Bimodal sensing of guidance cues in mechanically distinct microenvironments

Contact guidance due to extracellular matrix architecture is a key regulator of carcinoma invasion and metastasis, yet our understanding of how cells sense guidance cues is limited. Here, using a platform with variable stiffness that facilitates uniaxial or biaxial matrix cues, or competing E-cadherin adhesions, we demonstrate distinct mechanoresponsive behavior. Through disruption of traction forces, we observe a profound phenotypic shift towards a mode of dendritic protrusion and identify bimodal processes that govern guidance sensing. In contractile cells, guidance sensing is strongly dependent on formins and FAK signaling and can be perturbed by disrupting microtubule dynamics, while low traction conditions initiate fluidic-like dendritic protrusions that are dependent on Arp2/3. Concomitant disruption of these bimodal mechanisms completely abrogates the contact guidance response. Thus, guidance sensing in carcinoma cells depends on both environment architecture and mechanical properties and targeting the bimodal responses may provide a rational strategy for disrupting metastatic behavior

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize

Biophysical mechanisms of single-cell interactions with microtopographical cues

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures (“micropegs”) can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling

Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos

Asymmetric positioning of the mitotic spindle in C. elegans embryos is mediated by force-generating complexes that are anchored at the plasma membrane and that pull on microtubules growing out from the spindle poles. Although asymmetric distribution of the force generators is thought to underlie asymmetric positioning of the spindle, the number and location of the force generators has not been well defined. In particular, it has not been possible to visualize individual force generating events at the cortex. We discovered that perturbation of the acto-myosin cortex leads to the formation of long membrane invaginations that are pulled from the plasma membrane toward the spindle poles. Several lines of evidence show that the invaginations, which also occur in unperturbed embryos though at lower frequency, are pulled by the same force generators responsible for spindle positioning. Thus, the invaginations serve as a tool to localize the sites of force generation at the cortex and allow us to estimate a lower limit on the number of cortical force generators within the cell

Function and Dynamics of Tetraspanins during Antigen Recognition and Immunological Synapse Formation

Tetraspanin-enriched microdomains (TEMs) are specialized membrane platforms driven by protein protein interactions that integrate membrane receptors and adhesion molecules. Tetraspanins participate in antigen recognition and presentation by antigen-presenting cells (APCs) through the organization of pattern-recognition receptors (PRRs) and their downstream induced signaling, as well as the regulation of MHC-II-peptide trafficking. T lymphocyte activation is triggered upon specific recognition of antigens present on the APC surface during immunological synapse (IS) formation. This dynamic process is characterized by a defined spatial organization involving the compartmentalization of receptors and adhesion molecules in specialized membrane domains that are connected to the underlying cytoskeleton and signaling molecules. Tetraspanins contribute to the spatial organization and maturation of the IS by controlling receptor clustering and local accumulation of adhesion receptors and integrins, their downstream signaling, and linkage to the actin cytoskeleton. This review offers a perspective on the important role of TEMs in the regulation of antigen recognition and presentation and in the dynamics of IS architectural organization.The cost of this publication has been paid in part by FEDER funds.S

Cardiomyocytes Sense Matrix Rigidity through a Combination of Muscle and Non-muscle Myosin Contractions

British Heart Foundatio

The biophysical aspects of intercellular adhesion (role of e-cadherins in membrane-cytoskeleton interactions)

Le cytosquelette cortical est impliqué dans tous processus impliquant la surface cellulaire. Il est associé à la membrane soit de manière transitoire ou durable. La E-cadhérine est une molécule d adhérence qui stimule le développement de contact intercellulaire et augmente l énergie d adhérence intercellulaire en induisant le remodelage du cytosquelette d actine. J ai utilisé la technique d étirement hydrodynamique de nano-tubes membranaires à partir de cellules pour analyser les interactions membrane-cytosquelette. J ai réalisé des étirements de tubes soit à partir de billes recouvertes de polylysine (extrusion aspécifique) ou d anticorps spécifiques de la E-cadhérine et à partir de cellules exprimant ou non des cadhérines. Les résultats oobtenus montre que l extrusion aspécifique de tube de membrane est gouvernée par deux modes principaux d écoulement de composants membranaires selon leur relation avec le cytosquelette cortical sous-jacent : (1) le détachement du cortex sous la membranes à proximité du tube, et (2) la dissipation visqueuse dans la zone distale. Les résultats provenant des extrusions spécifiques de tubes de membranes montrent que l engagement des E-cadhérines augmente fortement l énergie d adhésion membrane-cytosquelette cortical. Cependant, cela se produit dans une zone strictement localisée au site adhésif. Après détachement des interactions membrane-cytosquelette, une récupération complète de leur adhésion est observée en 30 secondes montrant le rôle crucial des cadhérines dans la dynamique du remodelage cortical. Parfois lors de l extrusion spécifique il y a formation de protubérance cylindrique d un diamètre bien supérieur à celui d un tube de membrane classique et d environ 10-20 m de long, appelée tube cortical géant . Il permet de mesurer le module de courbure du cortex cellulaire kc=2 .4.10-16J. Ces résultats montrent le rôle crucial de la e-cadhérine dans le contrôle de l interaction membrane-cytosquelette.Cytoskeleton cortex is the basic cellular structure, determining cell viscoelastic properties and membrane dynamics. It is involved in every cell-surface associated process. Cadherins are transmembrane adhesion receptors spatially associated with actin cytoskeleton remodelling and promoting cell-cell adhesion and increasing adhesion energy. I used the tether extrusion technique, which consists of a hydrodynamic pulling of membrane tube from the cell surface, to directly measure the membrane-cytoskeleton interaction and adhesion energy. I performed specific or unspecific tether extrusions to compare the effect of E-cadherin-induced modification of cortex-membrane interactions. The results show that membrane tether unspecific extrusion is ruled by two main modes of the flow of membrane components through cytoskeleton related membrane-supporting network : (1) the detachment of the flowing membrane apart the cortex in the proximal zone of the tether s neck and (2) the viscous permeation of molecules in the flowing membrane at the distal zone. For specific extrusion, the initial stage of tube elongation is extremely resistant to the hydrodynamic force, followed by the regular extrusion resistance, comparable to unspecific case. Together, these data show that E-cadherin engagement largely increases the energy of the plasma membrane-cortex adhesion, but restricted at the cell-bead interface zone. In some cases of specific extrusion, a giant cortical tube is formed corresponding to a cylindrical protrusion of 4-5 m in diameter and 10-20 m in length. It reveals the presence of the membrane-supporting cytoskeleton inside the tube and its contractile activity. This phenomenon allows measuring the cell cortex curvature modulus Kc =2.4.10-16J and also displays the strong anchoring of the cortex to the cell surface through E-cadherins. The results show the E-cadherin-promoted cortex reorganization and reveal its restriction to the adhesive zone.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Microtubule-Actomyosin Mechanical Cooperation during Contact Guidance Sensing

Summary: Cancer cell migration through and away from tumors is driven in part by migration along aligned extracellular matrix, a process known as contact guidance (CG). To concurrently study the influence of architectural and mechanical regulators of CG sensing, we developed a set of CG platforms. Using flat and nanotextured substrates with variable architectures and stiffness, we show that CG sensing is regulated by substrate stiffness and define a mechanical role for microtubules and actomyosin-microtubule interactions during CG sensing. Furthermore, we show that Arp2/3-dependent lamellipodia dynamics can compete with aligned protrusions to diminish the CG response and define Arp2/3- and Formins-dependent actin architectures that regulate microtubule-dependent protrusions, which promote the CG response. Thus, our work represents a comprehensive examination of the physical mechanisms influencing CG sensing. : Aligned extracellular matrix architectures in tumors direct migration of invasive cancer cells. Tabdanov et al. show that the mechanical properties of aligned extracellular matrix environments influence invasive cell behavior and define a mechanical role for microtubules and actomyosin-microtubule interactions during sensing of contact guidance cues that arise from aligned extracellular matrix. Keywords: contact guidance, carcinoma metastasis, microtubule

Mechanosensing in T lymphocyte activation.

Mechanical forces play an increasingly recognized role in modulating cell function. This report demonstrates mechanosensing by T cells, using polyacrylamide gels presenting ligands to CD3 and CD28. Naive CD4 T cells exhibited stronger activation, as measured by attachment and secretion of IL-2, with increasing substrate elastic modulus over the range of 10-200 kPa. By presenting these ligands on different surfaces, this report further demonstrates that mechanosensing is more strongly associated with CD3 rather than CD28 signaling. Finally, phospho-specific staining for Zap70 and Src family kinase proteins suggests that sensing of substrate rigidity occurs at least in part by processes downstream of T-cell receptor activation. The ability of T cells to quantitatively respond to substrate rigidly provides an intriguing new model for mechanobiology