scholarly journals Cdk1-dependent phosphoinhibition of a formin-F-BAR interaction opposes cytokinetic contractile ring formation

2018 ◽  
Vol 29 (6) ◽  
pp. 713-721 ◽  
Author(s):  
Alaina H. Willet ◽  
K. Adam Bohnert ◽  
Kathleen L. Gould
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Site ◽  
Chromosome Segregation ◽  
Binding Motif ◽  
Contractile Ring ◽  
General Role ◽  
Bar Domain ◽  
Mitotic Kinase ◽  
M Phase ◽  
Direct Binding

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). A single essential formin, Cdc12, localizes to the cell middle upon mitotic onset and nucleates the F-actin of the CR. Cdc12 medial recruitment is mediated in part by its direct binding to the F-BAR scaffold Cdc15. Given that Cdc12 is hyperphosphorylated in M phase, we explored whether Cdc12 phosphoregulation impacts its association with Cdc15 during mitosis. We found that Cdk1, a major mitotic kinase, phosphorylates Cdc12 on six N-terminal residues near the Cdc15-binding site, and phosphorylation on these sites inhibits its interaction with the Cdc15 F-BAR domain. Consistent with this finding, a cdc12 mutant with all six Cdk1 sites changed to phosphomimetic residues (cdc12-6D) displays phenotypes similar to cdc12-P31A, in which the Cdc15-binding motif is disrupted; both show reduced Cdc12 at the CR and delayed CR formation. Together, these results indicate that Cdk1 phosphorylation of formin Cdc12 antagonizes its interaction with Cdc15 and thereby opposes Cdc12’s CR localization. These results are consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete.

scholarly journals MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif

2020 ◽  
Vol 295 (52) ◽  
pp. 18266-18275
Author(s):  
Sebastian Kiehstaller ◽  
Christian Ottmann ◽  
Sven Hennig
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Viral Infections ◽  
Binding Mode ◽  
Aminopeptidase N ◽  
Binding Motif ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Phosphorylated Peptides ◽  
Direct Binding

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

scholarly journals The F-BAR Cdc15 promotes contractile ring formation through the direct recruitment of the formin Cdc12

2015 ◽  
Vol 208 (4) ◽  
pp. 391-399 ◽  
Author(s):  
Alaina H. Willet ◽  
Nathan A. McDonald ◽  
K. Adam Bohnert ◽  
Michelle A. Baird ◽  
John R. Allen ◽  
...  
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Ring Formation ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Contractile Ring ◽  
Bar Domain ◽  
Medial Cortex ◽  
N Terminus

In Schizosaccharomyces pombe, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). Nucleation of F-actin for the CR requires a single formin, Cdc12, that localizes to the cell middle at mitotic onset. Although genetic requirements for formin Cdc12 recruitment have been determined, the molecular mechanisms dictating its targeting to the medial cortex during cytokinesis are unknown. In this paper, we define a short motif within the N terminus of Cdc12 that binds directly to the F-BAR domain of the scaffolding protein Cdc15. Mutations preventing the Cdc12–Cdc15 interaction resulted in reduced Cdc12, F-actin, and actin-binding proteins at the CR, which in turn led to a delay in CR formation and sensitivity to other perturbations of CR assembly. We conclude that Cdc15 contributes to CR formation and cytokinesis via formin Cdc12 recruitment, defining a novel cytokinetic function for an F-BAR domain.

scholarly journals Multifunctional Centromere Binding Factor 1 Is Essential for Chromosome Segregation in the Human Pathogenic YeastCandida glabrata

2001 ◽  
Vol 21 (15) ◽  
pp. 4875-4888 ◽  
Author(s):  
Tanja Stoyan ◽  
Gernot Gloeckner ◽  
Stephan Diekmann ◽  
John Carbon
Keyword(s):  
Binding Site ◽  
Chromosome Segregation ◽  
Leucine Zipper ◽  
Fold Increase ◽  
Plasmid Segregation ◽  
Plasmid Loss ◽  
Helix Loop Helix ◽  
Binding Factor ◽  
Direct Binding

ABSTRACT The CBF1 (centromere binding factor 1) gene ofCandida glabrata was cloned by functional complementation of the methionine biosynthesis defect of aSaccharomyces cerevisiae cbf1 deletion mutant. TheC. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 × 109 M−1). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4′,6′-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G2/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

scholarly journals Anillin-mediated Targeting of Peanut to Pseudocleavage Furrows Is Regulated by the GTPase Ran

2008 ◽  
Vol 19 (9) ◽  
pp. 3735-3744 ◽  
Author(s):  
Rosalind V. Silverman-Gavrila ◽  
Karen G. Hales ◽  
Andrew Wilde
Keyword(s):  
Binding Site ◽  
Chromosome Segregation ◽  
Inhibitory Action ◽  
Mutant Form ◽  
Biochemical Assays ◽  
Transport Receptors ◽  
Importin Α ◽  
Direct Binding ◽  
Cytoskeleton Disruption ◽  
Gtpase Ran

During early development in Drosophila, pseudocleavage furrows in the syncytial embryo prevent contact between neighboring spindles, thereby ensuring proper chromosome segregation. Here we demonstrate that the GTPase Ran regulates pseudocleavage furrow organization. Ran can exert control on pseudocleavage furrows independently of its role in regulating the microtubule cytoskeleton. Disruption of the Ran pathway prevented pseudocleavage furrow formation and restricted the depth and duration of furrow ingression of those pseudocleavage furrows that did form. We found that Ran was required for the localization of the septin Peanut to the pseudocleavage furrow, but not anillin or actin. Biochemical assays revealed that the direct binding of the nuclear transport receptors importin α and β to anillin prevented the binding of Peanut to anillin. Furthermore, RanGTP reversed the inhibitory action of importin α and β. On expression of a mutant form of anillin that lacked an importin α and β binding site, inhibition of Ran no longer restricted the depth and duration of furrow ingression in those pseudocleavage furrows that formed. These data suggest that anillin and Peanut are involved in pseudocleavage furrow ingression in syncytial embryos and that this process is regulated by Ran.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

2008 ◽  
Vol 19 (6) ◽  
pp. 2661-2672 ◽  
Author(s):  
Soomin Shim ◽  
Samuel A. Merrill ◽  
Phyllis I. Hanson
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Essential Role ◽  
Multivesicular Body ◽  
The Other ◽  
Aaa Atpase ◽  
Direct Binding ◽  
Novel Interactions

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

scholarly journals Assembly of normal actomyosin rings in the absence of Mid1p and cortical nodes in fission yeast

2008 ◽  
Vol 183 (6) ◽  
pp. 979-988 ◽  
Author(s):  
Yinyi Huang ◽  
Hongyan Yan ◽  
Mohan K. Balasubramanian
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Ring Structure ◽  
Contractile Ring ◽  
Ring Assembly ◽  
Cell Biol ◽  
In Cells

Cytokinesis in many eukaryotes depends on the function of an actomyosin contractile ring. The mechanisms regulating assembly and positioning of this ring are not fully understood. The fission yeast Schizosaccharomyces pombe divides using an actomyosin ring and is an attractive organism for the study of cytokinesis. Recent studies in S. pombe (Wu, J.Q., V. Sirotkin, D.R. Kovar, M. Lord, C.C. Beltzner, J.R. Kuhn, and T.D. Pollard. 2006. J. Cell Biol. 174:391–402; Vavylonis, D., J.Q. Wu, S. Hao, B. O'Shaughnessy, and T.D. Pollard. 2008. Science. 319:97–100) have suggested that the assembly of the actomyosin ring is initiated from a series of cortical nodes containing several components of this ring. These studies have proposed that actomyosin interactions bring together the cortical nodes to form a compacted ring structure. In this study, we test this model in cells that are unable to assemble cortical nodes. Although the cortical nodes play a role in the timing of ring assembly, we find that they are dispensable for the assembly of orthogonal actomyosin rings. Thus, a mechanism that is independent of cortical nodes is sufficient for the assembly of normal actomyosin rings.

scholarly journals Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

1996 ◽  
Vol 16 (1) ◽  
pp. 86-93 ◽  
Author(s):  
R Kovelman ◽  
P Russell
Keyword(s):  
Dna Replication ◽  
Schizosaccharomyces Pombe ◽  
Replication Checkpoint ◽  
Activation Assay ◽  
M Phase ◽  
Dna Replication Checkpoint ◽  
High Level ◽  
Tyrosyl Phosphorylation ◽  
In Cells

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.

A checkpoint that monitors cytokinesis in Schizosaccharomyces pombe

2000 ◽  
Vol 113 (7) ◽  
pp. 1223-1230 ◽  
Author(s):  
J. Liu ◽  
H. Wang ◽  
M.K. Balasubramanian
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mutant Allele ◽  
Significant Proportion ◽  
S Phase ◽  
Genetic Screens ◽  
Beta Glucan ◽  
Binucleate Cells ◽  
Number Of Genes ◽  
M Phase ◽  
Ring Constriction

Cell division in Schizosaccharomyces pombe is achieved through the use of a medially positioned actomyosin ring. A division septum is formed centripetally, concomitant with actomyosin ring constriction. Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly. These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei. Recently, we have shown that a mutant allele of the S. pombe drc1(+)/cps1(+) gene, which encodes a 1,3-(beta)-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype. drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring. Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G(2) nuclei are significantly delayed for entry into the M phase. In this study we have investigated the mechanism that causes cps1-191 to block with two G(2) nuclei. We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth. Rather, the failure to complete some aspect of cytokinesis may prevent the G(2)/M transition of the two interphase-G(2) nuclei. The G(2)/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1 gene and also by the dominant cdc2 allele cdc2-1w, but not the cdc2-3w allele. Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis. We conclude that an F-actin and Wee1p dependent checkpoint blocks G(2)/M transition until previous cytokinesis is completed.

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