Biology subjects

McCroskey, S.

Publications and source records attributed to McCroskey, S..

8 recordsLinked to original sources

bioRxiv2026

Nascent protein retention at polysomes reduces kinetic barriers to self-assembly

Living proteomes are necessarily far from equilibrium. It is paradoxical, then, that reducing the translation of new proteins -- which should promote equilibration -- instead prolongs life. We investigated the impact of translational flux to nucleation barriers that preserve the solubility of proteins destined to form amyloids or other assemblies. By manipulating translation initiation rates directly or indirectly, across yeast and human cells, and across a variety of supersaturable proteins, we find that accelerating translation initiation broadly accelerates nucleation irrespective of their global concentrations. We showed that this effect was confined to polysomes and was enhanced by N-terminal placement or other features that retained the nascent aggregating domain at polysomes. Finally, we show that intrinsically disordered regions with high tendencies to self-associate are specifically positioned to do so co-translationally, providing evidence that cotranslational nucleation has shaped proteome evolution.

cell biology↗

bioRxiv2026

Pericentromeric repeat copy number tunes heterochromatin dosage to control chromosome segregation and gene expression in fission yeast

Centromeres are essential for chromosome segregation, yet in many genomes they are composed entirely of rapidly evolving repetitive DNA, embedded in other repetitive DNA that forms pericentromeric heterochromatin. Due to the difficulties of manipulating these repeat-rich regions, how the relative size of pericentromeric repeat regions influences chromosome segregation remains an open question. Here, we take advantage of the tractable Schizosaccharomyces pombe system by combining population-level analysis, complete long-read assemblies, and engineered near-isogenic strains to test how pericentromeric repeat copy number affects chromosome biology in its native context. We find that pericentromeric dh/dg arrays on chromosome 3 vary almost tenfold in size among natural S. pombe isolates, ranging from 35 to 265 kb. We converted this natural diversity into an experimental system of nearly isogenic strains that primarily differ in pericentromere size (35 to >350 kb). We found that pericentromere size does not alter baseline growth under standard conditions. However, larger pericentromeres alter transcriptional output and sensitize cells to spindle stress. We show that this spindle-stress phenotype depends on heterochromatin: loss of the H3K9 methyltransferase Clr4 abolishes size-dependent differences, whereas artificial targeting of the Chromosomal Passenger Complex to heterochromatin partially rescues the defect. Thus, we find that larger pericentromeres act as sinks for limiting regulatory factors, weakening their effective concentration at centromeres and compromising faithful chromosome segregation under stress. These results establish that naturally occurring copy-number variation within repetitive pericentromeric DNA is not merely noise, but a functional source of variation in chromosome segregation and gene regulation. Our work provides an experimentally tractable framework for understanding how repeat expansion in centromere-proximal heterochromatin influences chromosome behavior across eukaryotes.

genetics↗

bioRxiv2021

Diverse mating phenotypes impact the spread of wtf meiotic drivers in S. pombe

Meiotic drivers are genetic loci that break Mendels law of segregation to be transmitted into more than half of the offspring produced by a heterozygote. The success of a driver relies on outcrossing because drivers gain their advantage in heterozygotes. It is, therefore, curious that Schizosaccharomyces pombe, a species reported to rarely outcross, harbors many meiotic drivers. To address this paradox, we measured mating phenotypes in S. pombe natural isolates. We found that the propensity to inbreed varies between natural isolates and can be affected both by cell density and by the available sexual partners. Additionally, we found that the observed level of inbreeding slows, but does not prevent, the spread of a wtf meiotic driver in the absence of additional fitness costs. These analyses reveal parameters critical to understanding the evolution of S. pombe and help explain the success of meiotic drivers in this species.

evolutionary biology↗

bioRxiv2021

Proteome plasticity in response to persistent environmental change

Temperature is a variable component of the environment and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses resulting in the refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here, we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to the reduction of temperature sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and importantly, cellular functions. We postulate that in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

molecular biology↗

bioRxiv2020

High-throughput identification of nuclear envelope protein interactions in Schizosaccharomyces pombe using an arrayed membrane yeast-two hybrid library

The nuclear envelope (NE) contains a specialized set of integral membrane proteins that maintain nuclear shape and integrity and influence chromatin organization and gene expression. Advances in proteomics techniques and studies in model organisms have identified hundreds of proteins that localize to the NE. However, the function of many of these proteins at the NE remains unclear, in part due to a lack of understanding of the interactions that these proteins participate in at the NE membrane. To assist in the characterization of NE transmembrane protein interactions we developed an arrayed library of integral and peripheral membrane proteins in the fission yeast Schizosaccharomyces pombe for high-throughput screening using the split-ubiquitin based membrane yeast two hybrid sys-tem. We used this approach to characterize protein interactions for three conserved proteins that localize to the inner nu-clear membrane: Cut11/Ndc1, Lem2, and Ima1/Samp1/NET5. Additionally, we determined how the interaction network for Cut11 is altered in canonical temperature-sensitive cut11 mutants. This library and screening approach is readily applicable to characterizing the interactomes of integral membrane proteins localizing to various subcellular compartments.

cell biology↗

bioRxiv2020

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids rescues SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that acquisition of lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes. Author SummaryPloidy is the number of whole sets of chromosomes in a species. Most eukaryotes alternate between a diploid (two copy) and haploid (one copy) state during their life and sexual cycle. However, as part of normal human development, specific tissues increase their DNA content. This gain of entire sets of chromosomes is known as polyploidization, and it is observed in invertebrates, plants and fungi, as well. Polyploidy is thought to improve fitness under stressful conditions and promote evolutionary diversity, but how ploidy is determined is poorly understood. Here, we use budding yeast to investigate mechanisms underlying the ploidy of wild-type cells and specific mutants that affect the centrosome, a conserved structure involved in chromosome segregation during cell division. Our work suggests that different scaling relationships (allometry) between the genome and cellular structures underlies alterations in ploidy. Furthermore, mutations in cellular structures with incompatible allometric relationships with the genome may drive genomic changes such duplications, which are underly the evolution of many species including both yeasts and humans.

genetics↗

bioRxiv2020

The wtf4 meiotic driver utilizes controlled protein aggregation to generate selective cell death

Meiotic drivers are parasitic loci that force their own transmission into greater than half of the offspring of a heterozygote. Many drivers have been identified, but their molecular mechanisms are largely unknown. The wtf4 gene is a meiotic driver in Schizosaccharomyces pombe that uses a poison-antidote mechanism. Here, we show that the Wtf4 proteins can function outside of gametogenesis and in a distantly related species, Saccharomyces cerevisiae. The Wtf4poison protein forms dispersed, toxic aggregates. The similar Wtf4antidote protein also forms aggregates but is sequestered within or near vacuoles and is mostly benign. The Wtf4antidote can co-assemble with the Wtf4poison and promote its trafficking to vacuoles. We show that neutralization of the Wtf4poison requires both co-assembly with the Wtf4antidote and aggregate sequestration, as mutations that disrupt either of these processes results in cell death. This work reveals that wtf parasites can exploit protein aggregate management pathways to selectively destroy gametes.

cell biology↗

bioRxiv2018

Asi1 regulates the distribution of proteins at the inner nuclear membrane in Saccharomyces cerevisiae

Inner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape and stability. Mechanisms that drive changes in the INM proteome are poorly understood in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation (ERAD) and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most pronounced effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggests that Asi1 not only removes mis-targeted proteins at the INM, but it also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33. Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the NE through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.

cell biology↗