Biology subjects

Chang, F.

Publications and source records attributed to Chang, F..

27 records · Page 2Linked to original sources

bioRxiv2020

Starvation induces shrinkage of the bacterial cytoplasm

Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. colis inner membrane from the cell wall. Shrinkage was accompanied by a [~]17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon-source quality. Robust recovery from starvation required the Tol-Pal system, highlighting the importance of envelope coupling during recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic. Significance statementBacterial cells constantly face nutrient fluctuations in their natural environments. While previous studies have identified gene expression changes upon nutrient depletion, it is much less well known how cellular morphology and cytoplasmic properties respond to shifts in nutrient availability. Here, we discovered that switching fast-growing Escherichia coli cells to nutrient-free conditions results in substantial shrinkage of the inner membrane away from the cell wall, especially at the new pole. Shrinkage was primarily driven by loss of cytoplasmic water contents. Shrinkage was also exhibited by cells naturally entering stationary phase, highlighting its biological relevance across starvation conditions. The membrane-spanning Tol-Pal system was critical for robust entry into and recovery from shrinkage, indicating the importance of cell-envelope homeostasis in surviving nutrient starvation.

microbiology↗

bioRxiv2020

Variations of intracellular density during the cell cycle arises from tip-growth regulation in fission yeast

Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density varies during the cell cycle in fission yeast. Density decreased during G2, increased in mitosis and cytokinesis, and rapidly dropped at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages led to continued increases or decreases in density. Spatially heterogeneous patterns of density suggested links between density regulation and tip growth, and septum bending away from higher-density daughters linked density to intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.

biophysics↗

bioRxiv2019

Girdin: an essential component of pre-replicative complex in human cells

A central event in the initiation of DNA replication in eukaryotes is the assembly of pre-replicative complex (pre-RC) on specific chromatin sites known as DNA replication origins. The pre-RC assembly process differs between budding and fission yeasts. In fission yeast, Sap1 directly participates in pre-RC assembly, together with the four initiation factors: ORC, Cdc18/Cdc6, Cdt1, and MCM. In metazoans, the nature of DNA replication origins is not defined and the mechanism of pre-RC assembly remains incompletely known. In this study, Girdin was identified as an essential replication initiation factor in human cells. Similar to the activity of Sap1, human Girdin binds to DNA origins, interacts with ORC, and is required for pre-RC assembly due to its essential role in recruitment of Cdc6 to DNA origins. Thus, DNA origins in human or metazoans are defined as including two elements, one bound by ORC and the other bound by Girdin.

biochemistry↗

bioRxiv2019

Reassessment of the basis of cell size control based on analysis of cell-to-cell variability

Fundamental mechanisms governing cell size control and homeostasis are still poorly understood. The relationship between sizes at division and birth in single cells is used as a metric to categorize the basis of size homeostasis [1-3]. Cells dividing at a fixed size regardless of birth size (sizer) are expected to show a division-birth slope of 0, whereas cells dividing after growing for a fixed size increment (adder) have an expected slope of +1 [4]. These two theoretical values are, however, rarely experimentally observed. Here we investigate possible reasons for this discrepancy. Rod-shaped fission yeast Schizosaccharomyces pombe cells divide at a fixed surface area [5, 6], and exhibit a division-birth slope of 0.23{+/-}0.04, significantly different from the sizer value of zero. To understand whether fission yeast uses a mix of sizer and adder control, or whether size sensing is simply imprecise, we developed a mathematical model of sizer control, including the relevant sources of variation. Our results support pure sizer control and show that deviation from zero slope is exaggerated by measurement of an inappropriate geometrical quantity (e.g., length instead of area), combined with cell-to-cell radius variability. The model predicts that mutants with greater errors in size sensing or septum positioning paradoxically appear to behave as better sizers. Furthermore, accounting for cell width variability, we show that pure sizer control can in some circumstances reproduce the apparent adder behaviour observed in E. coli. These findings demonstrate that refined analysis of geometric variation can lead to new insights into cell size control.

cell biology↗

bioRxiv2018

Decoupling of rates of protein synthesis from cell expansion leads to supergrowth

Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis, or the consequences when this homeostasis is perturbed. Here, we identified conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to global accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of \"supergrowth\" in which rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a novel mechanism for global proteome homeostasis based on modulation of volume growth and dilution.

cell biology↗

bioRxiv2018

Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of Tumor Overexpressed Gene (TOG) domains. The underlying mechanism of these arrays as microtubule polymerases remains unknown. Here, we describe the biochemical and structural basis for TOG domain arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 arrays, each with distinct exchange rates. X-ray structures reveal pseudo-dimeric square-shaped assemblies in which four TOG domains position four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking confirms square assemblies form in solution, and inactivation of their interfaces destabilizes square organizations without influencing tubulin binding. Using an approach to modulate tubulin polymerization, we determined a X-ray structure showing an unfurled assembly in which TOG1 and TOG2 uniquely bind two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies, which then unfurl facilitating their concerted polymerization into protofilaments.

biochemistry↗

bioRxiv2018

Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

The XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four {beta}-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two {beta}-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that {beta}-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.

biochemistry↗

bioRxiv2018

Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin - SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 - correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly bind euchromatic DNA replication origins and modify their local chromatin environment.

genetics↗

bioRxiv2017

Dynamical localization of a thylakoid membrane binding protein is required for acquisition of photosynthetic competency

Vipp1 is highly conserved and essential for photosynthesis, but its function is unclear as it does not participate directly in light-dependent reactions. We analyzed Vipp1 localization in live cyanobacterial cells and show that Vipp1 is highly dynamic, continuously exchanging between a diffuse fraction that is uniformly distributed throughout the cell and a punctate fraction that is concentrated at high curvature regions of the thylakoid located at the cell periphery. Experimentally perturbing the spatial distribution of Vipp1 by relocalizing it to the nucleoid causes a severe growth defect during the transition from non-photosynthetic (dark) to photosynthetic (light) growth. However, the same perturbation of Vipp1 in dark alone or light alone growth conditions causes no growth or thylakoid morphology defects. We propose that the punctuated dynamics of Vipp1 at the cell periphery in regions of high thylakoid curvature enable acquisition of photosynthetic competency, perhaps by facilitating biogenesis of photosynthetic complexes involved in light-dependent reactions of photosynthesis.

cell biology↗