scholarly journals Tea2p Is a Kinesin-like Protein Required to Generate Polarized Growth in Fission Yeast

2000 ◽  
Vol 151 (1) ◽  
pp. 15-28 ◽  
Author(s):  
Heidi Browning ◽  
Jacqueline Hayles ◽  
Juan Mata ◽  
Lauren Aveline ◽  
Paul Nurse ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Specific Marker ◽  
Polarized Growth ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Morphological Defects ◽  
Morphological Mutants ◽  
Cytoplasmic Microtubules ◽  
Microtubule Growth ◽  
Punctate Pattern

Cytoplasmic microtubules are critical for establishing and maintaining cell shape and polarity. Our investigations of kinesin-like proteins (klps) and morphological mutants in the fission yeast Schizosaccharomyces pombe have identified a kinesin-like gene, tea2+, that is required for cells to generate proper polarized growth. Cells deleted for this gene are often bent during exponential growth and initiate growth from improper sites as they exit stationary phase. They have a reduced cytoplasmic microtubule network and display severe morphological defects in genetic backgrounds that produce long cells. The tip-specific marker, Tea1p, is mislocalized in both tea2-1 and tea2Δ cells, indicating that Tea2p function is necessary for proper localization of Tea1p. Tea2p is localized to the tips of the cell and in a punctate pattern within the cell, often coincident with the ends of cytoplasmic microtubules. These results suggest that this kinesin promotes microtubule growth, possibly through interactions with the microtubule end, and that it is important for establishing and maintaining polarized growth along the long axis of the cell.

The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers

1998 ◽  
Vol 111 (3) ◽  
pp. 295-301 ◽  
Author(s):  
A. Huyett ◽  
J. Kahana ◽  
P. Silver ◽  
X. Zeng ◽  
W.S. Saunders
Keyword(s):  
Molecular Motors ◽  
Motor Proteins ◽  
Single Gene ◽  
Intermediate Phenotype ◽  
Microtubule Network ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cytoplasmic Microtubule ◽  
Spindle Poles ◽  
Microtubule Motors ◽  
Microtubule Growth

Microtubules provide the substrate for intracellular trafficking by association with molecular motors of the kinesin and dynein superfamilies. Motor proteins are generally thought to function as force generating units for transport of various cargoes along the microtubule polymer. Recent work suggests additional roles for motor proteins in changing the structure of the microtubule network itself. We report here that in the budding yeast Saccharomyces cerevisiae microtubule motors have antagonistic effects on microtubule numbers and lengths. As shown previously, loss of the Kar3p motor stimulates cytoplasmic microtubule growth while loss of Kip2p leads to a sharp reduction in cytoplasmic microtubule numbers. Loss of both the Kip2p and Kar3p motors together in the same cell produces an intermediate phenotype, suggesting that these two motors act in opposition to control cytoplasmic microtubule density. A Kip2p-GFP fusion from single gene expression is most concentrated at the spindle poles, as shown previously for an epitope tagged Kar3p-HA, suggesting both of these motors act from the minus ends of the microtubules to influence microtubule numbers.

scholarly journals Mto2p, a Novel Fission Yeast Protein Required for Cytoplasmic Microtubule Organization and Anchoring of the Cytokinetic Actin Ring

2005 ◽  
Vol 16 (6) ◽  
pp. 3052-3063 ◽  
Author(s):  
Srinivas Venkatram ◽  
Jennifer L. Jennings ◽  
Andrew Link ◽  
Kathleen L. Gould
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Microtubule Organization ◽  
Actin Ring ◽  
Medial Region ◽  
Cytoplasmic Microtubule ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules

Microtubules regulate diverse cellular processes, including chromosome segregation, nuclear positioning, and cytokinesis. In many organisms, microtubule nucleation requires γ-tubulin and associated proteins present at specific microtubule organizing centers (MTOCs). In fission yeast, interphase cytoplasmic microtubules originate from poorly characterized interphase MTOCs and spindle pole body (SPB), and during late anaphase from the equatorial MTOC (EMTOC). It has been previously shown that Mto1p (Mbo1p/Mod20p) function is important for the organization/nucleation of all cytoplasmic microtubules. Here, we show that Mto2p, a novel protein, interacts with Mto1p and is important for establishing a normal interphase cytoplasmic microtubule array. In addition, mto2Δ cells fail to establish a stable EMTOC and localize γ-tubulin complex members to this medial structure. As predicted from these functions, Mto2p localizes to microtubules, the SPB, and the EMTOC in an Mto1p-dependent manner. mto2Δ cells fail to anchor the cytokinetic actin ring in the medial region of the cell and under conditions that mildly perturb actin structures, these rings unravel in mto2Δ cells. Our results suggest that the Mto2p and the EMTOC are critical for anchoring the cytokinetic actin ring to the medial region of the cell and for proper coordination of mitosis with cytokinesis.

scholarly journals Physarum plasmodia do contain cytoplasmic microtubules!

1991 ◽  
Vol 100 (3) ◽  
pp. 509-520 ◽  
Author(s):  
I. Salles-Passador ◽  
A. Moisand ◽  
V. Planques ◽  
M. Wright
Keyword(s):  
Electron Microscopy ◽  
Cold Treatment ◽  
Three Dimensional ◽  
Immunofluorescent Staining ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Thin Sections ◽  
Cytoplasmic Microtubules ◽  
Oriented Parallel ◽  
Physarum Plasmodia

It has been claimed that the plasmodium of the myxomycete Physarum polycephalum constitutes a very unusual syncytium, devoid of cytoplasmic microtubules. In contrast, we have observed a cytoplasmic microtubule network, by both electron microscopy and immunofluorescence in standard synchronous plasmodia, either in semi-thin sections or in smears, and in thin plasmodia, used as a convenient model. Cytoplasmic microtubules could be seen after immunofluorescent staining with three different monospecific monoclonal anti-tubulin antibodies. The immunolabelling was strictly restricted to typical microtubules as shown by electron microscopy. These cytoplasmic microtubules were entirely and reversibly disassembled by cold treatment and by either of two microtubule poisons: methyl benzimidazole carbamate and griseofulvin. The microtubule network, present in all strains that have been studied, contains single microtubules and microtubule bundles composed of two to eight microtubules. Cytoplasmic microtubules form a dense and complex three-dimensional network, distinct from the microfilamentous domains and from the nuclei. The orientation of the microtubule network varies according to the plasmodial domain examined. Generally microtubules show no special orientation except in plasmodial veins where they are oriented parallel to the long axis of the veins. Differences between our observations and those of previous workers who failed to find cytoplasmic microtubules in plasmodia are discussed. We propose that they reflect difficulties of observation mainly due to the fluorescent background. In contrast with the previous view, the discovery of a microtubule cytoplasmic cytoskeleton in Physarum plasmodia raises several questions concerning its relationships with other cellular organelles and its dynamics during different cell cycle events.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals Organization of the flagellar apparatus and associate cytoplasmic microtubules in the quadriflagellate alga Polytomella agilis.

1976 ◽  
Vol 69 (1) ◽  
pp. 106-125 ◽  
Author(s):  
D L Brown ◽  
A Massalski ◽  
R Patenaude
Keyword(s):  
Anterior Part ◽  
Flagellar Apparatus ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Body Region ◽  
Basal Bodies ◽  
Cytoplasmic Microtubule ◽  
Large Numbers ◽  
Attachment Sites ◽  
Cytoplasmic Microtubules

The organization of microtubular systems in the quadriflagellate unicell Polytomella agilis has been reconstructed by electron microscopy of serial sections, and the overall arrangement confirmed by immunofluorescent staining using antiserum directed against chick brain tubulin. The basal bodies of the four flagella are shown to be linked in two pairs of short fibers. Light microscopy of swimming cells indicates that the flagella beat in two synchronous pairs, with each pair exhibiting a breast-stroke-like motion. Two structurally distinct flagellar rootlets, one consisting of four microtubules in a 3 over 1 pattern and the other of a striated fiber over two microtubules, terminate between adjacent basal bodies. These rootlets diverge from the basal body region and extend toward the cell posterior, passing just beneath the plasma membrane. Near the anterior part of the cell, all eight rootlets serve as attachment sites for large numbers of cytoplasmic microtubules which occur in a single row around the circumference of the cell and closely parallel the cell shape. It is suggested that the flagellar rootless may function in controlling the patterning and the direction of cytoplasmic microtubule assembly. The occurrence of similar rootlet structures in other flagellates is briefly reviewed.

scholarly journals Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin.

1984 ◽  
Vol 98 (3) ◽  
pp. 1017-1025 ◽  
Author(s):  
W C Thompson ◽  
D J Asai ◽  
D H Carney
Keyword(s):  
Differential Staining ◽  
Hypotonic Medium ◽  
Cytoplasmic Microtubule ◽  
Alpha Tubulin ◽  
Apparent Correlation ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
Fibroblastic Cells ◽  
In Cells

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.

Variations in the distribution and migration of centriole duplexes in mitotic PtK2 cells studied by immunofluorescence microscopy

1980 ◽  
Vol 43 (1) ◽  
pp. 177-194 ◽  
Author(s):  
J.E. Aubin ◽  
M. Osborn ◽  
K. Weber
Keyword(s):  
Nuclear Membrane ◽  
Immunofluorescence Microscopy ◽  
Cytoplasmic Microtubule ◽  
Migration Patterns ◽  
Large Variability ◽  
Membrane Breakdown ◽  
Large Populations ◽  
Tubulin Antibodies ◽  
Cytoplasmic Microtubules ◽  
And Migration

The localization and migration of centriole duplexes have been studied in PtK2 cells by indirect immunofluorescence microscopy using specific tubulin antibodies. The study demonstrated the usefulness of the immunofluorescence technique to quantitate studies of centriole migration and concomitant events such as cytoplasmic microtubule breakdown in large populations of cells. Centriole duplex locations in normal and Colcemid-treated interphase populations have been compared with duplex locations in prophase cells. A higher percentage of duplexes were found close to the nucleus in prophase than in interphase cells, but approximately 5% of the duplexes remained in the cytoplasm far removed from the nucleus in prophase and throughout the course of duplex separation. Duplex separation occurred along a wide variety of paths and duplexes did not have to be closely juxtaposed to the nuclear envelope for separation to occur. Some duplexes separated in the cytoplasm with no detectable nuclear attachment, with spindles forming far to the side of the condensing chromosomes. The timing of duplex separation did not always coincide either with chromosome condensation or with nuclear membrane breakdown, and in a small percentage of the cells separation occurred as late as prometaphase. These data suggest that normal spindle formation can occur despite the large variability in initial and final centriole duplex location, their migration patterns, and the timing of the different events. Breakdown of cytoplasmic microtubules began in prophase and progressed until prometaphase; the last cytoplasmic microtubules disappeared soon after the loss of the nuclear membrane.

scholarly journals Fission Yeast Mto1 Regulates Diversity of Cytoplasmic Microtubule Organizing Centers

2010 ◽  
Vol 20 (21) ◽  
pp. 1959-1965 ◽  
Author(s):  
Itaru Samejima ◽  
Victoria J. Miller ◽  
Sergio A. Rincon ◽  
Kenneth E. Sawin
Keyword(s):  
Fission Yeast ◽  
Cytoplasmic Microtubule ◽  
Microtubule Organizing Centers

Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes in fission yeast

2001 ◽  
Vol 114 (13) ◽  
pp. 2427-2435 ◽  
Author(s):  
Bunshiro Goto ◽  
Koei Okazaki ◽  
Osami Niwa
Keyword(s):  
Fission Yeast ◽  
De Novo ◽  
Nuclear Periphery ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Limited Area ◽  
Cytoplasmic Microtubule ◽  
Random Arrangement ◽  
Triplex Structure ◽  
Microtubular System

Chromosomes are not packed randomly in the nucleus. The Rabl orientation is an example of the non-random arrangement of chromosomes, centromeres are grouped in a limited area near the nuclear periphery and telomeres are located apart from centromeres. This orientation is established during mitosis and maintained through subsequent interphase in a range of species. We report that a Rabl-like configuration can be formed de novo without a preceding mitosis during the transition from the sexual phase to the vegetative phase of the life cycle in fission yeast. In this process, each of the dispersed centromeres is often associated with a novel Sad1-containing body that is contacting a cytoplasmic microtubule laterally (Sad1 is a component of the spindle pole body (SPB)). The Sad1-containing body was colocalized with other known SPB components, Kms1 and Spo15 but not with Cut12, indicating that it represents a novel SPB-related complex. The existence of the triplex structure (centromere-microtubule-Sad1 body) suggests that the clustering of centromeres is controlled by a cytoplasmic microtubular system. Accordingly, when microtubules are destabilized, clustering is markedly reduced.

scholarly journals Phosphoregulation of the cytokinetic protein Fic1 contributes to fission yeast growth polarity establishment

2020 ◽  
Vol 133 (18) ◽  
pp. jcs244392
Author(s):  
K. Adam Bohnert ◽  
Anthony M. Rossi ◽  
Quan-Wen Jin ◽  
Jun-Song Chen ◽  
Kathleen L. Gould
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Behavior ◽  
Polarized Growth ◽  
Yeast Growth ◽  
Multiple Protein ◽  
A Cell ◽  
Growth Polarity ◽  
Polarity Regulation ◽  
Cellular Polarization

ABSTRACTCellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.This article has an associated First Person interview with Anthony M. Rossi, joint first author of the paper.

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