Biology subjects

Garcia-Manyes, S.

Publications and source records attributed to Garcia-Manyes, S..

4 recordsLinked to original sources

bioRxiv2024

Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes

Differentiation of cultured human epidermal stem cells is regulated by interactions with the underlying substrate. Whereas differentiation is typically stimulated when keratinocytes are prevented from spreading, we previously identified two micron-scale topographical substrates that regulate differentiation of spread cells. On one substrate (S1), individual cells interact with small circular topographies, and differentiation is stimulated; on the other (S2), cells interact with larger triangular topographies, and differentiation is inhibited. By scanning electron microscopy we visualised substrate interactions at higher resolution than previously and using live cell imaging we established that induction of the differentiation marker involucrin did not involve transient cell rounding on S1. Bulk gene expression profiling did not reveal any differences between cells on S1 and S2 prior to the selective upregulation of differentiation markers at 12h on S1 and cell stiffness was lower on both S1 and S2 than on flat substrates. Nevertheless, cells on S2 differed from cells on flat and S1 substrates because they exhibited reduced cell volume, prompting us to explore whether cell volume could regulate differentiation independent of culture substrate. Treatment with polyethylene glycol (PEG) reduced cell volume and inhibited differentiation regardless of whether keratinocytes were seeded on flat, S1 or S2 substrates, micropatterned islands or in suspension. Conversely, treatment with deionised water increased cell volume and stimulated differentiation of substrate adherent keratinocytes. On flat substrates treatment with the Ca2+ chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester or an inhibitor of the water channel aquaporin 3 blocked induction of differentiaton by deionised water, whereas the gadolinium3+, a stretch-activated calcium channel blocker, did not. Our studies identify a new mechanism by which keratinocyte-niche interactions regulate initiation of differentiation.

cell biology↗

bioRxiv2023

Enzyme-less nanopore detection of post-translational modifications within long polypeptides

Means to sequence DNA and RNA quickly and cheaply have revolutionized biology and medicine. The ability to analyse cellular proteins and their millions of variants would be an advance of comparable importance, but requires a fresh technical approach. We use electroosmosis for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1200 residues by a protein nanopore. By monitoring the ionic current carried by the nanopore, we locate post-translational modifications deep within the polypeptide chains, and thereby lay the groundwork for obtaining inventories of the proteoforms in cells and tissues.

biochemistry↗

bioRxiv2022

How talin allosterically activates vinculin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single molecule magnetic tweezer experiments, Molecular Dynamics simulations, actin bundling assays, and adhesion assembly experiments in live cells, we here discover a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at 7-10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The allosteric vinculin mutant is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

biophysics↗

bioRxiv2020

Molecular fluctuations as a ruler of force-induced protein conformations

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

biophysics↗