PubMed Health⌕ Search

Biomedical subjects

Kenneth E Sawin

Publications and source records attributed to Kenneth E Sawin.

12 recordsLinked to original sources
PubMed2006

Cytoplasmic microtubule organization in fission yeast.

During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently-tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization.

Anaphase↗
PubMed2006

Noncore components of the fission yeast gamma-tubulin complex.

Relatively little is known about the in vivo function of individual components of the eukaryotic gamma-tubulin complex (gamma-TuC). We identified three genes, gfh1+, mod21+, and mod22+, in a screen for fission yeast mutants affecting microtubule organization. gfh1+ is a previously characterized gamma-TuC protein weakly similar to human gamma-TuC subunit GCP4, whereas mod21+ is novel and shows weak similarity to human gamma-TuC subunit GCP5. We show that mod21p is a bona fide gamma-TuC protein and that, like gfh1Delta mutants, mod21Delta mutants are viable. We find that gfh1Delta and mod21Delta mutants have qualitatively normal microtubule nucleation from all types of microtubule-organizing centers (MTOCs) in vivo but quantitatively reduced nucleation from interphase MTOCs, and this is exacerbated by mutations in mod22+. Simultaneous deletion of gfh1p, mod21p, and alp16p, a third nonessential gamma-TuC protein, does not lead to additive defects, suggesting that all three proteins contribute to a single function. Coimmunoprecipitation experiments suggest that gfh1p and alp16p are codependent for association with a small "core" gamma-TuC, whereas mod21p is more peripherally associated, and that gfh1p and mod21p may form a subcomplex independently of the small gamma-TuC. Interestingly, sucrose gradient analysis suggests that the major form of the gamma-TuC in fission yeast may be a small complex. We propose that gfh1p, mod21p, and alp16 act as facultative "noncore" components of the fission yeast gamma-TuC and enhance its microtubule-nucleating ability.

Cell Shape↗
PubMed2005

Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast.

The fission yeast cell-polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy-terminus and the membrane protein mod5p. Here, we show that a tea1p-related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein-protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism.

Cell Polarity↗
PubMed2005

Meiosis: organizing microtubule organizers.

During meiosis in fission yeast, the zygote nucleus undergoes microtubule-driven oscillatory movements that ultimately serve to promote genetic recombination. An essential component of this is a meiosis-specific consolidation of microtubule-organizing activity to the the spindle pole body, driven by the novel coiled-coil protein mcp6/hrs1p.

Cell Cycle Proteins↗
PubMed2005

Fission yeast mto2p regulates microtubule nucleation by the centrosomin-related protein mto1p.

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Delta strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Delta defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the gamma-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Delta mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to "satellite" particles on interphase microtubules. In mto1Delta mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Delta mutants, cytoplasmic forms of the gamma-tubulin complex are mislocalized, and the gamma-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the gamma-tubulin complex.

Antibodies, Monoclonal↗
PubMed2004

Microtubule nucleation at non-spindle pole body microtubule-organizing centers requires fission yeast centrosomin-related protein mod20p.

BACKGROUND: 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 gamma-tubulin complex to non-spindle pole body MTOCs and physically interacts with the gamma-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 gamma-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.

Amino Acid Sequence↗
PubMed2004

The spindle gets bigger.

Although cortical nonmuscle myosin II has long been implicated in cytokinetic aspects of cell division, there has never been strong evidence that it plays a role in the organization of the mitotic spindle. ([this issue of Cell]) use a number of methods to show that cortical myosin II is in fact important for spindle assembly in higher eukaryotic cells, specifically for the complete separation of centrosomes after nuclear envelope breakdown.

Actins↗
PubMed2004

Role of microtubules and tea1p in establishment and maintenance of fission yeast cell polarity.

Microtubules and the protein tea1p have important roles in regulating cell polarity in the fission yeast Schizosaccharomyces pombe. Here, using combinations of drugs, environmental perturbations and genetic mutants, we demonstrate that once a cell polarity axis is established, microtubules have at best a minor role in maintaining the cortical actin cytoskeleton and the rate and direction of cell growth. In addition, we find that after perturbations that disrupt cell polarity and the cortical actin cytoskeleton, microtubules are not required for re-establishment of polarity per se. However, after such perturbations, the distribution of cytoplasmic microtubules plays an important role in dictating the position of sites of polarity re-establishment. Furthermore, this influence of microtubule distribution on site selection during polarity re-establishment requires the presence of tea1p, suggesting that tea1p is crucial for coupling microtubule distribution to the regulation of cell polarity. Our results suggest a model in which, at the cellular level, two distinct and separable mechanisms contribute to how tea1p regulates site selection during polarity re-establishment. First, tea1p remaining at cell tips after cortical depolarization can serve as a cortical landmark for microtubule-independent site selection; second, tea1p newly targeted to the cell cortex by association with microtubules can promote the formation of polarity axes de novo.

Benzimidazoles↗
PubMed2003

Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips.

Microtubules have a central role in eukaryotic cell polarity, in part through interactions between microtubule end-binding proteins and the cell cortex. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth. The tea1p protein is transported to cell tips by association with growing microtubule plus ends; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5Delta cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wild-type cells is associated with the plasma membrane at cell tips. However, in tea1Delta cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity.

Cell Membrane↗
PubMed2002

Cell polarity: following formin function.

Mutation of a novel fission yeast formin, for3p, leads to marked changes in both the actin and microtubule cytoskeleton, as well as a surprising asymmetric pattern of cell growth. At the same time, new work in budding yeast implicates formins directly in actin filament assembly.

Actin Cytoskeleton↗