Biology subjects

Purkayastha, P.

Publications and source records attributed to Purkayastha, P..

5 recordsLinked to original sources

bioRxiv2023

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT. SummaryChanges in the availability of cellular GTP resulting from physiologically relevant processes, including cell migration and cell spreading, alter the rates of Ran-dependent nuclear import and export. Altered rates of nucleocytoplasmic transport regulate RNA localization and protein synthesis.

cell biology↗

bioRxiv2022

Laboratory evolution of Mycobacterium smegmatis in the presence of a fluoroquinolone leads to extreme drug resistance phenotype through the over-expression of Msmeg_5659-61 efflux pump.

Resistance to multiple drugs is one of the significant barriers in the treatment of tuberculosis (TB). Knowledge of mechanisms of resistance is important to design effective treatment strategies. While mutations in genes coding for drug targets are thought to be the primary source of drug resistance, absence of mutations in these genes in many clinical strains suggests additional mechanisms of resistance. In this study, we employ adaptive laboratory evolution of Mycobacterium smegmatis to understand alternate mechanisms of drug resistance to norfloxacin, a fluoroquinolone (FQ). Results show that, in addition to fluoroquinolones, the evolved strain, Norr, is resistant to first-line drugs, rifampicin and isoniazid, and a second-line drug (amikacin), exhibiting extreme drug resistance phenotype. However, mutations were absent in any of the drug target genes. Drug uptake studies revealed that resistance is an attribute of decreased intracellular accumulation, primarily due to increased efflux. Further, drug transport kinetics demonstrate the involvement of efflux mediated resistance, which was found to be reversed in the presence of efflux pump inhibitors (EPIs). Gene transcript analysis suggests differential upregulation of multiple efflux pumps across the genome of the mutant. Overexpression of one of the upregulated efflux pumps Msmeg_5659-5661, partially explains the XDR phenotype of the mutant, while also suggesting that the contribution of other efflux pumps is significant. Whole-genome sequencing (WGS) of Norr reveals that a mutation in soxR, a transcriptional regulator, could be responsible for the upregulation of the Msmeg_5659-5661 efflux pump by direct regulation, and other efflux pumps via indirect regulation. Thus, the present work demonstrates that high resistance to multiple drugs can arise even when the Mycobacterium was subjected to a single selection pressure. Further, alterations in drug transport is an important mechanism that leads to resistance to multiple drugs simultaneously.

systems biology↗

bioRxiv2022

Loss of mechanical homeostasis drives epithelial acinar eversion

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. While some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternate pathway for altered polarity. Because mechanical alterations are common in human cancer including an upregulation of RhoA mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of {beta}1 integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model which balances tension at cell-cell interfaces revealed a five-fold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

biophysics↗

bioRxiv2021

Substrate stiffness controls the cell cycle of human mesenchymal stem cells via cellular traction

The microenvironment of human mesenchymal stem cells (hMSCs) regulates their self-renewal and differentiation properties. Previously it was shown that hMSCs remained quiescent on soft (0.25 kPa) polyacrylamide (PA) gels but re-entered into cell cycle on a stiff (7.5 kPa) gel. However, how cells behave on intermediate stiffness and what intracellular factors transmit mechanical changes to cell interior thereby regulating cell cycle remained unknown. In this work we demonstrated that PA gels between 1 and 5 kPa act as a mechanical switch in regulating cell cycle of hMSCs. By experiments on cell-cycle exit and re-entry, we found that hMSCs demonstrated a sharp transition from quiescence to proliferation between 1 and 5 kPa. Further studies with ROCK inhibitor Y-27632 revealed that contractile proteins, but not cell spread area, accounts for the sensitivity of hMSCs towards substrate stiffness and hence correlates with their changes in cell cycle. These observations therefore suggest that substrate stiffness regulates hMSC proliferation through contractile forces as generated by cellular contractile proteins in a unique pattern which is distinct from other cell types as studied. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/469207v1_ufig1.gif" ALT="Figure 1"> View larger version (83K): org.highwire.dtl.DTLVardef@a4fff3org.highwire.dtl.DTLVardef@9fb415org.highwire.dtl.DTLVardef@e7d633org.highwire.dtl.DTLVardef@9fba04_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Natural selection imposed by mechanical properties of a biomaterial results in novel cellular phenotypes

Mechanical properties such as substrate stiffness are a ubiquitous feature of a cells environment. Many types of animal cells exhibit canonical phenotypic plasticity when grown on substrates of differing stiffness, in vitro and in vivo. Whether such plasticity is a multivariate optimum due to hundreds of millions of years of animal evolution, or instead is a compromise between conflicting selective demands, is unknown. We addressed these questions by means of experimental evolution of populations of mouse fibroblasts propagated for ~90 cell generations on soft or stiff substrates. The ancestral cells grow twice as fast on stiff substrate as on soft substrate and exhibit the canonical phenotypic plasticity. Soft-selected lines derived from a genetically diverse ancestral population increased growth rate on soft substrate to the ancestral level on stiff substrate and evolved the same multivariate phenotype. The pattern of plasticity in the soft-selected lines was opposite of the ancestral pattern, suggesting that reverse plasticity underlies the observed rapid evolution. Conversely, growth rate and phenotypes did not change in selected lines derived from clonal cells. Overall, our results suggest that the changes were the result of genetic evolution and not phenotypic plasticity per se. Whole-transcriptome analysis revealed consistent differentiation between ancestral and soft-selected populations, and that both emergent phenotypes and gene expression tended to revert in the soft-selected lines. However, the selected populations appear to have achieved the same phenotypic outcome by means of at least two distinct transcriptional architectures related to mechano-transduction and proliferation.

evolutionary biology↗