A serine-to-alanine mutant on Ser4 of Cof1, for 10 min at 4 C, were washed once in cold water, and were centrifuged again

A serine-to-alanine mutant on Ser4 of Cof1, for 10 min at 4 C, were washed once in cold water, and were centrifuged again. that actin cable assembly is regulated in a cell-cycleCdependent manner both in vivo and in vitro. = 10 individual experiments) and HU- (= 9), nocodazole- (= 4), and = 4). (cells before extract preparation blocks actin cable formation by GST-Bni1 FH1-COOHCcoated beads. Tasidotin hydrochloride 1NM-PP1 at a final concentration of 20 M was added for 30 min before sample preparation. ((= 3). (are compared by anti-Pgk1 Western blot. (Scale bars, 5 m.) (and and and gene (53). The endogenous promoter was replaced by Tasidotin hydrochloride a methionine promoter, allowing expression to be turned off upon addition of methionine-supplemented medium. Similar to extracts prepared from HU- and nocodazole-treated cells, extracts from arrested cells also initiated actin cable formation on Bni1 FH1-COOHCcoated beads (Fig. 1 and cells expressing epitope tagged-cyclins (Table S1) and Abp140-3GFP. cells were first arrested at late anaphase/telophase Tasidotin hydrochloride by incubation at 37 C for 180 min (54). Then they were Tasidotin hydrochloride released by shifting to 25 C, and cells were harvested every 30 min for parallel immunoblotting and actin cable-assembly assay. These studies revealed that the mitotic cyclin Clb2 is highly enriched in the extracts producing robust actin cable formation from GST-Bni1 FH1-COOHCfunctionalized beads (Fig. S2). However, the percentage of beads containing actin cables was lower (42%) than in extracts made from HU- or nocodazole-arrested cells. The most plausible explanation for the lower assembly in mutant extracts is that the temperature-sensitive mutant is not fully reversible. We next tested whether Cdk1 kinase activity is required for actin cable assembly from Bni1 FH1-COOH beads. To address this question, endogenous Cdk1 was replaced by an analog-sensitive allele of Cdk1 (cells were synchronized by HU addition and then were treated with 20 M of the ATP analog 1-NM-PP1 for 30 min to inhibit the Cdk1 kinase activity specifically or as a Tasidotin hydrochloride control were treated with DMSO before extract preparation. Inhibition of Cdk1 activity caused actin cable-assembly activity to be abolished completely (Fig. 1 and cells was not caused by a difference in soluble protein levels in the cytoplasmic extract, as shown by a phosphoglycerate kinase 1 (Pgk1) loading control (Fig. 1= 52 from 16 bundles, measured within 1 m from the bead boundary). This dimension is consistent with the reported in vivo cable thickness (90C100 nm) at the G2/M stage in fission yeast (47). Because it was difficult to discern the ends of filaments, we could not calculate their individual lengths. Because Bni1 has not been reported to have a bundling activity (50), the appearance of bundles in the cytoplasmic extracts suggests the presence of bundling factors. Recapitulation of Mouse monoclonal to MUM1 Regulatory Protein Dependence During Actin Cable Reconstitution. To test how faithfully the Bni1 FH1-COOHCdependent actin cable-reconstitution system recapitulates the in vivo function of actin-regulatory proteins, we generated extracts from four mutants in which actin-interacting proteins were knocked out or rendered dysfunctional. These included the actin cable-specific stabilizing protein Tpm1, the barbed end-capping protein (Cap2), the depolymerization factor cofilin (Cof1), and the actin cable regulator Bud6. HU-arrested cells expressing Abp140-3GFP were used for actin cable-reconstitution assays. Mutants of different actin-interacting proteins showed distinct actin cable phenotypes (Fig. 2 completely abolished cable formation (Fig. 2extracts were more numerous (>2.5 fold) than in WT extracts (Fig. 2 and mutant cells (Fig. 2and extracts, however, reconstituted cables disassembled more slowly upon LatA addition (Fig. S3 and extracts did not show obvious defects in actin cable assembly from beads (Fig. 2extracts relative to WT extracts is because Bud6 levels are depleted in our WT extracts. The actin elongation rate in extracts also was similar to that in WT extracts (17 versus 18 subunits/s) (Fig. S3extracts and that the cables showed similar geometry (Fig. S3cells expressing Abp140-3GFP. (showing actin cables emanating from fluorescent beads. (< 0.05). We normalized protein extract samples by loading equal protein in each lane (Fig. 3 and and were resolved by.