Cell Division: Mid-Level Management

When a fission yeast cell divides, the anillin-like protein mid1p helps to position the contractile ring in the cell middle. Recent experiments from two groups have shown how the cell-polarity factor pom1p negatively regulates the distribution of mid1p

Microtubule Nucleation at Non-Spindle Pole Body Microtubule-Organizing Centers Requires Fission Yeast Centrosomin-Related Protein mod20p

AbstractBackground: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase.Results: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the γ-tubulin complex to non-spindle pole body MTOCs and physically interacts with the γ-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin.Conclusions: Mod20p appears to act by recruiting components of the γ-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells

Two distinct regions of Mto1 are required for normal microtubule nucleation and efficient association with the gamma-tubulin complex in vivo

Cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe involves the interacting proteins Mto1 and Mto2, which are thought to recruit the γ-tubulin complex (γ-TuC) to prospective microtubule organizing centers. Mto1 contains a short amino-terminal region (CM1) that is conserved in higher eukaryotic proteins implicated in microtubule organization, centrosome function and/or brain development. Here we show that mutations in the Mto1 CM1 region generate mutant proteins that are functionally null for cytoplasmic microtubule nucleation and interaction with the γ-TuC (phenocopying mto1Δ), even though the Mto1-mutant proteins localize normally in cells and can bind Mto2. Interestingly, the CM1 region is not sufficient for efficient interaction with the γ-TuC. Mutation within a different region of Mto1, outside CM1, abrogates Mto2 binding and also impairs cytoplasmic microtubule nucleation and Mto1 association with the γ-TuC. However, this mutation allows limited microtubule nucleation in vivo, phenocopying mto2Δ rather than mto1Δ. Further experiments suggest that Mto1 and Mto2 form a complex (Mto1/2 complex) independent of the γ-TuC and that Mto1 and Mto2 can each associate with the γ-TuC in the absence of the other, albeit extremely weakly compared to when both Mto1 and Mto2 are present. We propose that Mto2 acts cooperatively with Mto1 to promote association of Mto1/2 complex with the γ-TuC

Comment on "A centrosome-independent role for gamma-TuRC proteins in the spindle assembly checkpoint"

Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature

Microtubule-independent movement of the fission yeast nucleus

Movement of the cell nucleus typically involves the cytoskeleton and either polymerization-based pushing forces or motor-based pulling forces. In the fission yeast Schizosaccharomyces pombe, nuclear movement and positioning are thought to depend on microtubule polymerization-based pushing forces. Here, we describe a novel, microtubule-independent, form of nuclear movement in fission yeast. Microtubule-independent nuclear movement is directed towards growing cell tips, and it is strongest when the nucleus is close to a growing cell tip, and weakest when the nucleus is far from that tip. Microtubule-independent nuclear movement requires actin cables but does not depend on actin polymerization-based pushing or myosin V-based pulling forces. The vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) Scs2 and Scs22, which are critical for endoplasmic reticulum–plasma membrane contact sites in fission yeast, are also required for microtubule-independent nuclear movement. We also find that in cells in which microtubule-based pushing forces are present, disruption of actin cables leads to increased fluctuations in interphase nuclear positioning and subsequent altered septation. Our results suggest two non-exclusive mechanisms for microtubule-independent nuclear movement, which may help illuminate aspects of nuclear positioning in other cells

Fission yeast NDR/LATS kinase Orb6 regulates exocytosis via phosphorylation of exocyst complex

Summary: NDR/LATS kinases regulate multiple aspects of cell polarity and morphogenesis from yeast to mammals. Fission yeast NDR/LATS kinase Orb6 has been proposed to control cell polarity by regulating the Cdc42 guanine nucleotide exchange factor Gef1. Here, we show that Orb6 regulates polarity largely independently of Gef1 and that Orb6 positively regulates exocytosis. Through Orb6 inhibition in vivo and quantitative global phosphoproteomics, we identify Orb6 targets, including proteins involved in membrane trafficking. We confirm Sec3 and Sec5, conserved components of the exocyst complex, as substrates of Orb6 both in vivo and in vitro, and we show that Orb6 kinase activity is important for exocyst localization to cell tips and for exocyst activity during septum dissolution after cytokinesis. We further find that Orb6 phosphorylation of Sec3 contributes to exocyst function in concert with exocyst protein Exo70. We propose that Orb6 contributes to polarized growth by regulating membrane trafficking at multiple levels. : NDR/LATS kinases are known primarily for their role in controlling cell and tissue proliferation and morphogenesis, e.g., via regulation of transcription in the Hippo pathway. Using fission yeast S. pombe as a model system, Tay et al. show that the NDR/LATS kinase Orb6 is a major regulator of exocytosis. Keywords: Orb6, NDR/LATS kinase, Cdc42, phosphoproteomics, exocytosis, exocyst, Sec3, phosphorylation, fission yeast, Schizosaccharomyces pomb

Activation of the γ-Tubulin Complex by the Mto1/2 Complex

SummaryThe multisubunit γ-tubulin complex (γ-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the γ-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the γ-TuC and recruits it to several different types of cytoplasmic MTOC sites [5–10]. Here, we show that the Mto1/2 complex activates γ-TuC-dependent microtubule nucleation independently of localizing the γ-TuC. This was achieved through the construction of a “minimal” version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and γ-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing ∼13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of γ-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast γ-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes