Biology subjects

Arceo, X. G.

Publications and source records attributed to Arceo, X. G..

4 recordsLinked to original sources

bioRxiv2024

Quantitative comparison of single-cell RNA sequencing versus single-molecule RNA imaging for quantifying transcriptional noise

Stochastic fluctuations (noise) in transcription generate substantial cell-to-cell variability. However, how best to quantify genome-wide noise, remains unclear. Here we utilize a small-molecule perturbation (IdU) to amplify noise and assess noise quantification from numerous scRNA-seq algorithms on human and mouse datasets, and then compare to noise quantification from single-molecule RNA FISH (smFISH) for a panel of representative genes. We find that various scRNA-seq analyses report amplified noise, without altered mean-expression levels, for [~]90% of genes and that smFISH analysis verifies noise amplification for the vast majority of genes tested. Collectively, the analyses suggest that most scRNA-seq algorithms are appropriate for quantifying noise including a simple normalization approach, although all of these systematically underestimate noise compared to smFISH. From a practical standpoint, this analysis argues that IdU is a globally penetrant noise-enhancer molecule--amplifying noise without altering mean-expression levels--which could enable investigations of the physiological impacts of transcriptional noise. MOTIVATIONCell-to-cell variability in isogenic populations is predominantly attributed to the stochastic fluctuations (i.e., noise) in transcription. However, the quantification of this noise, particularly on a genome-wide scale, remains an open question. To address this general question, here we utilize a small-molecule perturbation reported to amplify transcriptional noise. Previous single-cell RNA-sequencing (scRNA-seq) analysis indicated that the pyrimidine nucleobase 5'-iodo-2'-deoxyuridine (IdU) amplifies noise but technical drawbacks of scRNA-seq may have obscured the penetrance and degree of noise amplification. Consequently, here we assess numerous scRNA-seq algorithms, on two different scRNA-seq datasets for a human and mouse cell type, for their ability to quantify noise amplification and then compare the results to noise quantification from single-molecule RNA FISH (smFISH) imaging for a panel of representative genes. The specific questions addressed are whether IdU amplifies noise in a globally or partially penetrant manner, and what scRNA-seq algorithm is most appropriate for quantifying transcriptional noise. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/607289v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f9b17aorg.highwire.dtl.DTLVardef@1f2d410org.highwire.dtl.DTLVardef@1407cfaorg.highwire.dtl.DTLVardef@cd2bc9_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

systems biology↗

bioRxiv2022

Complexes of tubulin oligomers and tau form an intervening network cross-bridging microtubules into bundles

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) that are implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. While the role of tau in MT bundling is poorly understood, tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in response to varying concentrations of divalent cations (Mg2+ or Ca2+) in dissipative reaction mixtures containing {beta}-tubulin, full-length tau, and GTP at 37{degrees}C. A concentration-time kinetic phase diagram generated by synchrotron small-angle X-ray scattering (SAXS) reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. Remarkably, SAXS analysis combined with TEM of plastic embedded samples provides direct evidence of an intervening network (IN) of complexes of tubulin oligomers and tau ({approx}5 nm wide filaments), which stabilize MT bundles. In this model, {beta}-tubulin oligomers in the IN are crosslinked by taus MT binding repeats, which also link {beta}-tubulin oligomers to {beta}-tubulin within the MT lattice. The finding of a new role for tubulin revises current dogma where cross-bridging of MTs is attributed entirely to interactions between MAPs. The tubulin-tau complexes of the IN should enhance the barrier properties of MT fascicles in preventing tau missorting to the somatodendritic compartment as happens during neurodegeneration. Furthermore, tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau promoting nucleation and growth of tau fibrils in tauopathies. Significance StatementA cell free model of microtubule (MT) bundles of the axon-initial-segment (known as MT fascicles) was studied in physiologically relevant buffer conditions. MT fascicles have a role in retaining neuronal protein tau, a key protein stabilizing MTs, in the axon. X-ray scattering and electron microscopy led to the discovery of complexes of tubulin oligomers and tau as building blocks of an intervening network that cross-bridge MTs into stable bundles with precisely the same linear geometry observed in-vivo in neurons. Significantly, changes to the chemical structure of tau because of abnormal interactions with cellular enzymes, would be predicted to disrupt the intervening tubulin-tau network and the MT-fascicles barrier function, promoting leakage of tau to the somatodendritic compartment and neuron degradation.

biophysics↗

bioRxiv2022

Mitochondrial mRNA localization is governed by translation kinetics and spatial transport

For many nuclear-encoded mitochondrial genes, mRNA localizes to the mitochondrial surface co-translationally, aided by the association of a mitochondrial targeting sequence (MTS) on the nascent peptide with the mitochondrial import complex. For a subset of these co-translationally localized mRNAs, their localization is dependent on the metabolic state of the cell, while others are constitutively localized. To explore the differences between these two mRNA types we developed a stochastic, quantitative model for MTS-mediated mRNA localization to mitochondria in yeast cells. This model includes translation, applying gene-specific kinetics derived from experimental data; and diffusion in the cytosol. Even though both mRNA types are co-translationally localized we found that the steady state number, or density, of ribosomes along an mRNA was insufficient to differentiate the two mRNA types. Instead, conditionally-localized mRNAs have faster translation kinetics which modulate localization in combination with changes to diffusive search kinetics across metabolic states. Our model also suggests that the MTS requires a maturation time to become competent to bind mitochondria. Our work indicates that yeast cells can regulate mRNA localization to mitochondria by controlling mitochondrial volume fraction (influencing diffusive search times) and gene translation kinetics (adjusting mRNA binding competence) without the need for mRNA-specifc binding proteins. These results shed light on both global and gene-specific mechanisms that enable cells to alter mRNA localization in response to changing metabolic conditions.

biophysics↗

bioRxiv2019

Mitochondrial volume fraction controls translation of nuclear-encoded mitochondrial proteins

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. This control is particularly complicated for mitochondria, as they must coordinate gene expression from both the nuclear and mitochondrial genome. We have found that cells are able to use this dynamic morphology to post-transcriptionally coordinate protein expression with the metabolic demands of the cell through enhanced mRNA localization to the mitochondria. As yeast switch to respiratory metabolism, they increase their mitochondrial volume fraction that is, the ratio of mitochondrial volume to intracellular volume which drives the localization of nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through artificial tethering experiments, we show that this mitochondrial localization is sufficient to increase protein production, whereas sequestering mRNAs away from the mitochondrial surface decreases protein production, and those cells are deficient in growth in respiratory conditions. Furthermore, we find that this mRNA sensitivity to mitochondrial volume fraction is driven by the speed of translation downstream of the mitochondrial targeting sequence (MTS), as local ribosome stalling through a stretch of polyprolines in the nascent peptide can drive constitutive localization of mRNAs to the mitochondria. This points to a mechanism by which organelle volume fraction provides feedback to regulate organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome.

cell biology↗