Lis1 is an initiation factor for dynein-driven organelle transport

The molecular motor cytoplasmic dynein is responsible for most minus-end–directed, microtubule-based transport in eukaryotic cells. It is especially important in neurons, where defects in microtubule-based motility have been linked to neurological diseases. For example, lissencephaly is caused by mutations in the dynein-associated protein Lis1. In this paper, using the long, highly polarized hyphae of the filamentous fungus Aspergillus nidulans, we show that three morphologically and functionally distinct dynein cargos showed transport defects in the genetic absence of Lis1/nudF, raising the possibility that Lis1 is ubiquitously used for dynein-based transport. Surprisingly, both dynein and its cargo moved at normal speeds in the absence of Lis1 but with reduced frequency. Moreover, Lis1, unlike dynein and dynactin, was absent from moving dynein cargos, further suggesting that Lis1 is not required for dynein-based cargo motility once it has commenced. Based on these observations, we propose that Lis1 has a general role in initiating dynein-driven motility

Characterization of the Mutagenic Spectrum of 4-Nitroquinoline 1-Oxide (4-NQO) in Aspergillus nidulans by Whole Genome Sequencing

4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO−based genetic screens have been used to study diverse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO−induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing

The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation

We thank Cowen lab members for helpful discussions. We also thank David Rogers (University of Tennessee) for sharing microarray analysis of the CAS5 homozygous mutant, and Li Ang (University of Macau) for assistance in optimizing the ChIP-Seq experiments. J.L.X. is supported by a Canadian Institutes of Health Research Doctoral award and M.D.L. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072). B.T.G. holds an Ontario Graduate Scholarship. C.B. and B.J.A. are supported by the Canadian Institutes of Health Research Foundation Grants (FDN-143264 and -143265). D.J.K. is supported by a National Institute of Allergy and Infectious Diseases grant (1R01AI098450) and J.D.L.C.D. is supported by the University of Rochester School of Dentistry and Medicine PREP program (R25 GM064133). A.S. is supported by the Creighton University and the Nebraska Department of Health and Human Services (LB506-2017-55). K.H.W. is supported by the Science and Technology Development Fund of Macau S.A.R. (FDCT; 085/2014/A2). L.E.C. is supported by the Canadian Institutes of Health Research Operating Grants (MOP-86452 and MOP-119520), the Natural Sciences and Engineering Council (NSERC) of Canada Discovery Grants (06261 and 462167), and an NSERC E.W.R. Steacie Memorial Fellowship (477598).Peer reviewedPublisher PD

Hsf1 and Hsp90 orchestrate temperature-dependent global transcriptional remodelling and chromatin architecture in Candida albicans

We thank Karim Gharbi and Urmi Trivedi for their assistance with RNA sequencing, carried out in the GenePool genomics facility (University of Edinburgh). We also thank Susan Fairley and Eduardo De Paiva Alves (Centre for Genome Enabled Biology and Medicine, University of Aberdeen) for help with the initial bioinformatics analysis. We thank Aaron Mitchell for kindly providing the ALS3 mutant, Julian Naglik for the gift of TR146 cells, and Jon Richardson for technical assistance. We thank the Genomics and Bioinformatics core of the Faculty of Health Sciences for Next Generation Sequencing and Bioinformatics support, the Information and Communication Technology Office at the University of Macau for providing access to a High Performance Computer and Jacky Chan and William Pang for their expert support on the High Performance Computer. Finally, we thank Amanda Veri for generating CaLC2928. M.D.L. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072), R.A.F. by a Wellcome Trust-Massachusetts Institute of Technology (MIT) Postdoctoral Fellowship, L.E.C. by a Canada Research Chair in Microbial Genomics and Infectious Disease and by Canadian Institutes of Health Research Grants MOP-119520 and MOP-86452, A.J. P.B. was supported by the UK Biotechnology and Biological Sciences Research Council (BB/F00513X/1) and by the European Research Council (ERC-2009-AdG-249793-STRIFE), KHW is supported by the Science and Technology Development Fund of Macau S.A.R (FDCT) (085/2014/A2) and the Research and Development Administrative Office of the University of Macau (SRG2014-00003-FHS) and R.T.W. by the Burroughs Wellcome fund and NIH R15AO094406. Data availability RNA-sequencing data sets are available at ArrayExpress (www.ebi.ac.uk) under accession code E-MTAB-4075. ChIP-seq data sets are available at the NCBI SRA database (http://www.ncbi.nlm.nih.gov) under accession code SRP071687. The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author upon request.Peer reviewedPublisher PD

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this record. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (65) partner repository with the data set identifier PXD018967. ChIP-seq results were submitted to NCBI under accession number SUB7547091.Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C_2 H_2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. IMPORTANCE: In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.Medical Research Council (MRC)Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Technical University of Munich-Institute for Advanced Study (TUMIAS

A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here, we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics, but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified 7 mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct velocity requirements

Aspergillus Myosin-V Supports Polarized Growth in the Absence of Microtubule-Based Transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV

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A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocit

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

<div><p>The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen <i>Candida albicans</i> is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of <i>C</i>. <i>albicans HSF1</i> induces morphogenesis in the absence of external cues. <i>HSF1</i> depletion compromises Hsp90 function, thereby driving filamentation. <i>HSF1</i> overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.</p></div