The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

This is the publisher's version, also available electronically from "http://www.molbiolcell.org".The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-α-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew ≈5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10× reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells

Mitochondria and nuclei move by different mechanisms in Aspergillus nidulans

This is the publisher's version, also available electronically from "http://jcb.rupress.org".We have examined the effects of the antimicrotubule agent benomyl and several mutations on nuclear and mitochondrial movement in germlings of the filamentous fungus Aspergillus nidulans. While, as previously reported, benomyl inhibited nuclear division and movement, it did not inhibit mitochondrial movement. To test the effects of benomyl more rigorously, we germinated two benomyl super-sensitive, beta-tubulin mutants at a benomyl concentration 50-100 times greater than that required to inhibit colony formation completely. Again nuclear division and movement were inhibited, but mitochondrial movement was not. We also examined conditionally lethal beta-tubulin mutations that disrupt microtubule function under restrictive conditions. Nuclear division and movement were inhibited but, again, mitochondrial movement was not. Finally we examined the effects of five heat-sensitive mutations that inhibit nuclear movement but not nuclear division at restrictive temperatures. These mutations strongly inhibited nuclear movement at a restrictive temperature but did not inhibit mitochondrial movement. These data demonstrate that the mechanisms of nuclear and mitochondrial movement in Aspergillus nidulans are not identical and suggest that mitochondrial movement does not require functional microtubules

γ-Tubulin complexes in microtubule nucleation and beyond

Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases

Isolation of mip (microtubule interacting protein) mutations of Aspergillus nidulans

This is the publisher's version, also available electronically from "http://mcb.asm.org".We identified four mutations in two previously undescribed loci involved in microtubule function in Aspergillus nidulans as extragenic suppressors of benA33, a heat-sensitive beta-tubulin mutation. Three of the four mutations map to a locus closely linked to riboB on linkage group VIII; we designated this locus mipA (for microtubule-interacting protein). We were not able to map the remaining suppressor because of chromosomal rearrangements. However, since it recombines with riboB at a significantly higher frequency than the mipA alleles, it is unlikely to be in mipA; thus, we designated it mipB1. The mip mutations are not allelic to the previously identified loci that encode alpha- and beta-tubulin, and it is likely that mipA and mipB encode previously unidentified nontubulin proteins involved in microtubule function. Each of the mip mutations suppresses the heat sensitivity conferred by benA33 and suppresses the blockage of nuclear division and movement conferred by this mutation at high temperatures. Interactions between mipA and benA are allele specific. All of the mipA mutations are cryptic in a wild-type benA background but cause cold sensitivity in combination with benA33. These mutations also confer cold sensitivity in combination with benA31 and benA32 and reduce the resistance conferred by these mutations to the antimicrotubule agent benomyl but do not suppress the heat sensitivity conferred by these alleles. Finally, the mipA alleles suppress the heat sensitivity conferred by benA11, benA17, and benA21 but do not confer cold sensitivity in combination with these alleles

The use of beta-D-glucanase as a substitute for Novozyme 234 in immunofluorescence and protoplasting

Novozym 234 has been used for many years to prepare protoplasts of Aspergillus nidulans and other fungi for transformation. It has also been very useful in immunofluorescence studies for partially digesting walls of fixed hyphae or germlings to allow antibodies to penetrate into the cytoplasm. In recent years, the availability of Novozym 234 has become problematic, and we have searched for combinations of available enzymes that are suitable for protoplasting and immunofluorescence studies in A. nidulans

Alanine-scanning mutagenesis of Aspergillus γ-tubulin yields diverse and novel phenotypes

This is the publisher's version, also available electronically from "www.molbiolcell.org".We have created 41 clustered charged-to-alanine scanning mutations of the mipA, γ-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 γ-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 γ-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 γ-tubulin and these data give a good initial indication of the functionally important regions of the molecule

A mutation in γ-tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein Pkl1p

This is the publisher's version, also available electronically from "http://www.molbiolcell.org".Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp)Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component

Identification and molecular genetic analysis of the cichorine gene cluster in Aspergillus nidulans

We recently demonstrated that the phytotoxin cichorine is produced by Aspergillus nidulans. Through a set of targeted deletions, we have found a cluster of seven genes that are required for its biosynthesis. Two of the deletions yielded molecules that give information about the biosynthesis of this metabolite

Assembly of a heptameric STRIPAK complex is required for coordination of light-dependent multicellular fungal development with secondary metabolism in Aspergillus nidulans

This work is licensed under a Creative Commons Attribution 4.0 International License.Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively