under the agarose, but are unable to pull the cell body underneath. Consistent with a role for myosin II in general cortical stability, GFP-myosin dynamically localizes to the lateral and posterior cortex of cells moving under agarose. Cells lacking the essential light chain of myosin II (mlcE -), have no measurable myosin II motor activity, yet were able to move normally under all agarose concentrations. Mutants lacking either ABP-120 or α-actinin were also able to move under agarose at rates similar to wild-type cells. We hypothesize that myosin stabilizes the actin cortex through its cross-linking activity rather than its motor function and this activity is necessary and sufficient for the maintenance of cortical integrity of cells undergoing movement in a restrictive environment. The actin cross-linkers α-actinin and ABP-120 do not appear to play as major a role as myosin II in providing this cortical integrity. Research Article 3762 measurable actin activated ATPase activity (Chen et al., 1995;Xu et al., 1996). While not generally thought of as an actin cross-linking protein, myosin II minifilaments presumably also have this capability (Wachsstock et al., 1994;Humphrey et al., 2002). Mutants lacking myosin II (mhcA -) are able to accomplish both random and chemotactic motility; however, they move slowly and have defects in pseudopod extension (Peters et al., 1988;Wessels et al., 1988). Although mhcA -cells are able to aggregate, they are unable to complete the developmental program .The developmental defect of mhcA -cells appears to be due to their inability to move in a restrictive environment . During early development, cells acquire surface adhesion proteins and so movement occurs while cells are continually making and breaking adhesive contacts with their neighbors as well as the substratum. Unlike movement on a planar substratum, this form of motility is analogous to the movement of metastatic cancer cells away from a primary tumor, or the extravasation of immune...

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“…Since some of the fungal myosins I do not require phosphorylation to fulfill their cellular tasks (22,23), we decided to first investigate if the S. cerevisiae Myo5p TEDS site was phosphorylated in vivo. In order to address this matter, the wild type Myo5p or a mutant bearing a TEDS serine to alanine substitution (Myo5-S357Ap) was immunoprecipitated from 32 PO 4 3Ϫ -radiolabeled yeast cells.…”

Section: Resultsmentioning

confidence: 99%

“…Consistently, the yeast MYO3 TEDS site serine to alanine mutant (myo3-S357A) fails to complement the synthetic lethal phenotype of a double myo3⌬ myo5⌬ null strain (20,21). Interestingly, however, not all fungal myosin I functions require phosphorylation of this residue and/or full ATPase activity, since a TEDS site to alanine mutation in the Aspergillus nidulans MYOA only causes slight defects when compared with the null mutation (22,23).…”

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confidence: 94%

TEDS Site Phosphorylation of the Yeast Myosins I Is Required for Ligand-induced but Not for Constitutive Endocytosis of the G Protein-coupled Receptor Ste2p

Grosshans

Grötsch

Mukhopadhyay³

et al. 2006

Journal of Biological Chemistry

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The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.Myosins I constitute a well characterized and ubiquitous group of unconventional myosins, which participate in a variety of cellular processes, including endocytosis, phagocytosis, cell motility, secretion, and cell polarity (1, 2). As other myosins, myosins I bear an N-terminal actin-activated ATPase, which can translocate actin filaments in vitro (3). A short positively charged tail that binds acidic phospholipids and targets the myosin to the appropriate cellular membranes follows the conserved motor domain (1, 2, 4, 5). The fungal and the protozoal myosins I bear an additional C-terminal extension that participates in the activation of Arp2/3-dependent actin polymerization (6 -10). In the yeast Saccharomyces cerevisiae, two highly homologous genes encode myosins I: MYO3 and MYO5. Deletion of either gene does not result in any obvious phenotype, whereas, depending on the strain background, a double knock-out is lethal or very sick, suggesting functional redundancy (11,12). Genetic analysis indicates that the yeast myosins I participate in the polarization of the actin cytoskeleton and in endocytosis. Cortical actin patches, which concentrate in the daughter cells in wild type budding yeast, partially redistribute to mother cells in a double myo3⌬ myo5⌬ null mutant generated in a permissive strain background (11, 12). Besides, Myo3p and Myo5p have a well established function in the ligand-induced internalization of the ␣-factor pheromone receptor Ste2p. Ste2p is a G protein-coupled receptor that triggers the mating response in the presence of ␣-factor (13). Binding of the pheromone to its receptor accelerates its internalization and degradation rate as part of a pheromone desensitization program (14). Temperature-sens...

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Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)

Cited by 35 publications

References 45 publications

Functional Characterization of Myosin I Tail Regions in Candida albicans

Functional Characterization of Myosin I Tail Regions in Candida albicans

Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments

TEDS Site Phosphorylation of the Yeast Myosins I Is Required for Ligand-induced but Not for Constitutive Endocytosis of the G Protein-coupled Receptor Ste2p

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