Biology subjects

Crocker, A. W.

Publications and source records attributed to Crocker, A. W..

8 recordsLinked to original sources

bioRxiv2026

Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization. Although many condensates contain and regulate RNA, research has primarily focused on protein interactions. Here, we investigate RNA-protein interactions underlying cell cycle-regulating condensates in the multinucleate fungus Ashbya gossypii. These condensates form through interactions between G1 cyclin mRNA CLN3 and RNA-binding protein Whi3, which was predicted by homology to recognize a five-nucleotide motif repeated within the transcript. Natural variation in motif number in Ashbya strains led us to hypothesize that binding site valence may influence condensate properties. Using unbiased binding assays, we determined the preference of Ashbya Whi3 protein for specific primary RNA sequence and mutated individual Whi3-binding sites within CLN3 mRNA. Mutants exhibited distinct condensate properties despite having the same valence in terms of binding site number. Mutations altered the saturation concentration (Csat) and dense phase concentration of RNA and protein in cell-free reconstitution experiments. A subset of mutants, showed reduced number of condensates and deregulation of the cell cycle in cells. We also find that enhanced availability of single-stranded RNA can compensate for loss of binding sites. Together, these data indicate that differences in RNA protein binding-site context and not simply valence plays a critical role in determining condensate properties.

cell biology↗

bioRxiv2025

Mechanisms of nuclear segregation in a multinucleate multibudding yeast

Budding yeasts present an especially challenging geometry for segregation of chromosomes, which must be delivered across the narrow mother-bud neck into the bud. Studies in the model yeast Saccharomyces cerevisiae have revealed an elaborate set of mechanisms that selectively orient one mitotic spindle pole towards the bud and then drive spindle elongation along the mother-bud axis, ensuring nuclear segregation between mother and bud. It is unclear how these pathways might be adapted to yield similar precision in more complex cell geometries. Here we provide the first description of the dynamics of mitosis in a multi-nucleate, multibudding yeast, Aureobasidium pullulans, and identify many unexpected differences from uninucleate yeasts. Mitotic spindles do not orient along the mother-bud axis prior to anaphase, and accurate nuclear segregation often occurs after spindle disassembly. Cortical Num1-dynein forces pull highly mobile nuclei into buds, and once a nucleus enters a bud, it discourages others from entering, ensuring that most daughters inherit only one nucleus.

cell biology↗

bioRxiv2024

Negative feedback equalizes polarity sites in a multi-budding yeast

Morphogenesis in fungi and animals is directed by polarization of small GTPases Cdc42 and Rac. In the budding yeast Saccharomyces cerevisiae competition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeast Aureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1, and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.

cell biology↗

bioRxiv2022

In host evolution of Exophiala dermatitidis in cystic fibrosis lung micro-environment

Individuals with cystic fibrosis (CF) are susceptible to chronic lung infections that lead to inflammation and irreversible lung damage. While most respiratory infections that occur in CF are caused by bacteria, some are dominated by fungi such as the slow-growing black yeast Exophiala dermatitidis. Here, we analyze isolates of E. dermatitidis cultured from two samples, collected from a single subject two years apart. One isolate genome was sequenced using long-read Nanopore technology as an in-population reference to use in comparative single nucleotide polymorphism (SNP) and insertion-deletion (INDEL) variant analyses of twenty-three isolates. We then used population genomics and phylo-genomics to compare the isolates to each other as well as the type strain E. dermatitidis NIH/UT8656. Within the CF lung population, three E. dermatitidis clades were detected, each with varying mutation rates. Overall, the isolates were highly similar suggesting that they were recently diverged. All isolates were MAT 1-1, which was consistent with their high relatedness and the absence of evidence for mating or recombination between isolates. Phylogenetic analysis grouped sets of isolates into clades that contained isolates from both early and late time points indicating there are multiple persistent lineages. Functional assessment of variants unique to each clade identified alleles in genes that encode transporters, cytochrome P450 oxidoreductases, iron acquisition and DNA repair processes. Consistent with the genomic heterogeneity, isolates showed some stable phenotype heterogeneity in melanin production, subtle differences in antifungal minimum inhibitory concentrations, growth on different substrates. The persistent population heterogeneity identified in lung-derived isolates is an important factor to consider in the study of chronic fungal infections, and the analysis of changes in fungal pathogens over time may provide important insights into the physiology of black yeasts and other slow-growing fungi in vivo.

microbiology↗

bioRxiv2021

Calprotectin-mediated zinc chelation inhibits Pseudomonas aeruginosa protease activity in cystic fibrosis sputum

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to control cultures, but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc-starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to post-translationally inhibit zinc metalloprotease activity and thereby impact the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. ImportanceThe factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc-starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

microbiology↗

bioRxiv2019

Pseudomonas aeruginosa ethanol oxidation by AdhA in low oxygen environments

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its co-existence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation, and the accompanying decrease in pH, promotes P. aeruginosa survival in LB-grown stationary phase cultures. The transcription of adhA is elevated by hypoxia and in anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants have higher levels of Anr-regulated transcripts in low oxygen conditions compared to their wild type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than WT that is dependent on both anr and adhA. The large increase in AdhA activity similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification in anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa.\n\nImportanceEthanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol is relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase, AdhA, is responsible for ethanol catabolism and acetate accumulation in low oxygen conditions and that it is regulated by Anr.

microbiology↗

bioRxiv2019

The CAFA challenge reports improved protein function prediction and new functional annotations for hundreds of genes through experimental screens

The Critical Assessment of Functional Annotation (CAFA) is an ongoing, global, community-driven effort to evaluate and improve the computational annotation of protein function. Here we report on the results of the third CAFA challenge, CAFA3, that featured an expanded analysis over the previous CAFA rounds, both in terms of volume of data analyzed and the types of analysis performed. In a novel and major new development, computational predictions and assessment goals drove some of the experimental assays, resulting in new functional annotations for more than 1000 genes. Specifically, we performed experimental whole-genome mutation screening in Candida albicans and Pseudomonas aureginosa genomes, which provided us with genome-wide experimental data for genes associated with biofilm formation and motility (P. aureginosa only). We further performed targeted assays on selected genes in Drosophila melanogaster, which we suspected of being involved in long-term memory. We conclude that, while predictions of the molecular function and biological process annotations have slightly improved over time, those of the cellular component have not. Term-centric prediction of experimental annotations remains equally challenging; although the performance of the top methods is significantly better than expectations set by baseline methods in C. albicans and D. melanogaster, it leaves considerable room and need for improvement. We finally report that the CAFA community now involves a broad range of participants with expertise in bioinformatics, biological experimentation, biocuration, and bioontologies, working together to improve functional annotation, computational function prediction, and our ability to manage big data in the era of large experimental screens.

bioinformatics↗

bioRxiv2018

Profiling of bacterial and fungal microbial communities in cystic fibrosis sputum using RNA

Here, we report an approach to detect diverse bacterial and fungal taxa in complex samples by direct analysis of community RNA in one step using NanoString probe sets. We designed rRNA-targeting probe sets to detect forty two bacterial and fungal genera or species common in cystic fibrosis (CF) sputum, and demonstrated taxon-specificity of these probes as well as a linear response over more than three logs of input RNA. Culture-based analyses correlated qualitatively with relative abundance data on bacterial and fungal taxa obtained by NanoString and the analysis of serial samples demonstrated the use of this method to simultaneously detect bacteria and fungi and to detect microbes at low abundance without an amplification step. The relative abundances of bacterial taxa detected by analysis of RNA correlated with the relative abundances of the same taxa as measured by sequencing of the V4V5 region of the 16S rRNA gene amplified from community DNA from the same sample. We propose that this method may complement other methods designed to understand dynamic microbial communities, may provide information on bacteria and fungi in the same sample with a single assay, and, with further development, may provide quick and easily-interpreted diagnostic information on diverse bacteria and fungi at the genus or species level.\n\nImportanceHere we demonstrate the use of an RNA-based analysis of specific taxa of interest, including bacteria and fungi, within microbial communities. This multiplex method may be useful as a means to identify samples with specific combinations of taxa and to gain information on how specific populations vary over time and space or in response to perturbation. A rapid means to measure bacterial and fungal populations may aid in the study of host response to changes in microbial communities.

microbiology↗