Biology subjects

Budnar, S.

Publications and source records attributed to Budnar, S..

7 recordsLinked to original sources

bioRxiv2023

Cortical ductility governs cell-cell adhesion mechanics

This paper challenges our understanding of cell-cell adhesion by emphasising the role of mechanical dissipation at the cellular level. We have developed new microdevices to measure the energy dissipated during the rupture of junctions between cell-cell doublets. Using a synthetic cadherin approach, we decoupled the role of cadherin binding energy, signalling and downstream regulation of cytoskeletal architecture. This yielded a phase diagram in which cell junctions transition from a ductile to a brittle fracture mode based on their ratio of cortical tension and shape relaxation time. We recapitulated our results using a descriptive mechanical simulation approach. Our results shift our understanding of cell-cell adhesion from the current focus on bond energy and tension to the key role played by energy dissipation in the cytoskeleton during junction deformation and its active mechanosensitive regulation.

cell biology↗

bioRxiv2021

Enhanced RhoA signaling stabilizes E-cadherin in migrating epithelial monolayers.

Epithelia migrate as physically coherent populations of cells. Earlier studies revealed that mechanical stress accumulates in these cellular layers as they move. These stresses are characteristically tensile in nature and have often been inferred to arise when moving cells pull upon the cell-cell adhesions that hold them together. We now report that epithelial tension at adherens junctions between migrating cells also reflects an increase in RhoA-mediated junctional contractility. We find that active RhoA levels were stimulated by p114 RhoGEF at the junctions between migrating MCF-7 monolayers, and this is accompanied by increased levels of actomyosin and mechanical tension. By applying a strategy to restore active RhoA specifically at adherens junctions by manipulating its scaffold, anillin, we found that this junctional RhoA signal was necessary to stabilize junctional E-cadherin during epithelial migration. We suggest that stabilization of E-cadherin by RhoA serves to increase cell-cell adhesion against the mechanical stresses of migration.

cell biology↗

bioRxiv2021

Asymmetric Contraction of Adherens Junctions arises through RhoA and E-cadherin feedback

Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular "clutch" at tricellular vertices which stabilizes vertex motion upon tensional load.

cell biology↗

bioRxiv2019

Snail induces epithelial cell extrusion through transcriptional control of RhoA contractile signalling and cell matrix adhesion

Cell extrusion is a morphogenetic process that is implicated in epithelial homeostasis and highly dependent on the cellular insult and context. Minorities of cells expressing HRasV12 or undergoing apoptosis are typically extruded apically in vertebrate cells. However, basal extrusion (delamination) predominates when mammalian cells express oncogenic KRas and during development in Drosophila. To explore if the morphogenetic transcription factor, Snail, induces extrusion, we inducibly expressed a metabolically stabilized Snail6SA transgene mosaically in confluent MCF-7 monolayers. We found that the morphogenetic impact of Snail6SA was critically influenced by the proportion of cells in which it was expressed. When expressed in small clusters (<3 cells) within confluent monolayers, Snail6SA expression induced apical cell extrusion. In contrast, confluent cultures of Snail6SA expressing cells were retained in the monolayer to eventually undergo basal extrusion (delamination). Transcriptomic profiling revealed that Snail6SA did not substantively alter the balance of epithelial:mesenchymal genes. However, we identified a gene transcriptional network that led to the upregulation of RhoA signalling, actomyosin contractility and reduced basal adhesion in Snail6SA expressing cells. We show that this was necessary to drive both apical extrusion and basal delamination. Thus, contractile upregulation by RhoA along with weakened basal adhesion provides a pathway for Snail to influence epithelial morphogenesis independently of classic Epithelial to Mesenchymal Transition (EMT).

cell biology↗

bioRxiv2019

Temporal gating of SIRT1 functions by O-GlcNAcylation prevents hyperglycemia and enables physiological transitions in liver

Inefficient fasted-to-refed transitions are known to cause metabolic diseases. Thus, identifying mechanisms that may constitute molecular switches during such physiological transitions become crucial. Specifically, whether nutrients program a relay of interactions in master regulators, such as SIRT1, and affect their stability is underexplored. Here, we elucidate nutrient-dependent O-GlcNAcylation of SIRT1, within its N-terminal domain, as a key determinant of hepatic glucose- and fat-metabolism, and insulin signaling. SIRT1 glycosylation dictates interactions with PPAR/FOXO1/PGC1/SREBP1, to exert a temporal control over transcription of genes during fasted-to-refed transitions. Interestingly, glycosylation-dependent cytosolic export of SIRT1 promotes a transient interaction with AKT and subsequent proteasomal degradation. Loss of glycosylation discomposes these interactions and enhances stability of SIRT1 even upon refeeding, which causes insulin resistance, hyperglycemia and hepatic-inflammation. Aberrant glycosylation of SIRT1 is associated with aging and/or metabolic diseases. Thus, nutrient-dependent glycosylation constrains spatio-temporal dynamics of SIRT1 and gates its functions to maintain metabolic homeostasis.

physiology↗

bioRxiv2018

Scaffolding of RhoA contractile signaling by anillin: a regulatory analogue of kinetic proofreading

Scaffolding is a fundamental principle of cell signaling commonly thought to involve multi-domain proteins that tether different components of a pathway together into a complex 1,2. We now report an alternative mechanism for scaffolding that is necessary for RhoA-mediated contractile signaling. We find that anillin binding stabilizes active, GTP-RhoA, and promotes contractility at both the epithelial zonula adherens (ZA) and the cytokinetic furrow. However, anillin does not conform to the classical picture of a multi-domain tether, since its RhoA-binding AH domain alone was sufficient to promote contractile signaling. Moreover, anillin competes with contractile effectors for a common site on RhoA, presenting the conundrum of how an inhibitory interaction can otherwise promote signaling. To explain this, we propose that inactivation of RhoA is non-Poissonian, having a rate that increases with time, unless the process is reset via transient binding to anillin. Repeated cycles of binding and un-binding therefore increase cortical residence times of non-sequestered GTP-RhoA and hence the probability of engaging contractile effectors. We identify the modification of the local lipid environment as a potential mechanism underlying such non-Poisson statistics, and demonstrate agreement with a minimal cellular system. Finally, we show that Myosin II anchors anillin at the cortex to form a feedback pathway that enhances RhoA signaling. This new paradigm of scaffolding is a regulatory analogue of kinetic proofreading and may be employed by other binding proteins that do not fit the classical picture.

cell biology↗

bioRxiv2018

A mechanosensitive RhoA pathway that protects epithelia against acute tensile stress.

Adherens junctions are tensile structures that couple epithelial cells together. Junctional tension can arise from cell-intrinsic application of contractility or from the cell-extrinsic forces of tissue movement. In all these circumstances, it is essential that epithelial integrity be preserved despite the application of tensile stress. In this study, we identify junctional RhoA as a mechanosensitive signaling pathway that responds to epithelial stress. The junctional specificity of this response is mediated by the heterotrimeric protein G12, which is recruited by E-cadherin and, in turn, recruits p114 RhoGEF to activate RhoA. Further, we identify Myosin VI as a key mechanosensor, based on its intrinsic capacity to anchor E-cadherin to F-actin when exposed to tensile load. Tension-activated RhoA signaling was necessary to preserve epithelial integrity, which otherwise undergoes fracture when monolayer stress is acutely increased by calyculin. Paradoxically, this homeostatic RhoA signaling pathway increases junctional actomyosin, a contractile response that might be expected to itself promote fracture. Simulations of a vertex-based model revealed that the protective effect of RhoA signaling can be explained through increased yield limit at multicellular vertices, where experiments showed p114 RhoGEF was necessary to increase E-cadherin and promote actin assembly and organization.

cell biology↗