Biology subjects

Sivasankar, S.

Publications and source records attributed to Sivasankar, S..

12 recordsLinked to original sources

bioRxiv2025

A dimerization-activated proximity labeling system for direct characterization of cadherin cis interactions

E-cadherins (Ecads) are essential transmembrane cell-cell adhesion proteins that mediate epithelial tissue formation and maintenance. Robust adhesion requires cis dimerization between neighboring Ecads on the cell surface and previous structural and biophysical studies have proposed conflicting models on the role of specific and nonspecific interactions in mediating cis dimerization. However, since these studies were carried out with isolated Ecad extracellular regions in cell-free systems, it is unknown if specific and nonspecific cis binding modes also occur with transmembrane Ecads in live cells, and if specific and nonspecific cis dimers recruit different sets of cytoplasmic proteins to Ecad junctions. To directly address these knowledge gaps, we developed a Dimerization Activated TuboID (DAT) proximity labeling system that reports on different modes of Ecad cis binding and their corresponding proteomes. Using DAT, fluorescence measurements, diffusion-reaction simulations, and adhesion assays, we demonstrate that Ecads in live cells form cis dimers via both specific and non-specific interactions and that cis dimerization does not require either prior trans contacts or an intact actin cytoskeleton. However, we show that the loss of specific cis interactions results in increased junctional instability and Ecad mobility, which leads to dysregulated peripheral protein interactions without affecting recruitment of core junctional proteins. Our study provides key mechanistic insights on cadherin cis interactions and also presents a toolkit that can be used to study a broad range of protein cis dimers in live cells.

biophysics↗

bioRxiv2025

P-cadherin and desmoglein-2 interact as strand-swap dimers and facilitate desmosome assembly.

Desmosomes are essential adhesive junctions that mediate the integrity of tension prone tissue like the heart and skin. The classical cadherin, P-cadherin (Pcad), is known to play an important role in forming desmosomes. However, the molecular mechanisms by which Pcads enable desmosome assembly are unknown. Here, we combine single molecule Atomic Force Microscopy, super-resolution and confocal imaging, mutagenesis assays, and atomistic simulations to resolve the structural mechanisms by which Pcads facilitate the formation of desmosomes. We show that Pcads interact with the desmosomal cadherin Desmoglien-2 (Dsg2) on opposing cells, by mutually swapping {beta}-strands terminated by conserved Tryptophan residues. A key determinant of this trans heterophilic strand-swap dimerization is the flexibility of a hinge on the swapped {beta} -strands: stiffening this hinge reduces the likelihood of heterophilic strand-swap dimer formation. We demonstrate that while desmosome formation is impaired in cells that either lack classical cadherins or that express strand-swap deficient Pcad, introduction of strand-swap competent Pcad into these cells rescues desmosome assembly. We show that the heterophilic dimers formed by Pcad and Dsg2 interact in a robust and persistent manner, which retains Pcad within the desmosome throughout the maturation process. SignificanceDesmosomes are essential intercellular junctions that mediate the integrity of tissue like the heart and skin. Desmosomal defects are associated with numerous skin disorders, heart diseases, and cancer. The adhesive protein P-cadherin is known to play an important role in forming desmosomes. However, the molecular mechanisms by which P-cadherins enable desmosome assembly are unknown. Here, we show that desmosome formation is facilitated by the direct interaction of P-cadherin with the transmembrane desmosomal adhesion protein Desmoglein-2. We demonstrate that P-cadherin and Desmoglien-2 interact robustly by swapping flexible portions of their protein backbone and that this molecular complex nucleates desmosome formation. Our results resolve the biophysical basis by which different adhesive proteins interact and mediate stable desmosome assembly.

biophysics↗

bioRxiv2024

Desmoplakin is a desmosomal mechanosensor

Desmosomes are essential cell-cell adhesion organelles that enable tension-prone tissue, like the skin and heart, to withstand mechanical stress. Desmosomal anomalies are associated with numerous epidermal disorders and cardiomyopathies. Despite their critical role in maintaining tissue resilience, an understanding of how desmosomes sense and respond to mechanical stimuli is lacking. Here, we use a combination of super-resolution imaging, FRET-based tension sensors, atomistic computer simulations, and biochemical assays to demonstrate that actomyosin forces induce a conformational change in desmoplakin, a critical cytoplasmic desmosomal protein. We show that in human breast cancer MCF7 cells, actomyosin contractility reorients keratin intermediate filaments and directs force to desmoplakin along the keratin filament backbone. These forces induce a conformational change in the N-terminal plakin domain of desmoplakin, converting this domain from a folded (closed) to an extended (open) conformation. Our findings establish that desmoplakin is mechanosensitive and responds to changes in cellular load by undergoing a force-induced conformational change.

cell biology↗

bioRxiv2024

Outside-in engineering of cadherin endocytosis using a conformation strengthening antibody.

P-cadherin, a crucial cell-cell adhesion protein which is overexpressed in numerous malignant cancers, is a popular target for drug delivery antibodies. However, molecular guidelines for engineering antibodies that can be internalized upon binding to P-cadherin are unknown. Here, we use a combination of biophysical, biochemical, and cell biological methods to demonstrate that trapping the cadherin extracellular region in an X-dimer adhesive conformation, triggers cadherin endocytosis via a novel outside-in signaling mechanism. We show that the monoclonal antibody CQY684 traps P-cadherin in an X-dimer conformation and strengthens this adhesive structure. Formation of stable X-dimers results in the dissociation of p120-catenin, a suppressor of cadherin endocytosis, from the X-dimer cytoplasmic region. This increases the turnover of P-cadherin and targets the cadherin-antibody complex to the lysosome. Our results establish a previously unknown outside-in signaling mechanism that provides fundamental insights into how cells regulate adhesion and that can be exploited by anti-cadherin antibodies for intracellular drug delivery.

biophysics↗

bioRxiv2023

Strengthening E-cadherin adhesion via antibody mediated binding stabilization.

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8 mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site directed mutagenesis and single molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped {beta}-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

biophysics↗

bioRxiv2022

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.

Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the resolution and accuracy of proximity labeling methods are limited by a lack of control over the enzymatic labeling process. Here, we present a high spatial and temporal resolution technology that can be activated on demand using light, for high accuracy proximity labeling. Our system, called Light Activated BioID (LAB), is generated by fusing the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. Using live cell imaging, immunofluorescence, western blotting, and mass spectrometry, we show that upon exposure to blue light, CRY2 and CIB1 dimerize, reconstitute the split-TurboID enzyme, and biotinylate proximate proteins. Turning off the light halts the biotinylation reaction. We validate LAB in different cell types and demonstrate that it can identify known binding partners of proteins while reducing background labeling and false positives.

biochemistry↗

bioRxiv2022

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using x-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif - the strand-swap dimer interface. Molecular dynamics simulations and single molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes, one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped {beta}-strand and its complimentary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

biophysics↗

bioRxiv2020

Robust scan synchronized force-fluorescence imaging.

Simultaneous atomic force microscope (AFM) and sample scanning confocal fluorescence microscope measurements are widely used to obtain mechanistic and structural insights into protein dynamics in live cells. However, the absence of a robust technique to synchronously scan both AFM and confocal microscope piezo stages makes it difficult to visualize force-induced changes in fluorescent protein distribution in cells. To address this challenge, we have built an integrated AFM-confocal fluorescence microscope platform that implements a synchronous scanning method which eliminates image artifacts from piezo motion ramping, produces intuitive, accurate pixel binning and enables the collection of a scanned image of a sample while applying force to the sample. As proof of principle, we use this instrument to monitor the redistribution of fluorescent E-cadherin, an essential transmembrane protein, in live cells, upon application of mechanical force.

biophysics↗

bioRxiv2020

Mapping transmembrane binding partners for E-cadherin ectodomains.

We combine proximity labeling and single molecule binding assays, to discover novel transmembrane protein interactions in cells. We first screen for candidate binding partners by tagging the extracellular and cytoplasmic regions of a bait protein with TurboID biotin ligase, and identify proximal proteins that are biotin-tagged on both their extracellular and intracellular regions. We then test direct binding interactions between the proximal proteins and the bait, using single molecule Atomic Force Microscope binding assays. Using this approach, we identify novel binding partners for the extracellular region of E-cadherin, an essential cell-cell adhesion protein. We show that the desmosomal proteins desmoglein-2 and desmocollin-3, the focal adhesion protein integrin-2{beta}1, and the receptor tyrosine kinase ligand ephrin-B1, all directly interact with E-cadherin ectodomains. Our discovery of previously unknown heterophilic E-cadherin binding interactions, suggest the existence of novel cadherin cross-talk in epithelial cells.

cell biology↗

bioRxiv2020

Inside-out regulation of E-cadherin conformation and adhesion

Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown. Cadherin intracellular regions associate with several actin-binding proteins including vinculin, which are believed to tune cell-cell adhesion by remodeling the actin cytoskeleton. Here, we show at the single molecule level, that vinculin association with the cadherin cytoplasmic region allosterically converts weak X-dimers into strong strand-swap dimers, and that this process is mediated by myosin II dependent changes in cytoskeletal tension. We also show that in epithelial cells, [~]70% of apical cadherins exist as strand-swap dimers while the remaining form X-dimers, providing two cadherin pools with different adhesive properties. Our results demonstrate, for the first time, the inside-out regulation of cadherin conformation and establish a mechanistic role for vinculin in this process. SIGNIFICANCE STATEMENTCadherin cell-cell adhesion proteins play key roles in the formation and maintenance of tissues. Their adhesion is carefully regulated to orchestrate complex movement of cells. While cadherin ectodomains bind in two conformations with different adhesive properties, the mechanisms by which cells regulate the conformation (and consequently adhesion) of individual cadherins are unknown. Here, we demonstrate that the association of intracellular vinculin to the cadherin cytoplasmic region, regulates cadherin adhesion by switching ectodomains from a weak binding to the strongly adhesive conformation. In contrast with the prevailing view which suggests that vinculin regulates adhesion solely by remodeling the cytoskeleton, we show that vinculin can directly modulate single cadherin ectodomain conformation and that this process is mediated by changes in cytoskeletal tension.

biophysics↗

bioRxiv2019

Pitching single focus confocal data analysis one photon at a time with Bayesian nonparametrics

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

biophysics↗

bioRxiv2018

An Alternative Framework for Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS), is a flexible and widely used tool routinely exploited for in vivo and in vitro applications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted from {micro}-s long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy based approaches, to probe processes several orders of magnitude faster in time and permits a reduction of phototoxic effects on living samples induced by long periods of light exposure.

biophysics↗