Biomedical subjects

Shamik Sen

Publications and source records attributed to Shamik Sen.

6 recordsLinked to original sources

PubMed2006

Matrix elasticity directs stem cell lineage specification.

Microenvironments appear important in stem cell lineage specification but can be difficult to adequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs) are shown here to specify lineage and commit to phenotypes with extreme sensitivity to tissue-level elasticity. Soft matrices that mimic brain are neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigid matrices that mimic collagenous bone prove osteogenic. During the initial week in culture, reprogramming of these lineages is possible with addition of soluble induction factors, but after several weeks in culture, the cells commit to the lineage specified by matrix elasticity, consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibition of nonmuscle myosin II blocks all elasticity-directed lineage specification-without strongly perturbing many other aspects of cell function and shape. The results have significant implications for understanding physical effects of the in vivo microenvironment and also for therapeutic uses of stem cells.

Biomarkers↗

PubMed2006

Membrane mobility and clustering of Integrin Associated Protein (IAP, CD47)--major differences between mouse and man and implications for signaling.

Integrin Associated Protein (IAP, CD47) is a ubiquitous integral membrane protein implicated in processes (in mice) that range from inhibiting clearance by phagocytes [Oldenborg et al., Science 2000; Gardai et al., Cell 2005] to neutrophil motility [Lindberg et al., Science 1996]. SIRPalpha is CD47's main receptor on phagocytes plus a number of other cell types, and SIRPalpha-CD47 interactions in clusters are believed to mediate signaling. However, considerable species differences in CD47 sequence as well as differences in CD47 extractability from mouse cells versus man motivate a characterization of mobility, clusterability, and kinetics under force of CD47-SIRPalpha. Despite similar levels of CD47 on red cells from mouse and man, we find an effective avidity of SIRPalpha-CD47 for mouse appears higher than for human. Both mouse and human CD47 show clustering by multivalent SIRPalpha complexes, but only mouse cells aggregate with CD47 concentrating at cell-cell contacts. This proves consistent with fluorescence imaged micro-deformation, which indicates near-complete mobility of CD47 on mouse cells compared to only about 30-40% mobility on normal human cells. To qualify the method, we also show that disrupting cellular F-actin dramatically increases the mobility of integral membrane proteins. Furthermore, atomic force microscopy probing of cell membranes with human SIRPalpha confirms the species-specific interactions and provides evidence of clustering and adhesion on short time scales, but it also shows surprisingly strong forces in detachment for a signaling complex. The results thus highlight major species differences in CD47-SIRPalpha interactions and CD47 integration, suggesting that signaling by CD47 in man may be qualitatively different from mouse.

Animals↗

PubMed2005

Species- and cell type-specific interactions between CD47 and human SIRPalpha.

CD47 on red blood cells (RBCs) reportedly signals "self" by binding SIRPalpha on phagocytes, at least in mice. Such interactions across and within species, from mouse to human, are not yet clear and neither is the relation to cell adhesion. Using human SIRPalpha1 as a probe, antibody-inhibitable binding to CD47 was found only with human and pig RBCs (not mouse, rat, or cow). In addition, CD47-mediated adhesion of human and pig RBCs to SIRPalpha1 surfaces resists sustained forces in centrifugation (as confirmed by atomic force microscopy) but only at SIRPalpha-coating densities far above those measurable on human neutrophils, monocytes, and THP-1 macrophages. While interactions strengthen with deglycosylation of SIRPalpha1, low copy numbers explain the absence of RBC adhesion to phagocytes under physiologic conditions and imply that the interaction being studied is not responsible for red cell clearance in humans. Evidence of clustering nonetheless suggests mechanisms of avidity enhancement. Finally, using the same CD47 antibodies and soluble SIRPalpha1, bone marrow-derived mesenchymal stem cells were assayed and found to display CD47 but not bind SIRPalpha1 significantly. The results thus demonstrate that SIRPalpha-CD47 interactions, which reportedly define self, exhibit cell type specificity and limited cross-species reactivity.

Animals↗

PubMed2005

Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments.

In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments.

Animals↗

PubMed2004

Adhesion-contractile balance in myocyte differentiation.

Tissue cells generally pull on their matrix attachments and balance a quasi-static contractility against adequate adhesion, but any correlation with and/or influence on phenotype are not yet understood. Here, we begin to demonstrate how differentiation state couples to actomyosin-based contractility through adhesion and substrate compliance. Myotubes are differentiated from myoblasts on collagen-patterned coverslips that allow linear fusion but prevent classic myotube branching. Post-fusion, myotubes adhere to the micro-strips but lock into a stress fiber-rich state and do not differentiate significantly further. In contrast, myotubes grown on top of such cells do progress through differentiation, exhibiting actomyosin striations within one week. A compliant adhesion to these lower cells is suggested to couple to contractility and accommodate the reorganization needed for upper cell striation. Contractility is assessed in these adherent cells by mechanically detaching one end of the myotubes. All myotubes, whether striated or not, shorten with an exponential decay. The cell-on-cell myotubes relax more, which implies a greater contractile stress. The non-muscle myosin II inhibitor blebbistatin inhibits relaxation for either case. Myotubes in culture are thus clearly prestressed by myosin II, and this contractility couples to substrate compliance and ultimately influences actomyosin striation.

Actins↗

PubMed2004

Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments.

Contractile myocytes provide a test of the hypothesis that cells sense their mechanical as well as molecular microenvironment, altering expression, organization, and/or morphology accordingly. Here, myoblasts were cultured on collagen strips attached to glass or polymer gels of varied elasticity. Subsequent fusion into myotubes occurs independent of substrate flexibility. However, myosin/actin striations emerge later only on gels with stiffness typical of normal muscle (passive Young's modulus, E approximately 12 kPa). On glass and much softer or stiffer gels, including gels emulating stiff dystrophic muscle, cells do not striate. In addition, myotubes grown on top of a compliant bottom layer of glass-attached myotubes (but not softer fibroblasts) will striate, whereas the bottom cells will only assemble stress fibers and vinculin-rich adhesions. Unlike sarcomere formation, adhesion strength increases monotonically versus substrate stiffness with strongest adhesion on glass. These findings have major implications for in vivo introduction of stem cells into diseased or damaged striated muscle of altered mechanical composition.

Acrylic Resins↗