Biology subjects

Kashina, A.

Publications and source records attributed to Kashina, A..

9 recordsLinked to original sources

bioRxiv2024

Amino acid-level differences in alpha tubulin sequences are uniquely required for meiosis

Tubulin is the major structural constituent of the microtubule cytoskeleton. Yeast Schizosaccharomyces pombe contain two - tubulins genes, nda2 and atb2, that are highly functionally distinct: nda2 deletion is lethal, while lack of atb2 does not interfere with cell viability. The functional determinants underlying this distinction are unknown. Here we used CRISPR-Cas9 gene editing to generate a yeast strain expressing Atb2 amino acid sequence utilizing Nda2 codon usage in the native Nda2 locus. Such Nda2-coded Atb2 (NCA) yeast, unlike Nda2 knockout, were viable and displayed no visible abnormalities in cell morphology or vegetative life cycle. However, these NCA yeast showed strong impairments in sporulation and meiosis, including major meiotic delays and high rates of abnormal chromosome segregation. Our data indicate that the amino acid sequence of Nda2 is uniquely required for normal meiosis, and identify a novel determinant that underlies functional distinction between closely related tubulin isoforms.

cell biology↗

bioRxiv2023

Fecal microbiomics biomarkers for Chronic Wasting Disease

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the US. Here we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the US, compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer. At each rank level, these taxa and taxa pairs could facilitate identification of around 70% of both the CWD-negative and the CWD-positive samples. Our results provide a potential tool for diagnostics and surveillance of CWD in the wild, as well as conceptual advances in our understanding of the disease. ImportanceThis is a comprehensive study that tests the connection between the composition of the gut microbiome in deer in response to Chronic Wasting Disease (CWD). We analyzed 50 fecal samples obtained from CWD-positive animals compared to 50 CWD-negative controls to identify CWD-dependent changes in the gut microbiome, matched with the analysis of fecal metabolites. Our results show promising trends suggesting that fecal microbial composition can directly correspond to CWD disease status. These results point to microbial composition of the feces as a potential tool for diagnostics and surveillance of CWD in the wild, including non-invasive CWD detection in asymptomatic deer and deer habitats, and enable conceptual advances in our understanding of the disease.

microbiology↗

bioRxiv2023

β-actin is essential for structural integrity and physiological function of the retina

Lack of non-muscle {beta}-actin gene (Actb) leads to early embryonic lethality in mice, however mice with {beta}- to{gamma} -actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated de-generation of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS. These defects are accompanied by changes in the F-actin binding of several key actin interacting partners, including ezrin, myosin, talin, and vinculin known to play central roles in modulating actin cytoskeleton and cell adhesion and mediating the phagocytosis of OS. Our data show that {beta}-actin protein is essential for maintaining normal retinal structure and function.

cell biology↗

bioRxiv2022

Functional interplay between arginyl-tRNA synthetases and arginyltransferase

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance between ATE1, RARS and translation are unknown. Here we addressed the functional interplay between these mechanisms using intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We find that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1s redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence between translation, arginyl-tRNA synthesis, and arginylation. Key PointsO_LIIntracellular arginylation depends on the physiological state of the cell, but does not compete with the translation machinery C_LIO_LIA fraction of ATE1 binds directly to both long and short Arg-tRNA synthetases (RARS) C_LIO_LIDisplacement of long RARS from the multi-tRNA synthetase complex increases cytosolic fraction and activity of ATE1 C_LI

cell biology↗

bioRxiv2021

Protein Posttranslational Signatures Identified in COVID-19 Patient Plasma

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a highly contagious virus of the coronavirus family that causes coronavirus disease-19 (COVID-19) in humans and a number of animal species. COVID-19 has rapidly propagated in the world in the past 2 years, causing a global pandemic. Here, we performed proteomic analysis of plasma samples from COVID-19 patients compared to healthy control donors in an exploratory study to gain insights into protein-level changes in the patients caused by SARS-CoV-2 infection and to identify potential proteomic and posttranslational signatures of this disease. Our results suggest a global change in protein processing and regulation that occurs in response to SARS-CoV-2, and the existence of a posttranslational COVID-19 signature that includes an elevation in threonine phosphorylation, a change in glycosylation, and a decrease in arginylation, an emerging posttranslational modification not previously implicated in infectious disease. This study provides a resource for COVID-19 researchers and, longer term, will inform our understanding of this disease and its treatment. Key PointsO_LIPlasma from COVID-19 patients exhibits prominent protein- and peptide-level changes C_LIO_LIProteins from COVID-19 patient plasma exhibit prominent changes in several key posttranslational modifications C_LI

biochemistry↗

bioRxiv2021

Differential N-terminal processing of beta and gamma actin in vivo

Actin is one of the most essential and abundant intracellular proteins, playing an essential physiological role as the major constituent of the actin cytoskeleton. Two cytoplasmic actins, beta- and gamma-actin, are encoded by different genes, but their amino acid sequences differ only by four conservative substitutions at the N-terminus, making it very difficult to dissect their individual regulation in vivo. The majority of actins are N-terminally acetylated, following the removal of N-terminal Met. Here, we analyzed beta and gamma cytoplasmic actin N-termini in vivo and found that beta actin, unlike gamma actin, specifically undergoes sequential removal of N-terminal amino acid Asp residues. This processing affects [~]1-3% of beta actin in different cell types. We identified candidate enzymes capable of mediating this type of processing, and used CRISPR/Cas-9 to delete them, individually or together, in mammalian cell lines. This deletion abolishes most of the beta actin N-terminal processing and results in changes in F-actin levels, cell spreading, filopodia formation, and cell migration, suggesting that the beta actin processing mediated by these enzymes is physiologically important to beta actin function. We propose that selective N-terminal processing of beta actin by sequential removal of Asp contributes to differentiating the functions of non-muscle actin isoforms in vivo.

cell biology↗

bioRxiv2021

Different translation dynamics of β- and γ-actin regulates cell migration

{beta}- and {gamma}-cytoplasmic actins are ubiquitously expressed in every cell type and are nearly identical at the amino acid level but play vastly different roles in vivo. Their essential roles in embryogenesis and cell migration critically depend on the nucleotide sequences of their genes, rather than their amino acid sequence. However it is unclear which gene elements underlie this effect. Here we address the specific role of the coding sequence in {beta}- and {gamma}-cytoplasmic actins intracellular functions, using stable cell lines with exogenously expressed actin isoforms and their "codon-switched" variants. When targeted to the cell periphery using the {beta}-actin 3'UTR, {beta}-actin and {gamma}-actin have differential effects on cell migration. These effects directly depend on the coding sequence. Single molecule measurements of actin isoform translation, combined with fluorescence recovery after photobleaching, demonstrate a pronounced difference in {beta}- and {gamma}-actins translation elongation rates, leading to changes in their dynamics at focal adhesions, impairments in actin bundle formation, and reduced cell anchoring to the substrate during migration. Our results demonstrate that coding sequence-mediated differences in actin translation play a key role in cell migration.

cell biology↗

bioRxiv2020

New mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy.

Fn1 fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live imaging and single-molecule localization microscopy (SMLM) are inconsistent with this traditional view and show that Fn1 fibrils are composed of roughly spherical nanodomains containing 6-11 Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, wherein nanodomains are spaced at the average periodicity of 105{+/-}17 nm. Periodical Fn1 nanodomain arrays are bona fide fibrils: they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. FUD, a bacterial peptide that binds Fn1 N-terminus and disrupts Fn1 fibrillogenesis does not disrupt the formation of Fn1 nanodomains, instead, it blocks the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis.

cell biology↗

bioRxiv2017

Diverse functions of closely homologous actin isoforms are defined by their nucleotide, rather than their amino acid sequence.

{beta}- and {gamma}-cytoplasmic-actin are nearly indistinguishable in their amino acid sequence, but are encoded by different genes that play non-redundant biological roles. The key determinants that drive their functional distinction are unknown. Here we tested the hypothesis that {beta}- and {gamma}-actin functions are defined by their nucleotide, rather than their amino acid sequence, using targeted editing of the mouse genome. Although previous studies have shown that disruption of {beta}-actin gene critically impacts cell migration and mouse embryogenesis, we demonstrate here that generation of a mouse lacking {beta}-actin protein by editing {beta}-actin gene to encode {gamma}-actin protein, and vice versa, does not affect cell migration and/or organism survival. Our data suggest that the essential in vivo function of {beta}-actin is provided by the gene sequence independent of the encoded protein isoform. We propose that this regulation constitutes a global \"silent code\" mechanism that controls the functional diversity of protein isoforms.

cell biology↗