scholarly journals Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii

2001 ◽  
Vol 114 (5) ◽  
pp. 975-986 ◽  
Author(s):  
C. Alberti-Segui ◽  
F. Dietrich ◽  
R. Altmann-Johl ◽  
D. Hoepfner ◽  
P. Philippsen
Keyword(s):  
Long Range ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Mitotic Division ◽  
Chain Gene ◽  
Ashbya Gossypii ◽  
Yeast Saccharomyces Cerevisiae ◽  
Filamentous Ascomycete ◽  
Nuclear Clusters ◽  
Actin Cables

We have followed the migration of GFP-labelled nuclei in multinucleate hyphae of Ashbya gossypii. For the first time we could demonstrate that the mode of long range nuclear migration consists of oscillatory movements of nuclei with, on average, higher amplitudes in the direction of the growing tip. We could also show that mitotic division proceeds at a constant rate of 0. 64 microm/minute which differs from the biphasic kinetics described for the yeast Saccharomyces cerevisiae. Furthermore we were able to identify the microtubule-based motor dynein as a key element in the control of long range nuclear migration. For other filamentous fungi it had already been demonstrated that inactivating mutations in dynein led to severe problems in nuclear migration, i.e. generation of long nuclei-free hyphal tips and clusters of nuclei throughout the hyphae. This phenotype supported the view that dynein is important for the movement of nuclei towards the tip. In A. gossypii the opposite seems to be the case. A complete deletion of the dynein heavy chain gene leads to nuclear clusters exclusively at the hyphal tips and to an essentially nucleus-free network of hyphal tubes and branches. Anucleate hyphae and branches in the vicinity of nuclear clusters show actin cables and polarized actin patches, as well as microtubules. The slow growth of this dynein null mutant could be completely reverted to wild-type-like growth in the presence of benomyl, which can be explained by the observed redistribution of nuclei in the hyphal network.

scholarly journals The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle.

1994 ◽  
Vol 125 (1) ◽  
pp. 143-158 ◽  
Author(s):  
J N McMillan ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Low Temperature ◽  
Mother Cell ◽  
Significant Proportion ◽  
Nuclear Migration ◽  
Cold Sensitivity ◽  
Chain Gene ◽  
Chromosome Loss ◽  
Lacz Gene ◽  
Yeast Saccharomyces Cerevisiae

JNM1, a novel gene on chromosome XIII in the yeast Saccharomyces cerevisiae, is required for proper nuclear migration. jnm1 null mutants have a temperature-dependent defect in nuclear migration and an accompanying alteration in astral microtubules. At 30 degrees C, a significant proportion of the mitotic spindles is not properly located at the neck between the mother cell and the bud. This defect is more severe at low temperature. At 11 degrees C, 60% of the cells accumulate with large buds, most of which have two DAPI staining regions in the mother cell. Although mitosis is delayed and nuclear migration is defective in jnm1 mutant, we rarely observe more than two nuclei in a cell, nor do we frequently observe anuclear cells. No loss of viability is observed at 11 degrees C and cells continue to grow exponentially with increased doubling time. At low temperature the large budded cells of jnm1 mutants exhibit extremely long astral microtubules that often wind around the periphery of the cell. jnm1 mutants are not defective in chromosome segregation during mitosis, as assayed by the rate of chromosome loss, or nuclear migration during conjugation, as assayed by the rate of mating and cytoduction. The phenotype of a jnm1 mutant is strikingly similar to that for mutants in the dynein heavy chain gene (Eshel, D., L. A. Urrestarazu, S. Vissers, J.-C. Jauniaux, J. C. van Vliet-Reedijk, R. J. Plants, and I. R. Gibbons. 1993. Proc. Natl. Acad. Sci. USA. 90:11172-11176; Li, Y. Y., E. Yeh, T. Hays, and K. Bloom. 1993. Proc. Natl. Acad. Sci. USA. 90:10096-10100). The JNM1 gene product is predicted to encode a 44-kD protein containing three coiled coil domains. A JNM1:lacZ gene fusion is able to complement the cold sensitivity and microtubule phenotype of a jnm1 deletion strain. This hybrid protein localizes to a single spot in the cell, most often near the spindle pole body in unbudded cells and in the bud in large budded cells. Together these results point to a specific role for Jnm1p in spindle migration, possibly as a subunit or accessory protein for yeast dynein.

scholarly journals The SH3/PH Domain Protein AgBoi1/2 Collaborates with the Rho-Type GTPase AgRho3 To Prevent Nonpolar Growth at Hyphal Tips of Ashbya gossypii

2006 ◽  
Vol 5 (10) ◽  
pp. 1635-1647 ◽  
Author(s):  
Philipp Knechtle ◽  
Jürgen Wendland ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Ashbya Gossypii ◽  
Ph Domain ◽  
Filamentous Ascomycete ◽  
Actin Cable ◽  
Single Deletion ◽  
Tip Extension ◽  
Green Fluorescent ◽  
Actin Cables ◽  
Polar Tip Growth

ABSTRACT Unlike most other cells, hyphae of filamentous fungi permanently elongate and lack nonpolar growth phases. We identified AgBoi1/2p in the filamentous ascomycete Ashbya gossypii as a component required to prevent nonpolar growth at hyphal tips. Strains lacking AgBoi1/2p frequently show spherical enlargement at hyphal tips with concomitant depolarization of actin patches and loss of tip-located actin cables. These enlarged tips can repolarize and resume hyphal tip extension in the previous polarity axis. AgBoi1/2p permanently localizes to hyphal tips and transiently to sites of septation. Only the tip localization is important for sustained elongation of hyphae. In a yeast two-hybrid experiment, we identified the Rho-type GTPase AgRho3p as an interactor of AgBoi1/2p. AgRho3p is also required to prevent nonpolar growth at hyphal tips, and strains deleted for both AgBOI1/2 and AgRHO3 phenocopied the respective single-deletion strains, demonstrating that AgBoi1/2p and AgRho3p function in a common pathway. Monitoring the polarisome of growing hyphae using AgSpa2p fused to the green fluorescent protein as a marker, we found that polarisome disassembly precedes the onset of nonpolar growth in strains lacking AgBoi1/2p or AgRho3p. AgRho3p locked in its GTP-bound form interacts with the Rho-binding domain of the polarisome-associated formin AgBni1p, implying that AgRho3p has the capacity to directly activate formin-driven actin cable nucleation. We conclude that AgBoi1/2p and AgRho3p support polarisome-mediated actin cable formation at hyphal tips, thereby ensuring permanent polar tip growth.

A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii

2000 ◽  
Vol 113 (24) ◽  
pp. 4563-4575 ◽  
Author(s):  
Y. Ayad-Durieux ◽  
P. Knechtle ◽  
S. Goff ◽  
F. Dietrich ◽  
P. Philippsen
Keyword(s):  
Protein Kinase ◽  
Filamentous Fungi ◽  
Fluorescent Protein ◽  
Ashbya Gossypii ◽  
Polarized Growth ◽  
Fast Growing ◽  
Filamentous Ascomycete ◽  
Green Fluorescent ◽  
Actin Cables ◽  
Developmental Switch

Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi, including Ashbya gossypii, polarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow-growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypii gene encodes a presumptive PAK (p21-activated kinase)-like kinase. Its closest homolog is the S. cerevisiae Cla4 protein kinase; the A. gossypii protein is therefore called AgCla4p. Agcla4 deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4 deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developed network of actin cables can be seen in Agcla4 deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.

scholarly journals Cytoplasmic dynein is involved in nuclear migration in Aspergillus nidulans.

1994 ◽  
Vol 91 (6) ◽  
pp. 2100-2104 ◽  
Author(s):  
X. Xiang ◽  
S. M. Beckwith ◽  
N. R. Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein

A Screen for Dynein Synthetic Lethals in Aspergillus nidulans Identifies Spindle Assembly Checkpoint Genes and Other Genes Involved in Mitosis

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 101-116
Author(s):  
Vladimir P Efimov ◽  
N Ronald Morris
Keyword(s):  
Aspergillus Nidulans ◽  
Spindle Assembly Checkpoint ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Spindle Assembly ◽  
Nuclear Migration ◽  
Cytoplasmic Dynein ◽  
Vesicle Transport ◽  
Synthetic Lethal ◽  
Checkpoint Genes

Abstract Cytoplasmic dynein is a ubiquitously expressed microtubule motor involved in vesicle transport, mitosis, nuclear migration, and spindle orientation. In the filamentous fungus Aspergillus nidulans, inactivation of cytoplasmic dynein, although not lethal, severely impairs nuclear migration. The role of dynein in mitosis and vesicle transport in this organism is unclear. To investigate the complete range of dynein function in A. nidulans, we searched for synthetic lethal mutations that significantly reduced growth in the absence of dynein but had little effect on their own. We isolated 19 sld (synthetic lethality without dynein) mutations in nine different genes. Mutations in two genes exacerbate the nuclear migration defect seen in the absence of dynein. Mutations in six other genes, including sldA and sldB, show a strong synthetic lethal interaction with a mutation in the mitotic kinesin bimC and, thus, are likely to play a role in mitosis. Mutations in sldA and sldB also confer hypersensitivity to the microtubule-destabilizing drug benomyl. sldA and sldB were cloned by complementation of their mutant phenotypes using an A. nidulans autonomously replicating vector. Sequencing revealed homology to the spindle assembly checkpoint genes BUB1 and BUB3 from Saccharomyces cerevisiae. Genetic interaction between dynein and spindle assembly checkpoint genes, as well as other mitotic genes, indicates that A. nidulans dynein plays a role in mitosis. We suggest a model for dynein motor action in A. nidulans that can explain dynein involvement in both mitosis and nuclear distribution.

scholarly journals From Function to Shape: A Novel Role of a Formin in Morphogenesis of the Fungus Ashbya gossypii

2006 ◽  
Vol 17 (1) ◽  
pp. 130-145 ◽  
Author(s):  
Hans-Peter Schmitz ◽  
Andreas Kaufmann ◽  
Michael Köhli ◽  
Pierre Philippe Laissue ◽  
Peter Philippsen
Keyword(s):  
Giant Cells ◽  
Polar Growth ◽  
Ashbya Gossypii ◽  
Essential Factor ◽  
Wild Type ◽  
Actin Cable ◽  
Lateral Branches ◽  
Actin Cables ◽  
Two Hybrid

Morphogenesis of filamentous ascomycetes includes continuously elongating hyphae, frequently emerging lateral branches, and, under certain circumstances, symmetrically dividing hyphal tips. We identified the formin AgBni1p of the model fungus Ashbya gossypii as an essential factor in these processes. AgBni1p is an essential protein apparently lacking functional overlaps with the two additional A. gossypii formins that are nonessential. Agbni1 null mutants fail to develop hyphae and instead expand to potato-shaped giant cells, which lack actin cables and thus tip-directed transport of secretory vesicles. Consistent with the essential role in hyphal development, AgBni1p locates to tips, but not to septa. The presence of a diaphanous autoregulatory domain (DAD) indicates that the activation of AgBni1p depends on Rho-type GTPases. Deletion of this domain, which should render AgBni1p constitutively active, completely changes the branching pattern of young hyphae. New axes of polarity are no longer established subapically (lateral branching) but by symmetric divisions of hyphal tips (tip splitting). In wild-type hyphae, tip splitting is induced much later and only at much higher elongation speed. When GTP-locked Rho-type GTPases were tested, only the young hyphae with mutated AgCdc42p split at their tips, similar to the DAD deletion mutant. Two-hybrid experiments confirmed that AgBni1p interacts with GTP-bound AgCdc42p. These data suggest a pathway for transforming one axis into two new axes of polar growth, in which an increased activation of AgBni1p by a pulse of activated AgCdc42p stimulates additional actin cable formation and tip-directed vesicle transport, thus enlarging and ultimately splitting the polarity site.

The microtubule motor cytoplasmic dynein is required for spindle orientation during germline cell divisions and oocyte differentiation in Drosophila

Development ◽  
1997 ◽  
Vol 124 (12) ◽  
pp. 2409-2419 ◽  
Author(s):  
M. McGrail ◽  
T.S. Hays
Keyword(s):  
Asymmetric Cell Division ◽  
Fruit Fly ◽  
Cytoplasmic Dynein ◽  
Spindle Orientation ◽  
Chain Gene ◽  
Animal Development ◽  
Dynein Motor ◽  
Microtubule Motor ◽  
Two Stages ◽  
Oocyte Differentiation

During animal development cellular differentiation is often preceded by an asymmetric cell division whose polarity is determined by the orientation of the mitotic spindle. In the fruit fly, Drosophila melanogaster, the oocyte differentiates in a 16-cell syncytium that arises from a cystoblast which undergoes 4 synchronous divisions with incomplete cytokinesis. During these divisions, spindle orientation is highly ordered and is thought to impart a polarity to the cyst that is necessary for the subsequent differentiation of the oocyte. Using mutations in the Drosophila cytoplasmic dynein heavy chain gene, Dhc64C, we show that cytoplasmic dynein is required at two stages of oogenesis. Early in oogenesis, dynein mutations disrupt spindle orientation in dividing cysts and block oocyte determination. The localization of dynein in mitotic cysts suggests spindle orientation is mediated by the microtubule motor cytoplasmic dynein. Later in oogenesis, dynein function is necessary for proper differentiation, but does not appear to participate in morphogen localization within the oocyte. These results provide evidence for a novel developmental role for the cytoplasmic dynein motor in cellular determination and differentiation.

Identification and molecular evolution of new dynein-like protein sequences in rat brain

1995 ◽  
Vol 108 (5) ◽  
pp. 1883-1893 ◽  
Author(s):  
Y. Tanaka ◽  
Z. Zhang ◽  
N. Hirokawa
Keyword(s):  
Rat Brain ◽  
Heavy Chain ◽  
Phylogenetic Trees ◽  
Cytoplasmic Dynein ◽  
Amino Acid Sequences ◽  
Adult Brain ◽  
Chain Gene ◽  
Evolutionary Analysis ◽  
Degenerate Primers ◽  
Dynein Heavy Chain

RT-PCR cloning was performed to find unknown members of the dynein superfamily expressed in rat brain. Six kinds of degenerate primers designed for the dynein catalytic domain consensuses were used for extensive PCR amplifications. We have sequenced 550 plasmid clones which turned out to include 13 kinds of new dynein-like sequences (DLP1-8, 9A/B, 10–12) and cytoplasmic dynein heavy chain. In these clones, alternative splicing was detected for a 105 nt-domain containing the CFDEFNRI consensus just downstream of the most N-terminal P-loop (DLP9A and 9B). By using these obtained sequences, initial hybridization studies were performed. Genomic Southern blotting showed each sequence corresponds to a single copy of the gene, while northern blotting of adult brain presented more than one band for some subtypes. We further accomplished molecular evolutionary analysis to recognize their phylogenetic origins for the axonemal and non-axonemal (cytoplasmic) functions. Different methods (UPGMA, NJ and MP) presented well coincident phylogenetic trees from 44 partial amino acid sequences of dynein heavy chain from various eukaryotes. The trunk for all the cytoplasmic dynein heavy chain homologues diverged directly from the root of the phylogenetic tree, suggesting that the first dynein gene duplication defined two distinct functions as respective subfamilies. Of particular interest, we found a duplication event of the cytoplasmic dynein heavy chain gene giving rise to another subtype, DLP4, located between the divergence of yeast and that of Dictyostelium. Such evolutionary topology builds up an inceptive hypothesis that there are at least two non-axonemal dynein heavy chains in mammals.

Mutations in the 8 kDa dynein light chain gene disrupt sensory axon projections in the Drosophila imaginal CNS

Development ◽  
1996 ◽  
Vol 122 (10) ◽  
pp. 2955-2963 ◽  
Author(s):  
R. Phillis ◽  
D. Statton ◽  
P. Caruccio ◽  
R.K. Murphey
Keyword(s):  
Light Chain ◽  
Cytoplasmic Dynein ◽  
Null Alleles ◽  
Chain Gene ◽  
Sensory Axon ◽  
Axon Pathfinding ◽  
Dynein Light Chain ◽  
Light Chain Gene ◽  
Point Of Entry ◽  
Mutant Female

Mutations in an 8 kDa (8x10(3) Mr) cytoplasmic dynein light chain disrupt sensory axon trajectories in the imaginal nervous system of Drosophila. Weak alleles are behaviorally mutant, female-sterile and exhibit bristle thinning and bristle loss. Null alleles are lethal in late pupal stages and alter neuronal anatomy within the imaginal CNS. We utilized P[Gal4] inserts to examine the axon projections of stretch receptor neurons and an engrailed-lacZ construct to characterize the anatomy of tactile neurons. In mutant animals both types of sensory neurons exhibited altered axon trajectories within the CNS, suggesting a defect in axon pathfinding. However, the alterations in axon trajectory did not prevent these axons from reaching their normal termination regions. In the alleles producing these neuronal phenotypes, expression of the cytoplasmic dynein 8 kDa light chain gene is completely absent. These results demonstrate a new function for the cytoplasmic dynein light chain in the regulation of axonogenesis and may provide a point of entry for studies of the role of cellular motors in growth cone guidance.

Search, capture and signal: games microtubules and centrosomes play

2001 ◽  
Vol 114 (2) ◽  
pp. 247-255 ◽  
Author(s):  
S.C. Schuyler ◽  
D. Pellman
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cytoplasmic Dynein ◽  
Cell Cycle Checkpoint ◽  
Cellular Polarity ◽  
Cycle Progression ◽  
Cycle Checkpoint ◽  
Dividing Cells ◽  
Actin Cables ◽  
Type V

Accurate distribution of the chromosomes in dividing cells requires coupling of cellular polarity cues with both the orientation of the mitotic spindle and cell cycle progression. Work in budding yeast has demonstrated that cytoplasmic dynein and the kinesin Kip3p define redundant pathways that ensure proper spindle orientation. Furthermore, it has been shown that the Kip3p pathway components Kar9p and Bim1p (Yeb1p) form a complex that provides a molecular link between cortical polarity cues and spindle microtubules. Recently, other studies indicated that the cortical localization of Kar9p depends upon actin cables and Myo2p, a type V myosin. In addition, a BUB2-dependent cell cycle checkpoint has been described that inhibits the mitotic exit network and cytokinesis until proper centrosome position is achieved. Combined, these studies provide molecular insight into how cells link cellular polarity, spindle position and cell cycle progression.

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