scholarly journals Role of fungal dynein in hyphal growth, microtubule organization, spindle pole body motility and nuclear migration

1998 ◽  
Vol 111 (11) ◽  
pp. 1555-1566 ◽  
Author(s):  
S. Inoue ◽  
B.G. Turgeon ◽  
O.C. Yoder ◽  
J.R. Aist
Keyword(s):  
Spindle Pole Body ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Wild Type ◽  
Interphase Nuclei ◽  
Spindle Pole Bodies ◽  
Body Motility ◽  
Tip Cells ◽  
Hyphal Tip

Cytoplasmic dynein is a microtubule-associated motor protein with several putative subcellular functions. Sequencing of the gene (DHC1) for cytoplasmic dynein heavy chain of the filamentous ascomycete, Nectria haematococca, revealed a 4,349-codon open reading frame (interrupted by two introns) with four highly conserved P-loop motifs, typical of cytoplasmic dynein heavy chains. The predicted amino acid sequence is 78.0% identical to the cytoplasmic dynein heavy chain of Neurospora crassa, 70.2% identical to that of Aspergillus nidulans and 24.8% identical to that of Saccharomyces cerevisiae. The genomic copy of DHC1 in N. haematococca wild-type strain T213 was disrupted by inserting a selectable marker into the central motor domain. Mutants grew at 33% of the wild-type rate, forming dense compact colonies composed of spiral and highly branched hyphae. Major cytological phenotypes included (1) absence of aster-like arrays of cytoplasmic microtubules focused at the spindle pole bodies of post-mitotic and interphase nuclei, (2) limited post-mitotic nuclear migration, (3) lack of spindle pole body motility at interphase, (4) failure of spindle pole bodies to anchor interphase nuclei, (5) nonuniform distribution of interphase nuclei and (6) small or ephemeral Spitzenkorper at the apices of hyphal tip cells. Microtubule distribution in the apical region of tip cells of the mutant was essentially normal. The nonuniform distribution of nuclei in hyphae resulted primarily from a lack of both post-mitotic nuclear migration and anchoring of interphase nuclei by the spindle pole bodies. The results support the hypothesis that DHC1 is required for the motility and functions of spindle pole bodies, normal secretory vesicle transport to the hyphal apex and normal hyphal tip cell morphogenesis.

scholarly journals Reorientation of Mispositioned Spindles in Short Astral Microtubule Mutantspc72Δ Is Dependent on Spindle Pole Body Outer Plaque and Kar3 Motor Protein

2002 ◽  
Vol 13 (4) ◽  
pp. 1366-1380 ◽  
Author(s):  
Dominic Hoepfner ◽  
Florian Schaerer ◽  
Arndt Brachat ◽  
Achim Wach ◽  
Peter Philippsen
Keyword(s):  
Spindle Pole Body ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Astral Microtubule ◽  
Spindle Pole Bodies ◽  
Microtubule Motor ◽  
Microtubule Formation ◽  
Mother Cells

Nuclear migration and positioning in Saccharomyces cerevisiae depend on long astral microtubules emanating from the spindle pole bodies (SPBs). Herein, we show by in vivo fluorescence microscopy that cells lacking Spc72, the SPB receptor of the cytoplasmic γ-tubulin complex, can only generate very short (<1 μm) and unstable astral microtubules. Consequently, nuclear migration to the bud neck and orientation of the anaphase spindle along the mother-bud axis are absent in these cells. However,SPC72 deletion is not lethal because elongated but misaligned spindles can frequently reorient in mother cells, permitting delayed but otherwise correct nuclear segregation. High-resolution time-lapse sequences revealed that this spindle reorientation was most likely accomplished by cortex interactions of the very short astral microtubules. In addition, a set of double mutants suggested that reorientation was dependent on the SPB outer plaque and the astral microtubule motor function of Kar3 but not Kip2/Kip3/Dhc1, or the cortex components Kar9/Num1. Our observations suggest that Spc72 is required for astral microtubule formation at the SPB half-bridge and for stabilization of astral microtubules at the SPB outer plaque. In addition, our data exclude involvement of Spc72 in spindle formation and elongation functions.

scholarly journals Conservative Duplication of Spindle Poles during Meiosis in Saccharomyces cerevisiae

2001 ◽  
Vol 183 (7) ◽  
pp. 2372-2375 ◽  
Author(s):  
Andreas Wesp ◽  
Susanne Prinz ◽  
Gerald R. Fink
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Spore Formation ◽  
Wild Type ◽  
Body Distribution ◽  
Spindle Poles ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Type Strains

ABSTRACT During sporulation in diploid Saccharomyces cerevisiae, spindle pole bodies acquire the so-called meiotic plaque, a prerequisite for spore formation. Mpc70p is a component of the meiotic plaque and is thus essential for spore formation. We show here that MPC70/mpc70 heterozygous strains most often produce two spores instead of four and that these spores are always nonsisters. In wild-type strains, Mpc70p localizes to all four spindle pole bodies, whereas in MPC70/mpc70 strains Mpc70p localizes to only two of the four spindle pole bodies, and these are always nonsisters. Our data can be explained by conservative spindle pole body distribution in which the two newly synthesized meiosis II spindle pole bodies of MPC70/mpc70 strains lack Mpc70p.

S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the spindle pole body and essential for its modification

2000 ◽  
Vol 113 (3) ◽  
pp. 545-554 ◽  
Author(s):  
S. Ikemoto ◽  
T. Nakamura ◽  
M. Kubo ◽  
C. Shimoda
Keyword(s):  
Life Cycle ◽  
Fusion Gene ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Spindle Pole ◽  
Wild Type ◽  
Membrane Formation ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Meiotic Spindles

Spindle pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of spindle pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of spindle pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with spindle pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.

Independent nuclear motility and hyphal tip growth

1995 ◽  
Vol 73 (S1) ◽  
pp. 122-125 ◽  
Author(s):  
James R. Aist
Keyword(s):  
Tip Growth ◽  
Spindle Pole ◽  
Life Cycles ◽  
Microtubule Organizing Center ◽  
Hyphal Tip Growth ◽  
Spindle Pole Bodies ◽  
Organizing Center ◽  
Key Words Actin ◽  
Tip Cells ◽  
Hyphal Tip

Independent nuclear motility is involved in many important aspects of fungal life cycles, including the following: nuclear division; population of hyphal tip cells, branches, and spores with nuclei; dikaryotization; and karyogamy. Spindle pole bodies are almost constantly in motion during all phases of the nuclear cycle, and they have been linked to most instances of independent nuclear motility. A role for microtubules in this process is now well established, and research is being focused on which set of them, astral or cytoplasmic, is utilized as well as on the microtubule-associated motor proteins that may generate the force. In some cases, F-actin may interact with the microtubules or even provide an alternative cytoskeleton supporting nuclear migration. Hyphal tip growth and independent nuclear motility are coordinated and interrelated processes, making the elucidation of the signals, processes, and structures involved an attractive area for further research. Key words: actin, microtubule, microtubule associated protein, microtubule organizing center, motility, nucleus.

scholarly journals Cytoplasmic microtubules and fungal morphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamate determined by freeze-substitution of hyphal tip cells.

1980 ◽  
Vol 87 (1) ◽  
pp. 55-64 ◽  
Author(s):  
R J Howard ◽  
J R Aist
Keyword(s):  
Intracellular Transport ◽  
Freeze Substitution ◽  
Ultrastructural Level ◽  
Spindle Pole ◽  
Voltage Electron ◽  
Spindle Pole Bodies ◽  
Fungal Morphogenesis ◽  
Cytoplasmic Microtubules ◽  
Tip Cells ◽  
Hyphal Tip

The effects of methyl benzimidazole-2-ylcarbamate (MBC), one of only a few agents that are active against microtubules of fungi, were analyzed at the ultrastructural level in freeze-substituted hyphal tip cells of Fusarium acuminatum. Nontreated and control cells had numerous microtubules throughout. After just 10 min of exposure to MBC, almost no cytoplasmic microtubules were present, except near spindle pole bodies. After 45 min of exposure to MBC, no microtubules were present in hyphal tip cells, but they were present in the relatively quiescent subapical cells. These observations suggested that there are different rates of turnover for cytoplasmic microtubules in apical and subapical cells and for microtubules near spindle pole bodies and that MBC acts by inhibiting microtubules assembly. A statistical analysis of the distribution of intracytoplasmic vesicles in thick sections of cells treated with MBC, D2O or MBC + D2O was obtained by use of a high-voltage electron microscope. More than 50% of the vesicles in the apical 30 micrometers of control cells were found to lie within 2 micrometers of the tip cell apex. MBC treatment caused this vesicle distribution to become uniform, resulting in a substantial increase in the number of vesicles in subapical regions. The reduction in the number of cytoplasmic microtubules, induced by MBC, apparently inhibited intracellular transport of these vesicles and rendered random the longitudinal orientation of mitochondria. In most cases, D2O appeared capable of preventing these MBC-effects through stabilization of microtubules. These observations support the "vesicle hypothesis" of tip growth and establish a transport role for cytoplasmic microtubules in fungal morphogenesis.

scholarly journals Deletion of nudC, a nuclear migration gene of Aspergillus nidulans, causes morphological and cell wall abnormalities and is lethal.

1997 ◽  
Vol 8 (9) ◽  
pp. 1735-1749 ◽  
Author(s):  
Y H Chiu ◽  
X Xiang ◽  
A L Dawe ◽  
N R Morris
Keyword(s):  
Cell Wall ◽  
Aspergillus Nidulans ◽  
Protein Function ◽  
Spindle Pole Body ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Spindle Pole ◽  
Polymer Composition ◽  
Gene Products ◽  
Fungal Cell

Nuclear migration is required for normal development in both higher and lower eukaryotes. In fungi this process is mediated by cytoplasmic dynein. It is believed that this motor protein is anchored to the cell membrane and moves nuclei by capturing and pulling on spindle pole body microtubules. To date, four genes have been identified and shown to be required for this process in Aspergillus nidulans. The nudA and nudG genes, respectively, encode the heavy and light chains of cytoplasmic dynein, and the nudF and nudC gene products encode proteins of 49 and 22 kDa. The precise biochemical functions of the nudF and nudC genes have not yet been identified. In this report we further investigate NUDC protein function by deleting the nudC gene. Surprisingly, although deletion of nudA and nudF affect nuclear migration, deletion of nudC profoundly affected the morphology and composition of the cell wall. Spores of the strain deleted for nudC grew spherically and lysed. The thickness of the cell wall was increased in the deletion mutant and wall polymer composition was abnormal. This phenotype could be repressed by growth on osmotically buffered medium at low temperature. Similar, but less severe, effects were also noted in a strain depleted for NUDC by down-regulation. These results suggest a possible relationship between fungal cell wall biosynthesis and nuclear migration.

scholarly journals Mutant Membrane Protein of the Budding Yeast Spindle Pole Body Is Targeted to the Endoplasmic Reticulum Degradation Pathway

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 567-578 ◽  
Author(s):  
Susan McBratney ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Degradation Pathway ◽  
Spindle Pole ◽  
Wild Type ◽  
Er Quality Control ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugation

Abstract Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.

Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast

1999 ◽  
Vol 112 (14) ◽  
pp. 2313-2321 ◽  
Author(s):  
L. Cerutti ◽  
V. Simanis
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Spindle Pole Body ◽  
Mitotic Cell ◽  
Spindle Pole ◽  
The Other ◽  
Septum Formation ◽  
Spindle Pole Bodies ◽  
Interphase Cells ◽  
Early Mitosis

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.

Fine structure of the haplosporidan Kernstab, a persistent, intranuclear mitotic apparatus

1975 ◽  
Vol 18 (2) ◽  
pp. 327-346
Author(s):  
F.O. Perkins
Keyword(s):  
Fine Structure ◽  
Nuclear Envelope ◽  
Light Microscope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Interphase Nuclei ◽  
Mitotic Apparatus ◽  
Pole Body

The fine structure of the haplosporidan mitotic apparatus is described from observations of plasmodial nuclei of Minchinia nelsoni, M. costalis, Minchinia sp., and Urosporidium crescens. The apparatus, which is the Kernstab of light-microscope studies, consists of a bundle of microtubules terminating in a spindle pole body (SPB) at each end of the bundle. A few microtubules extend from SPB to SPB, but most either extend from an SPB and terminate in the nucleoplasm or lie in the nucleoplasm, free of either SPB. The bundle lengthens during mitosis, increasing the SPB-to-SPB distance by a factor of 2 to 3 as compared to interphase nuclei. SPBs are not in contact with the nuclear envelope, being found always in the nucleoplasm which is delimited by the nuclear envelope throughout mitosis. The mitotic apparatus is persistent through interphase, at least in a form which is not significantly different from that found in mitotic nuclei.

scholarly journals The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

2013 ◽  
Vol 13 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Meera Govindaraghavan ◽  
Alisha A. Lad ◽  
Stephen A. Osmani
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cycle Phase ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Content Type ◽  
Mitotic Entry ◽  
Spindle Pole Bodies ◽  
Pore Complexes

ABSTRACTThe G2-M transition inAspergillus nidulansrequires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events.

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