A β-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly

Oakley, Berl R; Morris, Nicholas P.

doi:10.1016/0092-8674(81)90109-4

Cited by 176 publications

(116 citation statements)

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“…Horio and Oakley (2005) have studied the effects of the antimicrotubule agent benomyl on tip growth. From these and previous studies (e.g., Oakley and Morris, 1981), it is clear that benomyl is an excellent antimicrotubule agent in A. nidulans. It causes rapid microtubule disassembly (all microtubules disassemble in Ͻ10 min after addition to the culture chamber), it is rapidly reversible (microtubules begin to reassemble in Ͻ5 min after benomyl is washed out), and it is quite specific for microtubules (Oakley and Morris, 1981).…”

Section: Effects Of the Anti-actin Agents Cytochalasin A And Latruncumentioning

confidence: 99%

“…From these and previous studies (e.g., Oakley and Morris, 1981), it is clear that benomyl is an excellent antimicrotubule agent in A. nidulans. It causes rapid microtubule disassembly (all microtubules disassemble in Ͻ10 min after addition to the culture chamber), it is rapidly reversible (microtubules begin to reassemble in Ͻ5 min after benomyl is washed out), and it is quite specific for microtubules (Oakley and Morris, 1981). Treatment with benomyl caused a dramatic reduction in tip growth rate but not a complete stoppage of growth (Horio and Oakley, 2005).…”

Section: Effects Of the Anti-actin Agents Cytochalasin A And Latruncumentioning

confidence: 99%

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The Tip Growth Apparatus ofAspergillus nidulans

Taheri-Talesh

Horio

Araújo‐Bazán

et al. 2008

MBoC

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Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkö rper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1-2 m behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position. INTRODUCTIONPolarized cell growth occurs in most eukaryotic phyla, and it includes a plethora of important phenomena, such as neuronal growth cone extension in animals and pollen tube extension in vascular plants. It is particularly important in filamentous fungi where nearly all growth occurs by hyphal tip extension (reviewed by Momany, 2002). Given that some filamentous fungi are important fermentation organisms, the growth of which is of considerable economic importance, whereas others are serious plant, animal, and human pathogens, there is considerable interest in the mechanisms of tip growth in these organisms.A great deal of progress has been made in understanding fungal tip growth (summarized by Harris et al., 2005; Steinberg, 2007a,b;Riquelme et al., 2007), but key questions remain unanswered. There is general agreement that fungal tip growth involves the synthesis of cell wall components in the cell body, the incorporation of these components into vesicles, the transport of these vesicles to the cell tip, the fusion of these vesicles with the plasma membrane in the area of the cell tip (exocytosis) to release their contents, and the cross-linking of the components after release. It is clear that both microtubules and actin microfilaments play important roles in fungal tip growth, but their exact functions are not yet defined. The exact sites of exocytosis and endocytosis also remain to be determined. The positions of the site(s) of exocytosis are particularly important because fungal walls are relatively stiff structures, and once they have formed the shape of the hypha is established. Hyphal shape is thus determined to a very significant extent by where the wall precursors are released from the cytoplasm, i.e., by the positioning of the site(s) of exocyto...

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Section: Effects Of the Anti-actin Agents Cytochalasin A And Latruncumentioning

confidence: 99%

Section: Effects Of the Anti-actin Agents Cytochalasin A And Latruncumentioning

confidence: 99%

The Tip Growth Apparatus ofAspergillus nidulans

Taheri-Talesh

Horio

Araújo‐Bazán

et al. 2008

MBoC

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274

414

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“…Benomyl, for example, depolymerizes the microtubule cytoskeleton (Ovechkina et al, 1999) but does not block germ tube formation (Oakley and Morris, 1980). Numerous ␣-and ␤-tubulin mutations have been identified in A. nidulans that alter microtubule functions in a variety of ways (Oakley and Morris, 1981;Oakley et al, , 1987a, but germ tubes form in each case. It should be noted that mipAR63 and benA33 caused a slight inhibition of germ tube formation in our experiments but much less than mipAR243.…”

Section: Inhibition Of Germ Tube Formation By Mipar243mentioning

confidence: 99%

Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

Jung

Prigozhina

Oakley

et al. 2001

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We have created 41 clustered charged-to-alanine scanning mutations of the mipA, ␥-tubulin, gene of Aspergillus nidulans and have created strains carrying these mutations by two-step gene replacement and by a new procedure, heterokaryon gene replacement. Most mutant alleles confer a wild-type phenotype, but others are lethal or conditionally lethal. The conditionally lethal alleles exhibit a variety of phenotypes under restrictive conditions. Most have robust but highly abnormal mitotic spindles and some have abnormal cytoplasmic microtubule arrays. Two alleles appear to have reduced amounts of ␥-tubulin at the spindle pole bodies and nucleation of spindle microtubule assembly may be partially inhibited. One allele inhibits germ tube formation. The cold sensitivity of two alleles is strongly suppressed by the antimicrotubule agents benomyl and nocodazole and a third allele is essentially dependent on these compounds for growth. Together our data indicate that ␥-tubulin probably carries out functions essential to mitosis and organization of cytoplasmic microtubules in addition to its well-documented role in microtubule nucleation. We have also placed our mutations on a model of the structure of ␥-tubulin and these data give a good initial indication of the functionally important regions of the molecule.

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“…Thus, the low critical concentration of CMR795 can be interpreted as enhanced polymerization. Oakley and Morris have also detected, by indirect means, hyperstabilization of microtubules in Aspergillus nidulans resistant to the antimicrotubule drug benomyl (28).…”

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Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

Keates¹,

Sarangi²,

Ling³

1981

Proc. Natl. Acad. Sci. U.S.A.

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We have examined mutant lines of Chinese hamster ovary cells that have increased resistance to the antimicrotubule drug Colcemid. Analysis of the functional properties of purified microtubule protein indicates that increased tolerance to the drug in vivo is reflected in altered properties of microtubules and tubulin in vitro. In this study, we have examined one series of related mutants and have found different microtubule alterations associated with each selection step. These changes include decreased Colcemid-binding affinity, an altered electrophoretic pattern of tubulin subcomponents, increased resistance to Colcemid inhibition of polymerization in vitro and, in one case, a de-.creased critical concentration for microtubule assembly. Characterized mutants of the class described here will be useful for probing the regulation of microtubule assembly in vivo.Microtubules are a major component ofthe mitotic spindle and cytoskeleton of eukaryotic cells (1). A characteristic of microtubules is their ability to assemble and disassemble under different conditions. Considerable effort has been expended on elucidating the control mechanisms for microtubule assembly (1-3). Microtubules are formed by polymerization of the globular protein, tubulin, and appear to be regulated by association with a variety ofadditional proteins, the microtubule-associated proteins (MAPs) (2). Several workers have developed kinetic models to describe the microtubule elongation reaction in vitro (4, 5), but in vivo regulation of assembly, in particular with respect to initiation, is less well understood (3). We feel that a genetic approach may provide an effective probe of regulatory mechanisms in vivo.To this end, we have isolated mutant clones of Chinese hamster ovary (CHO) cells resistant to Colcemid, with the expectation that these cells would possess altered microtubules (6). We report here the characterization of the properties of microtubules isolated from a related series of Colcemid-resistant mutants. The selection procedures used were designed to avoid the membrane mutants that have reduced drug permeability previously isolated (7,8 Microtubule protein was isolated from cells grown in monolayer culture, which were washed twice in phosphate-buffered saline and three times in an isotonic buffer of 0.2 M 2-(N-morpholino) ethanesulfonic acid (MES), pH 6.6/1 mM MgCl2/1 mM ethylene glycol bis(,/3aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)/2 mM 2-mercaptoethanol. To the washed cell pellet was added 0.7 vol of 1 mM MgCl2/L mM EGTA/2 mM 2-mercaptoethanol/1.7 mM GTP/0. 1 mM L-1-tosylamido-2-phenylethyl chloromethyl ketone/0.1 mM N-tosyl-L-lysine chloromethyl ketone adjusted to pH 6.6 with NaOH. Cells were broken by ultrasonic cavitation for three 1-min bursts at 0C (MSE sonicator). The suspension was centrifuged at 81,000 X gma for 40 min at 0C. Glycerol was added to the supernatant as recommended by Nagle et al. (9) to a final concentration of 5 M, and'GTP was added to a final concentration of 2.7 mM. The mixture was incub...

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A β-tubulin mutation in Aspergillus nidulans that blocks microtubule function without blocking assembly

Cited by 176 publications

References 18 publications

The Tip Growth Apparatus ofAspergillus nidulans

The Tip Growth Apparatus ofAspergillus nidulans

Alanine-scanning Mutagenesis ofAspergillusγ-Tubulin Yields Diverse and Novel Phenotypes

Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants.

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