Biology subjects

Khadpekar, A.

Publications and source records attributed to Khadpekar, A..

3 recordsLinked to original sources

bioRxiv2019

Asamataxis: A cooperative relayed migration in response to subsurface inhomogeneity leads to long-range self-patterning of cells

Cells self-organize to give patterns that are essential for tissue functioning. While the effects of biochemical and mechanical cues are relatively well studied, the role of stiffness inhomogeneity on cellular patterning is unexplored. Using a rigid structure embedded in soft polyacrylamide (PAA) gel, we show that such mechanical inhomogeneity leads to long-range self-organized cellular patterns. Our results reveal that this patterning depends on cellular traction and cell morphology. Depending on a suitable combination of cellular morphology and traction, the information about the presence of embedded structure gets relayed outward. In response to this relay, the cells reorient their axis and migrate towards the embedded structure leading to the observed long-range (20-35 cell length) patterning. To predict the possibility of pattern formation, we present a dimensionless number f combining the governing parameters. We have also shown that the pattern can be tailor-made by pre-designing sub-surface structures, a potential tool for tissue engineering. This mechanism of directed migration driven long-range pattern formation in response to mechanical inhomogeneity may be involved during several pathophysiological conditions, a proposition that needs further investigation.\n\nOne Sentence SummarySubstrate inhomogeneity and cooperative cellular traction together lead to cellular migration and long-range pattern formation.

biophysics↗

bioRxiv2019

Operating regimes in a single enzymatic cascade at ensemble-level

Operating regime of a single enzymatic cascade such as ubiquitously conserved MAPK building block provides insights into the nature of the sensitivity of the steady-state dose response that maps the upstream kinase and the downstream activated substrate concentrations. Steady-state response can be viewed either at a single-cell level in an ensemble of cells or at population-average level. Four operating regimes, viz., hyperbolic, threshold-hyperbolic, signal transducing and saturated, have been identified using the population-average level dose response curve. However, cell-to-cell variability exists in enzymatic cascades. This variability is captured using reporter based experimentation at ensemble level which permits detection of snapshot(s) of ensemble-level distribution of phosphorylated proteins. As a result, often the corresponding underlying steady-state dose response curve may not be available. We consider the question if the underyling operating regime can be directly inferred from ensemble level snapshot upstream kinase (input) and downstream phosphorylated substrates (response) distributions. In order to address this question, we use mathematical model of a single enzymatic cascade based on quasi-steady state approximation superimposed with an input distribution constrained by single-cell level experimental measurements of the MAPK cascade in Jurkat E6.1 cells stimulated with Phorbol Myristate Acetate (PMA). We prove that, under steady-state conditions, a monotonic relationship between the RIQR (=ratio of the inter-quartile range of the response and input distributions) and Rm (=ratio of medians of the two distributions), both of which are experimental observables, can be used to identify the underlying operating regime. We also show that the identification of the unimodal vs bimodal nature of the response distributions can further lead to identification of the potential parameter range in the planes of Michaelis-Menten constants K1 and K2, the two key parameters that dictate the operating regimes. We implement the proposed method on the stimulus strength dependent steady-state single-cell level pMEK (input) and pERK (response) distributions in Jurkat E6.1 cells treated with PMA. While cells stimulated using low concentrations of PMA are likely to operate in hyperbolic regime, those exposed to higher concentrations may lie in signal-transducing regime. Authors summarySingle enzymatic cascade, a ubiquitously found key building block in biological signaling networks, exhibits different steady-state behaviour at population (ensemble) level compared to population-averaged response. Detection at ensemble level typically achieved by non-plasmid based reporter permits snapshot fluorescence distribution measurement. We ask if there are signatures of fluorescence distribution of input kinase and response activated protein that can help decipher the underlying dose-response belonging to four distinct operating regimes a single enzymatic cascade exhibits. Based on simultaneously measured snapshot input (pMEK) and response (pERK) protein levels in an ensemble of PMA stimulated immortalized cancer (Jurkat E6.1) cells, we superimpose pMEK data-guided upstream kinase distribution capturing cell-to-cell variability on the steady-state Michaelis-Menten (MM) kinetic model. Following extensive MM constants sampling and simulations, we systematically identify that monotonicity between RIQR, the ratio of the inter-quartile range of response and input distributions, and RM, the median ratio of the two distributions, enables regime identification of the measured pERK distribution. We further show qualitative assessment of the modality of the response distribution can constrain the parameter range within an operating regime. Both RIQR and RM being experimental observables makes the proposed method suitable for assessing signal modulation capability of an enzymatic cascade of interest.

systems biology↗

bioRxiv2018

Soft Substrate Maintains Proliferative and Multi-lineage Potential of Human Mesenchymal Stem Cells on Long Term Expansion

Human mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by {beta}-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.\n\nOne Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.\n\nSignificance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

bioengineering↗