Biology subjects

Chang, F.

Publications and source records attributed to Chang, F..

At least 19 recordsLinked to original sources

bioRxiv2026

Foxp1 Coordinates Early Temporal Identity and Neurogenic Progression in Retinal Progenitors

Neural diversity depends on both the temporal competence of progenitors to generate particular cell types and their progression into a neurogenic state. How these distinct features of progenitor development are mechanistically coordinated remains poorly understood. Here, we identify Foxp1 as a dual function transcriptional regulator that couples early progenitor identity to neurogenic progression in the developing retina. By defining its direct transcriptional targets, we show that Foxp1 coordinates complementary regulatory programs: it sustains early competence by activating early-stage retinal progenitor genes and repressing late temporal factors, while promoting neurogenic progression through activation of the proneural factors Atoh7 and Neurog2. We establish a cis-regulatory mechanism linking these functions: Foxp1 activates Atoh7 through its remote shadow enhancer, and deletion of this enhancer prevents Foxp1-dependent induction of Atoh7 and retinal ganglion cell (RGC) production. We further reveal that Neurog2 partially compensates for loss of Atoh7 function. This coordinated early progenitor program is temporally restricted by progressive silencing of Foxp1, involving sequential deposition of H2AK119ub1 followed by H3K27me3 at a distal conserved silencer essential for its repression. Together, these findings establish a mechanistic link between temporal competence and neurogenic progression, revealing how a shared transcriptional regulator coordinates progenitor identity with neuronal production through distinct cis-regulatory elements.

developmental biology↗

bioRxiv2026

Nuclear size and physical properties of the nucleoplasm are determined by colloid osmotic pressure at the nuclear envelope

The size of the nucleus scales with cell size, suggesting a universal scaling rule. Yet the biophysical determinants of nuclear size and the significance and consequences of altered nuclear-to-cell (N/C) ratios, which are observed across diverse pathological states and cell-fate transitions, remain poorly understood. Recent theoretical models propose that nuclear size arises from a balance of colloid osmotic pressures generated by macromolecules in the nucleoplasm and cytoplasm. Here we demonstrate that altering this osmotic balance through massive overexpression of an exogenous protein targeted to either the nucleoplasm or cytoplasm produces predictable changes in the N/C ratio in S. pombe. These quantitative perturbations show that nuclear size is set primarily by the number of proteins in the nucleus and cytoplasm, providing strong support for a pure osmotic pressure mechanism. Furthermore, cells with altered N/C ratios display tunable changes in nucleoplasmic crowding, nuclear condensate formation, nucleolar scaling and heterochromatin organization, establishing a causal link between nuclear size and gene regulatory processes. These findings reveal how cells exploit osmotic forces to set organelle dimensions, with broad implications for understanding how nuclear size shapes gene expression and cell identity in health and disease.

biophysics↗

bioRxiv2026

Klf4 overexpression remodels chromatin to reprogram late retinal progenitors toward an retinal ganglion cell-like fate

Developing neuron-replacement therapies for retinal ganglion cells (RGCs) lost to injury or disease requires a deeper understanding of how restriction to cell identity acquisition may be overcome. Previously, we showed that overexpression of Klf4 in late retinal progenitor cells (late RPCs), which are normally restricted from RGC production, is sufficient to produce cells that display a subset of canonical RGC properties including RGC-associated gene expression and morphological features. In the present study, we investigated the transcriptional and epigenetic mechanisms by which Klf4 overexpression influences the fate of cell types generated from late RPCs. scRNA-seq analysis revealed that Klf4 induces transcriptional changes, with some cells exhibiting gene expression profiles similar to those of resident RGCs. In addition, we observed widespread changes in chromatin accessibility, suggesting that KLF4 remodels the chromatin of late RPCs and influences their transcriptional profile. Our findings show KLF4-driven reprogramming of late RPCs, providing insight into progenitor competence and fate specification to an RGC-like identity. These results suggest that KLF4 could be a component in regenerative therapies due to its ability to reprogram and induce RGC genes outside of the normal RGC developmental window.

neuroscience↗

bioRxiv2025

Analyses of bent spindles reveal the mechanics of anaphase B in fission yeast

The mitotic spindle in the fission yeast Schizosaccharomyces pombe is a single bundle of microtubules which elongates to segregate the chromosomes during anaphase B. The mechanical properties of the spindle and the forces driving its elongation remain poorly defined. Here, we analyzed how spindles react to mechanical and genetic perturbations to uncover their mechanical properties. Treatment of cells with osmotic oscillations and blue light led to a consistent phenotype of spindle buckling and breakage in mid-anaphase. The stalling of pole separation and reduced rates of spindle elongation indicated that spindles elongate and buckle under increased mechanical load. The structural integrity of the bent spindles was dependent on Ase1 (PRC1), while the spindle elongation rate was dependent on motor proteins Klp9 (kinesin-6) and Cut7 (kinesin-5). Modeling of bent spindle shapes revealed that most spindles behave mechanically as a beam with a two-fold increase in rigidity in the midzone. Upon reaching a threshold size, bent spindles broke at a specific fragile site near the edge of the spindle midzone. Our findings in this simple fission yeast spindle are relevant to the mechanics of more complex metazoan spindles. Significance statementO_LIThe anaphase B spindle in S. pombe consists of a microtubule bundle that elongates to move the chromosomes apart. The various forces and mechanical properties of the spindle remain poorly quantified. C_LIO_LIThe authors establish a method to induce spindle buckling in mid-anaphase. Time-lapse imaging shows that these spindles elongate at reduced rates, buckle as a non-homogeneous beam under mechanical load, and break at a fragile site adjacent to the midzone. C_LIO_LIThese results provide quantitative and molecular insights into spindle force regulation and structural integrity that are relevant to mitosis in other cell types. C_LI

cell biology↗

bioRxiv2025

Role of Age in Mediating the Association Between the Vaginal Microbiota and Preterm Birth

The vaginal microbiota plays a crucial role in vaginal health and pregnancy outcomes. However, the influence of maternal characteristics, especially age, on the relationship between the vaginal microbiota and preterm birth is poorly understood. This study quantifies the effects of race, maternal age, and gestational age on vaginal microbiota composition. Our results demonstrate stronger associations between preterm birth and a less optimal vaginal microbiota in White and Asian women compared to Black or African American women. The microbiota difference between preterm and term births was more pronounced in older reproductive-age women, with Lactobacillus species increasing with age only in term births. Additionally, lower alpha diversity was observed in term pregnancies compared to preterm during both early and late pregnancy, particularly in women aged above 25 years. These findings highlight the mediating role of age in the relationship between the vaginal microbiota and preterm birth.

microbiology↗

bioRxiv2025

LGBTQ+ realities in the biological sciences

While scientific environments have been described as unwelcoming to the LGBTQ+ community, and fields such as physics have systematically documented these challenges, the climate in biology workplaces has not been assessed. We conducted the largest survey to date of LGBTQ+ biologists to examine how their sense of belonging and perception of climate in the biology workplace and professional societies compare to that of their straight and cis peers. We surveyed 1419 biologists across five professional societies, with 486 identifying as LGBTQ+. Trans and gender non-conforming (TGNC) biologists reported lower belonging and morale within the workplace, professional societies, and the biology community compared to cis, straight biologists. They also reported being less comfortable with the climate of various professional biology environments. While LGBTQ+ biologists report that their workplaces are moderately inclusive, over 20% of all LGBTQ+ biologists and nearly 40% of TGNC biologists experience exclusionary behavior at work. This landmark survey provides the first comprehensive analysis of the LGBTQ+ climate in biology, revealing specific challenges faced by TGNC scientists and identifying interventions to enhance inclusivity for scientists. Significance StatementThis landmark study includes the largest known sample of LGBTQ+ biologists and offers the first comprehensive description of the LGBTQ+ climate in biology, differentiating between the experiences of cisgender lesbian, gay, bisexual, and queer (LGBQ) biologists and transgender and gender non-conforming (TGNC) biologists. The study found that compared to non-LGBTQ+ biologists, TGNC participants report lower belonging, morale and comfort with the climate across biology workplaces, professional societies, and the biology community. While on average LGBTQ+ participants reported that their workplaces are moderately inclusive, over 20% of all LGBTQ+ biologists and nearly 40% of TGNC biologists report experiencing exclusionary behaviors at work. The study offers immediate implications for institutional policies and professional development in the biological sciences.

scientific communication and education↗

bioRxiv2024

Intracellular diffusion in the cytoplasm increases with cell size in fission yeast

Diffusion in the cytoplasm can greatly impact cellular processes, yet regulation of macromolecular diffusion remains poorly understood. There is increasing evidence that cell size affects the density and macromolecular composition of the cytoplasm. Here, we studied whether cell size affects diffusion at the scale of macromolecules tens of microns in diameter. We analyzed the diffusive motions of intracellular genetically-encoded multimeric 40 nm nanoparticles (cytGEMs) in the cytoplasm of the fission yeast Schizosaccharomyces pombe. Using cell size mutants, we showed that cytGEMs diffusion coefficients decreased in smaller cells and increased in larger cells. This increase in diffusion in large cells may be due to a decrease in the DNA-to-Cytoplasm ratio, as diffusion was not affected in large multinucleate cytokinesis mutants. In investigating the underlying causes of altered cytGEMs diffusion, we found that the proteomes of large and small cells exhibited size-specific changes, including the sub-scaling of ribosomal proteins in large cells. Comparison with a similar dataset from human cells revealed that features of size-dependent proteome remodeling were conserved. These studies demonstrate that cell size is an important parameter in determining the biophysical properties and the composition of the cytoplasm.

cell biology↗

bioRxiv2024

Microtubule dynamic instability is sensitive to specific biological viscogens in vitro

Cytoplasm is a viscous, crowded, and heterogeneous environment, and its local viscosity and degree of macromolecular crowding have significant effects on cellular reaction rates. Increasing viscosity slows down diffusion and protein conformational changes, while increasing macromolecular crowding speeds up reactions. As a model system for cellular reactions, microtubule dynamics are slowed down in vivo when cytoplasm concentration is increased by osmotic shifts, indicating a dominant role for viscosity in microtubule reaction pathways. In the cell, viscosity is determined by diverse species of "biological viscogens", including glycerol, trehalose, intermediate metabolites, proteins, polymers, organelles, and condensates. Here we show in vitro that microtubule dynamic instability is sensitive to specific viscogen species, particularly glycerol. We found that increasing viscosity with glycerol or trehalose slowed microtubule growth, slowed microtubule shrinkage, and increased microtubule lifetimes, similar to the "freezing" observed previously in vivo. Increasing viscosity with a globular protein, bovine serum albumin, increased microtubule growth rates, as its viscous effects may be balanced against its macromolecular crowding effects. At matched viscosities, glycerol had an outsized effect on microtubule lifetimes, rescues, and nucleation compared to other viscogens. Increasing viscosity did not, however, increase the intensity of EB3-GFP comets, indicating that GTP hydrolysis is unaffected by buffer conditions. We propose that glycerol exerts its distinct effect on microtubule dynamic instability by stabilizing the microtubule lattice after phosphate release. Effects of specific viscogens may modulate many cellular reaction rates within local environments of cytoplasm.

biochemistry↗

bioRxiv2023

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and {gamma}-IR. CP-8 and CP-23 effectively inhibited {gamma}-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23s efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/552990v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dcec56org.highwire.dtl.DTLVardef@1202a09org.highwire.dtl.DTLVardef@a7e7b6org.highwire.dtl.DTLVardef@15d8925_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Quantifying turgor pressure in budding and fission yeasts based upon osmotic properties

Walled cells, such as plants, fungi, and bacteria cells, possess a high internal hydrostatic pressure, termed turgor pressure, that drives volume growth and contributes to cell shape determination. Rigorous measurement of turgor pressure, however, remains challenging, and reliable quantitative measurements, even in budding yeast are still lacking. Here, we present a simple and robust experimental approach to access turgor pressure in yeasts based upon the determination of isotonic concentration using protoplasts as osmometers. We propose three methods to identify the isotonic condition - 3D cell volume, cytoplasmic fluorophore intensity, and mobility of a cytGEMs nano-rheology probe - that all yield consistent values. Our results provide turgor pressure estimates of 1.0 {+/-} 0.1 MPa for S. pombe, 0.49 {+/-} 0.01 MPa for S. japonicus, 0.5 {+/-} 0.1 MPa for S. cerevisiae W303a and 0.31 {+/-} 0.03 MPa for S. cerevisiae BY4741. Large differences in turgor pressure and nano-rheology measurements between the S. cerevisiae strains demonstrate how fundamental biophysical parameters can vary even among wildtype strains of the same species. These side-by-side measurements of turgor pressure in multiple yeast species provide critical values for quantitative studies on cellular mechanics and comparative evolution.

biophysics↗

bioRxiv2022

ZNF524 directly interacts with telomeric DNA and supports telomere integrity

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity.

cell biology↗

bioRxiv2022

A unified model for the dynamics of ATP-independent ultrafast contraction

In nature, several ciliated protists possess the remarkable ability to execute ultrafast motions using protein assemblies called myonemes, which contract in response to Ca2+ ions. Existing theories, such as actomyosin contractility and macroscopic biomechanical latches, do not adequately describe these systems, necessitating new models to understand their mechanisms. In this study, we image and quantitatively analyze the contractile kinematics observed in two ciliated protists (Vorticella sp and Spirostomum sp), and, based on the mechanochemistry of these organisms, we propose a minimal mathematical model that reproduces our observations as well as those published previously. Analyzing the model reveals three distinct dynamic regimes, differentiated by the rate of chemical driving and the importance of inertia. We characterize their unique scaling behaviors and kinematic signatures. Besides providing insights into Ca2+-powered myoneme contraction in protists, our work may also inform the rational design of ultrafast bioengineered systems such as active synthetic cells.

biophysics↗

bioRxiv2022

Spike-specific CXCR3+ TFH cells play a dominant functional role in supporting antibody responses in SARS-CoV-2 infection and vaccination

CD4+ T follicular helper (TFH) cells are required for high-quality antibody generation and maintenance. However, the longevity and functional role of these cells are poorly defined in COVID-19 convalescents and vaccine recipients. Here, we longitudinally investigated the dynamics and functional roles of spike-specific circulating TFH cells and their subsets in convalescents at the 2nd, 5th, 8th, 12th and 24th months after COVID-19 symptom onset and in vaccinees after two and three doses of inactivated vaccine. SARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, of which spike-specific CXCR3+ TFH cells but not spike-specific CXCR3- TFH cells and neutralizing antibodies were persistent for at least two years in more than 80% of convalescents who experienced symptomatic COVID-19, which was well coordinated between spike-specific TFH cell and antibody responses at the 5th month after infection. Inactivated vaccine immunization also induced spike-specific TFH cell and antibody responses; however, these responses rapidly declined after six months with a two-dose standard administration, and a third dose significantly promoted antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells exhibited better responsiveness than spike-specific CXCR3- TFH cells upon spike protein stimulation in vitro and showed superior capacity in supporting spike-specific antibody secreting cell (ASC) differentiation and antibody production than spike-specific CXCR3- TFH cells cocultured with autologous memory B cells. In conclusion, spike-specific CXCR3+ TFH cells played a dominant functional role in antibody elicitation and maintenance in SARS-CoV-2 infection and vaccination, suggesting that induction of CXCR3-biased spike-specific TFH cell differentiation will benefit SARS-CoV-2 vaccine development aiming to induce long-term protective immune memory. HighlightsO_LISARS-CoV-2 infection elicited robust spike-specific TFH cell and antibody responses, which persisted for at least two years in the majority of symptomatic COVID-19 convalescent patients. C_LIO_LIInactivated vaccine immunization also elicited spike-specific TFH cell and antibody responses, which rapidly declined over time, and a third dose significantly promoted antibody maturation and potency. C_LIO_LISpike-specific CXCR3+ TFH cells exhibited more durable responses than spike-specific CXCR3- TFH cells, correlated with antibody responses and showed superior capacity in supporting ASC differentiation and antibody production than spike-specific CXCR3- TFH cells. C_LI

immunology↗

bioRxiv2022

Benzo(a)pyrene regulates chaperone-mediated autophagy via heat shock protein 90

The Benzo(a)pyrene (Bap) exposure induced oxidative damage, DNA damage and autophagy. To explore the molecular mechanism of BaP-induced autophagy. In these studies, we first found that heat shock protein 90 (HSP90), heat shock cognate 70 (HSC70) and lysosomal-associated membrane protein type 2 receptor (Lamp-2a) expressions of C57BL mice lung tissue and A549 cells exposed to BaP were significant increase, as well as Bap induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by comet assay and {gamma}-H2AX foci analysis in A549 cells. Our results demonstrated BaP induced CMA and caused DNA damage. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90 shRNA lentivirus transduction in A549 cells. HSC70 and Lamp-2a expressions of these cells exposed to BaP were not significant increase, which showed that BaP inducted CMA was mediated by HSP90. Further, HSP90 shRNA prevented BaP induced of Bap which suggested BaP regulated CMA and caused DNA damage by HSP90. Our results elucidated a new mechanism of BaP regulated CMA through HSP90.

biochemistry↗

bioRxiv2022

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelganger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak non-ergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40-nm sizescale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels. SignificanceBiophysical properties of the cytoplasm influence many cellular processes, from differentiation to cytoskeletal dynamics, yet little is known about how tightly cells control these properties. We developed a combined experimental and computational approach to analyze cytoplasmic heterogeneity through the lens of diffusion. We find that the apparent cytoplasmic viscosity varies tremendously - over 100-fold within any individual cell, and over 10-fold among individual cells when comparing averages of all particles measured for each cell. The variance was largely independent of temperature, the cytoskeleton, cell cycle stage, and localization, but was magnified under hyperosmotic shock. This suggests that cytoplasmic heterogeneity contributes substantially to biological variability within and between cells, and has significant implications for any cellular process that depends on diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/491518v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@a6faeforg.highwire.dtl.DTLVardef@a49dc4org.highwire.dtl.DTLVardef@171d62borg.highwire.dtl.DTLVardef@a3c7bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

Control of nuclear size by osmotic forces in Schizosaccharomyces pombe

The size of the nucleus scales robustly with cell size so that the nuclear-to-cell volume ratio (N/C ratio) is maintained during cell growth in many cell types. The mechanism responsible for this scaling remains mysterious. Previous studies have established that the N/C ratio is not determined by DNA amount but is instead influenced by factors such as nuclear envelope mechanics and nuclear transport. Here, we developed a quantitative model for nuclear size control based upon colloid osmotic pressure and tested key predictions in the fission yeast Schizosaccharomyces pombe. This model posits that the N/C ratio is determined by the numbers of macromolecules in the nucleoplasm and cytoplasm. Osmotic shift experiments showed that the fission yeast nucleus behaves as an ideal osmometer whose volume is primarily dictated by osmotic forces. Inhibition of nuclear export caused accumulation of macromolecules and an increase in crowding in the nucleoplasm, leading to nuclear swelling. We further demonstrated that the N/C ratio is maintained by a homeostasis mechanism based upon synthesis of macromolecules during growth. These studies demonstrate the functions of colloid osmotic pressure in intracellular organization and size control.

biophysics↗

bioRxiv2021

nucGEMs probe the biophysical properties of the nucleoplasm

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

biophysics↗

bioRxiv2021

The ESX-1 secretion system senses bacterial contact and prepares mycobacteria for environmental adaptation

The ESX-1 system (6-kDa early secretory antigenic target (ESAT-6) secretion system-1) is essential for Mycobacterium tuberculosis pathogenesis and conjugal transfer in Mycobacterium smegmatis, yet little is known about how its function is regulated. Live-cell fluorescence microscopy showed natively expressed ESX-1 was organized into distinct foci predominantly observed at cell-cell contacts. These foci formed when two cells touched and required a fully assembled ESX-1 system in both bacteria, suggesting the generation of an ESX-1 megacomplex across multiple membranes. The emergence of ESX-1 foci and ESX-1 secretion was environmentally dependent: foci formed in low nitrogen environments in which secretion was suppressed, yet with increasing concentrations of nitrogen, ESX-1 systems diffused along the plasma membrane and secretion was activated. Genome-wide transcriptional profiling revealed ESX-1 dependent induction of genes required for the SOS response and error prone DNA replication in high nitrogen. Based on these findings, we propose a new model of ESX-1 function where ESX-1 localization and secretion are responsive to nitrogen levels and form an integral node in the mycobacterial response to neighboring cells and environmental adaptation.

microbiology↗