Srikanth Budnar

A mechanobiological perspective on cadherins and the actin-myosin cytoskeleton

Classical cadherin receptors mediate morphogenetic cell-cell interactions within many tissues of the body. Their biological impact often entails cooperation between cadherin adhesion and the actin cytoskeleton, but how this may occur and – even more urgently – how this leads to morphogenetic outcomes are questions that remain poorly understood. Here, we suggest that the emerging field of cadherin mechanobiology provides a useful new perspective from which to revisit these issues. We propose that the actin cytoskeleton can be considered as an active agent that mediates how cadherin junctions resist, sense and transduce forces between cells.

Role of tumor cell surface lysosome-associated membrane protein-1 (LAMP1) and its associated carbohydrates in lung metastasis

AbstractPurpose Expression of lysosome-associated membrane protein-1 (LAMP1) on the surface correlates with metastatic potential of B16 melanoma cells. Downregulation of their expression in high metastatic (B16F10) cells reduced their surface expression and metastatic potential. Present investigations explore if overexpression of LAMP1 on the surface of low metastatic (B16F1) cells augment their metastatic ability, and if so, how?MethodsB16F1 cells were transduced with lentiviral vector carrying mutant-LAMP1 (Y386A) (mutLAMP1). Surface expression of LAMP1 and carbohydrates was analyzed by flow cytometry, immunofluorescence and/or immunoprecipitation and Western blotting. Cell spreading and motility were assessed on components of extracellular matrix (ECM) (fibronectin) and basement membrane (BM) (matrigel), and galectin-3-coated coverslips/plates. Metastatic potential was assessed using experimental metastasis assay.ResultsPre-incubation with anti-LAMP1 antibodies significantly reduced lung metastasis of B16F10 cells. Overexpression of mutLAMP1 significantly increased its surface expression on B16F1 cells, resulting in increased cellular spreading and motility on fibronectin and matrigel. LAMP1 is the major carrier of poly-N-acetyllactosamine (polyLacNAc) on B16F10 cells. However, significantly higher expression of mutLAMP1 had no effect on galectin-3 binding on cell surface or on spreading or motility of cells on galectin-3-coated coverslips/plates. These cells also failed to show any gain in metastatic ability. This could be because LAMP1 from these cells carried significantly lower levels of polyLacNAc in comparison with B16F10 cells.ConclusionsPolyLacNAc on B16F10 cells and galectin-3 on lungs are the major participants in melanoma metastasis. Although surface LAMP1 promotes interactions with organ ECM and BM, carbohydrates on LAMP1 play a decisive role in dictating lung metastasis.

Pulsatile contractility of actomyosin networks organizes the cellular cortex at lateral cadherin junctions

The physical properties of cells reflect how the structure and dynamics of the actomyosin cortex are coupled to the plasma membrane. In epithelia, adhesive E-cadherin clusters associate with the cell cortex to assemble the junctional actomyosin that participates in epithelial morphogenesis. E-cadherin is present not only at the apical zonula adherens (ZA), but also distributed throughout the lateral adherens junction (LAJ) below the ZA. However, the organizational dynamics of the actomyosin network at the LAJs remains elusive. To address this, we used quantitative real-time imaging to characterize the dynamics of actomyosin contractility at lateral cadherin contacts. Here, we report that contractility is coordinated into smaller actomyosin rings that link cadherin clusters together within the larger cortical network at the lateral junctions. We conclude that Myosin II activity determines the contractility of actomyosin cables between cadherin clusters to propagate pulsatility across lateral cell-cell contacts.

N-glycans and metastasis in galectin-3 transgenic mice

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3(+/+) (wild type), gal-3(+/-) (hemizygous) and gal-3(-/-) (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3(+/-) mice which showed <50% expression of galectin-3 on the lungs also showed proportionate decrease in the number of B16F10 melanoma metastatic colonies affirming that galectin-3 and polyLacNAc interactions are indeed key determinants of lung metastasis. However, surprisingly, the number and size of metastatic colonies in gal-3(-/-) mice was very similar as that seen in gal-3(+/+) mice. The levels of lactose binding lectins on the lungs and the transcripts of other galectins (galectin-1, -8 and -9) which are expressed on lungs and have similar sugar binding specificities as galectins-3, remain unchanged in gal-3(+/+) and gal-3(-/-) mice. Further, inhibition of N-glycosylation with Swainsonine (SW) which drastically reduces metastasis of B16F10 cells in gal-3(+/+) mice, did not affect lung metastasis when assessed in gal-3(-/-) mice. Together, these results rule out the possibility of some other galectin taking over the function of galectin-3 in gal-3(-/-) mice. Chimeric mice generated to assess if absence of any effect on metastasis is due to compromised tumor immunity by replacing bone marrow of gal-3(-/-) mice with that from gal-3(+/+) mice, also failed to impact melanoma metastasis. As galectin-3 regulates several immune functions including maturation of different immune cells, compromised tumor immunity could be the major determinant of melanoma metastasis in gal-3(-/-) mice and warrants thorough investigation.

Tyrosine dephosphorylated cortactin downregulates contractility at the epithelial zonula adherens through SRGAP1

Contractile adherens junctions support cell−cell adhesion, epithelial integrity, and morphogenesis. Much effort has been devoted to understanding how contractility is established; however, less is known about whether contractility can be actively downregulated at junctions nor what function this might serve. We now identify such an inhibitory pathway that is mediated by the cytoskeletal scaffold, cortactin. Mutations of cortactin that prevent its tyrosine phosphorylation downregulate RhoA signaling and compromise the ability of epithelial cells to generate a contractile zonula adherens. This is mediated by the RhoA antagonist, SRGAP1. We further demonstrate that this mechanism is co-opted by hepatocyte growth factor to promote junctional relaxation and motility in epithelial collectives. Together, our findings identify a novel function of cortactin as a regulator of RhoA signaling that can be utilized by morphogenetic regulators for the active downregulation of junctional contractility.Epithelial cell-cell adhesions are contractile junctions, but whether contractility can be down-regulated is not known. Here the authors report how tyrosine dephosphorylation of the cytoskeletal scaffold, cortactin, recruits the RhoA antagonist SRGAP1 to relax adherens junctions in response to HGF.

Bistable front dynamics in a contractile medium: Travelling wave fronts and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.

Coronin 1B supports RhoA signaling at cell-cell junctions through Myosin II

ABSTRACT Non-muscle myosin II (NMII) motor proteins are responsible for generating contractile forces inside eukaryotic cells. There is also a growing interest in the capacity for these motor proteins to influence cell signaling through scaffolding, especially in the context of RhoA GTPase signaling. We previously showed that NMIIA accumulation and stability within specific regions of the cell cortex, such as the zonula adherens (ZA), allows the formation of a stable RhoA signaling zone. Now we demonstrate a key role for Coronin 1B in maintaining this junctional pool of NMIIA, as depletion of Coronin 1B significantly compromised myosin accumulation and stability at junctions. The loss of junctional NMIIA, upon Coronin 1B knockdown, perturbed RhoA signaling due to enhanced junctional recruitment of the RhoA antagonist, p190B Rho GAP. This effect was blocked by the expression of phosphomimetic MRLC-DD, thus reinforcing the central role of NMII in regulating RhoA signaling.

Functional Implications of O-GlcNAcylation-dependent Phosphorylation at a Proximal Site on Keratin 18.

Keratins 8/18 (K8/18) are phosphoglycoproteins and form the major intermediate filament network of simple epithelia. The three O-GlcNAcylation (Ser29, Ser30, and Ser48) and two phosphorylation (Ser33 and Ser52) serine sites on K18 are well characterized. Both of these modifications have been reported to increase K18 solubility and regulate its filament organization. In this report, we investigated the site-specific interplay between these two modifications in regulating the functional properties of K18, like solubility, stability, and filament organization. An immortalized hepatocyte cell line (HHL-17) stably expressing site-specific single, double, and triple O-GlcNAc and phosphomutants of K18 were used to identify the site(s) critical for regulating these functions. Keratin 18 mutants where O-GlcNAcylation at Ser30 was abolished (K18-S30A) exhibited reduced phosphorylation induced solubility, increased stability, defective filament architecture, and slower migration. Interestingly, K18-S30A mutants also showed loss of phosphorylation at Ser33, a modification known to regulate the solubility of K18. Further to this, the K18 phosphomutant (K18-S33A) mimicked K18-S30A in its stability, filament organization, and cell migration. These results indicate that O-GlcNAcylation at Ser30 promotes phosphorylation at Ser33 to regulate the functional properties of K18 and also impact cellular processes like migration. O-GlcNAcylation and phosphorylation on the same or adjacent sites on most proteins antagonize each other in regulating protein functions. Here we report a novel, positive interplay between O-GlcNAcylation and phosphorylation at adjacent sites on K18 to regulate its fundamental properties.

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. Here, we report a mechanosensitive signaling pathway that activates RhoA at adherens junctions to preserve epithelial integrity in response to acute tensile stress. We identify Myosin VI as the force sensor, whose association with E-cadherin is enhanced when junctional tension is increased by mechanical monolayer stress. Myosin VI promotes recruitment of the heterotrimeric Gα12 protein to E-cadherin, where it signals for p114 RhoGEF to activate RhoA. Despite its potential to stimulate junctional actomyosin and further increase contractility, tension-activated RhoA signaling is necessary to preserve epithelial integrity. This is explained by an increase in tensile strength, especially at the multicellular vertices of junctions, that is due to mDia1-mediated actin assembly.

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.