The intrinsically disordered region of the cytokinetic F-BAR protein Cdc15 performs a unique essential function in maintenance of cytokinetic ring integrity

Abstract

Successful separation of two daughter cells (i.e., cytokinesis) is essential for life. Many eukaryotic cells divide using a contractile apparatus called the cytokinetic ring (CR) that associates dynamically with the plasma membrane (PM) and generates force that contributes to PM ingression between daughter cells. In Schizosaccharomyces pombe, important membrane-CR scaffolds include the paralogous F-BAR proteins Cdc15 and Imp2. Their conserved protein structure consists of the archetypal F-BAR domain linked to an SH3 domain by an intrinsically disordered region (IDR). Functions have been assigned to the F-BAR and SH3 domains. In this study we probed the function of the central IDR. We found that the IDR of Cdc15 is essential for viability and cannot be replaced by that of Imp2, whereas the F-BAR domain of Cdc15 can be swapped with several different F-BAR domains, including that of Imp2. Deleting part of the IDR results in CR defects and abolishes calcineurin phosphatase localization to the CR. Together these results indicate that Cdc15's IDR has a nonredundant essential function that coordinates regulation of CR architecture.

FIGURE 1:

The IDR of Cdc15 is essential. (A) Table of cdc15 alleles, schematic of gene product (drawn to scale), and ability to rescue cdc15 null. Numbers indicate aa position. (B, C) Repres­entative tetrads from diploids of the indicated genotype that were induced to sporulate.

FIGURE 2:

Cdc15 F-BAR, but not IDR, can be replaced by domains of homologous proteins. (A) Table of cdc15 alleles, schematic of gene product (drawn to scale), and ability to rescue cdc15 null. Numbers indicate aa position. (B) Live-cell images of the indicated haploid strains. A single BF z-slice and sum projections of mNG images are shown. Images of the Fer and PSTPIP1 chimeras were acquired on a separate day. Bar, 5 µm.

FIGURE 3:

Cdc15 IDR deletion mutants have cytokinesis defects. (A) Schematic of Cdc15 indicating the aa boundaries of IDR regions. (B) BF images of the indicated strains. Bar, 5 µm. (C) Quantification of the number of nuclei (left) and septa (right) per cell of the indicated strains, determined from cells stained with DAPI and methyl blue to visualize DNA and septum, respectively. Results are the means from three biological replicates, n > 198 cells per replicate. Error bars are SD. *, p < 0.0001. Ordinary two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. (D) Table of cdc15 alleles, schematic of gene product, and ability to rescue cdc15 null. Schematics are drawn to scale. Black lines indicate deletion. (E) Duration of cytokinesis stages determined from imaging of strains expressing the indicated cdc15 allele plus rlc1-mNG and sid4-mNG to label the CR and SPB, respectively. Results are the means from three biological replicates, n > 5 cells per replicate, and the total number of cells analyzed for each strain is indicated on the graph. Error bars are SD. *, p < 0.0001. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. (F) Representative time-lapse series of the indicated strains. Images were acquired every 2 min. A single BF slice and max intensity z-projections of deconvolved mNG images are shown. Numbers indicate min from SPB separation. Bar, 5 µm. Double arrow indicates first frame when CR loses ringlike structure. Yellow arrowheads point out first and last frame with visible asymmetric septum deposition.

FIGURE 4:

Characterizing the CR defect in cdc15-∆2. (A) End-on imaging of the indicated strains. Numbers indicate minutes elapsed since the start of the series. Bar, 5 µm. (B) Z-slices of two representative cells of the indicated genotype. The pink dashed lines mark the middle slice. Z-slices were taken every 0.2 µm through the volume of the cell. Fluorescence images are deconvolved. Bar, 5 µm.

FIGURE 5:

Cell wall synthases are properly trafficked in cdc15-∆2. (A) Representative time-lapse series of the indicated strains. Sum z-projections are shown. Images were acquired every 2 min. Numbers indicate minutes from SPB separation. Bar, 5 µm. (B) Timing of GFP-Bgs1 arrival to the division site in the indicated strains also expressing Sid4-mNG and Rlc1-mCh. Error bars are SD. p = 0.67. Student’s t test. (C) Average peak fluorescence of GFP-Bgs1 in the indicated strains also expressing Sid4-mNG and Rlc1-mCh. Error bars are SD. p = 0.85. Student’s t test. (D) Timing of Rlc1 (×) and Bgs1 (•) peak intensity relative to SPB separation for the indicated strains determined from strains imaged as in (A). Results in A–D are the means from two biological replicates, n > 9 cells per replicate, and total number of cells analyzed for each strain is indicated on the graph.

FIGURE 6:

Calcineurin is not recruited to CR in cdc15-∆2. (A–C) Representative sum projections of the indicated strains. Bar, 5 µm. (A) At least 100 cells from 3 d of imaging were examined. (B, C) At least 50 cells were examined.