Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function

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.

Download Full-text

Alp7-Mto1 and Alp14 synergize to promote interphase microtubule regrowth from the nuclear envelope

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz038 ◽

2019 ◽

Vol 11 (11) ◽

pp. 944-955 ◽

Cited By ~ 2

Author(s):

Wenyue Liu ◽

Fan Zheng ◽

Yucai Wang ◽

Chuanhai Fu

Keyword(s):

Nuclear Envelope ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Microtubule Assembly ◽

Dependent Manner ◽

Microtubule Nucleation ◽

Microtubule Organizing Centers ◽

Evolutionarily Conserved ◽

Microtubule Growth ◽

In Cells

Abstract Microtubules grow not only from the centrosome but also from various noncentrosomal microtubule-organizing centers (MTOCs), including the nuclear envelope (NE) and pre-existing microtubules. The evolutionarily conserved proteins Mto1/CDK5RAP2 and Alp14/TOG/XMAP215 have been shown to be involved in promoting microtubule nucleation. However, it has remained elusive as to how the microtubule nucleation promoting factors are specified to various noncentrosomal MTOCs, particularly the NE, and how these proteins coordinate to organize microtubule assembly. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, efficient interphase microtubule growth from the NE requires Alp7/TACC, Alp14/TOG/XMAP215, and Mto1/CDK5RAP2. The absence of Alp7, Alp14, or Mto1 compromises microtubule regrowth on the NE in cells undergoing microtubule repolymerization. We further demonstrate that Alp7 and Mto1 interdependently localize to the NE in cells without microtubules and that Alp14 localizes to the NE in an Alp7 and Mto1-dependent manner. Tethering Mto1 to the NE in cells lacking Alp7 partially restores microtubule number and the efficiency of microtubule generation from the NE. Hence, our study delineates that Alp7, Alp14, and Mto1 work in concert to regulate interphase microtubule regrowth on the NE.

Download Full-text

Spindle Pole Body Duplication in Fission Yeast Occurs at the G1/S Boundary but Maturation Is Blocked until Exit from S by an Event Downstream of Cdc10+

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0255 ◽

2004 ◽

Vol 15 (12) ◽

pp. 5219-5230 ◽

Cited By ~ 31

Author(s):

Satoru Uzawa ◽

Fei Li ◽

Ye Jin ◽

Kent L. McDonald ◽

Michael B. Braunfeld ◽

...

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Specific Inhibitor ◽

Spindle Pole ◽

Feedback Mechanism ◽

G1 Arrest ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Pole Body ◽

In Cells

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1–2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase–specific inhibitor of SPB maturation whose expression is under control of cdc10+. This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.

Download Full-text

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.01319-08 ◽

2008 ◽

Vol 29 (2) ◽

pp. 602-611 ◽

Cited By ~ 13

Author(s):

Sanjay Kumar ◽

Joel A. Huberman

Keyword(s):

Fission Yeast ◽

Replication Fork ◽

S Phase ◽

Yeast Cells ◽

Methyl Methanesulfonate ◽

Cyclin Dependent Kinase ◽

Cdc25 Phosphatase ◽

Origin Firing ◽

Replication Intermediates ◽

In Cells

ABSTRACT To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G1) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.

Download Full-text

Efficient formation of bipolar microtubule bundles requires microtubule-bound γ-tubulin complexes

The Journal of Cell Biology ◽

10.1083/jcb.200410119 ◽

2005 ◽

Vol 169 (2) ◽

pp. 297-308 ◽

Cited By ~ 100

Author(s):

Marcel E. Janson ◽

Thanuja Gangi Setty ◽

Anne Paoletti ◽

P.T. Tran

Keyword(s):

Epithelial Cells ◽

Fission Yeast ◽

Linear Array ◽

Spatial Separation ◽

Wild Type ◽

Basic Form ◽

Yeast Protein ◽

Polarized Epithelial Cells ◽

In Cells

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal γ-tubulin complexes (γ-TuCs). In interphase mto2Δ cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from γ-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.

Download Full-text

Robust cullin-RING ligase function is established by a multiplicity of poly-ubiquitylation pathways

eLife ◽

10.7554/elife.51163 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Spencer Hill ◽

Kurt Reichermeier ◽

Daniel C Scott ◽

Lorena Samentar ◽

Jasmin Coulombe-Huntington ◽

...

Keyword(s):

Chain Extension ◽

Genome Wide ◽

A Genome ◽

Crispr Screen ◽

Extension Activity ◽

The Stability ◽

Genetic Buffering ◽

Ligase Function ◽

In Cells

The cullin-RING ligases (CRLs) form the major family of E3 ubiquitin ligases. The prototypic CRLs in yeast, called SCF enzymes, employ a single E2 enzyme, Cdc34, to build poly-ubiquitin chains required for degradation. In contrast, six different human E2 and E3 enzyme activities, including Cdc34 orthologs UBE2R1 and UBE2R2, appear to mediate SCF-catalyzed substrate polyubiquitylation in vitro. The combinatorial interplay of these enzymes raises questions about genetic buffering of SCFs in human cells and challenges the dogma that E3s alone determine substrate specificity. To enable the quantitative comparisons of SCF-dependent ubiquitylation reactions with physiological enzyme concentrations, mass spectrometry was employed to estimate E2 and E3 levels in cells. In combination with UBE2R1/2, the E2 UBE2D3 and the E3 ARIH1 both promoted SCF-mediated polyubiquitylation in a substrate-specific fashion. Unexpectedly, UBE2R2 alone had negligible ubiquitylation activity at physiological concentrations and the ablation of UBE2R1/2 had no effect on the stability of SCF substrates in cells. A genome-wide CRISPR screen revealed that an additional E2 enzyme, UBE2G1, buffers against the loss of UBE2R1/2. UBE2G1 had robust in vitro chain extension activity with SCF, and UBE2G1 knockdown in cells lacking UBE2R1/2 resulted in stabilization of the SCF substrates p27 and CYCLIN E as well as the CUL2-RING ligase substrate HIF1α. The results demonstrate the human SCF enzyme system is diversified by association with multiple catalytic enzyme partners.

Download Full-text

Two S. pombe septation phases differ in ingression rate, septum structure, and response to F-actin loss

The Journal of Cell Biology ◽

10.1083/jcb.201808163 ◽

2019 ◽

Vol 218 (12) ◽

pp. 4171-4194 ◽

Cited By ~ 5

Author(s):

Mariona Ramos ◽

Juan Carlos G. Cortés ◽

Mamiko Sato ◽

Sergio A. Rincón ◽

M. Belén Moreno ◽

...

Keyword(s):

Fission Yeast ◽

Dual Function ◽

Second Phase ◽

Glucan Synthase ◽

Two Phases ◽

Anaphase B ◽

The Relationship ◽

Contractile Structure ◽

In Cells

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

Download Full-text

Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events.

The Journal of Cell Biology ◽

10.1083/jcb.110.5.1617 ◽

1990 ◽

Vol 110 (5) ◽

pp. 1617-1621 ◽

Cited By ~ 25

Author(s):

I M Hagan ◽

P N Riddle ◽

J S Hyams

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Average Length ◽

Nuclear Migration ◽

Yeast Cells ◽

Reverse Direction ◽

Wild Type ◽

Spindle Elongation ◽

Anaphase B ◽

In Cells

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

Download Full-text

The Ran GTPase System in Fission Yeast Affects Microtubules and Cytokinesis in Cells That Are Competent for Nucleocytoplasmic Protein Transport

Molecular and Cellular Biology ◽

10.1128/mcb.22.24.8491-8505.2002 ◽

2002 ◽

Vol 22 (24) ◽

pp. 8491-8505 ◽

Cited By ~ 23

Author(s):

Sandra S. Salus ◽

Janos Demeter ◽

Shelley Sazer

Keyword(s):

Fission Yeast ◽

Protein Transport ◽

Temperature Sensitive ◽

Nucleotide Exchange ◽

Cellular Processes ◽

Nucleotide Exchange Factor ◽

Ring Structures ◽

Dependent Processes ◽

In Cells

ABSTRACT Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.

Download Full-text