Mridula Vishwanath

Dynamic Assessment of Fibroblast Mechanical Activity During Rac-Induced Cell Spreading in 3-D Culture

The goal of this study was to determine the morphological and sub‐cellular mechanical effects of Rac activation on fibroblasts within 3‐D collagen matrices. Corneal fibroblasts were plated at low density inside 100 µm thick fibrillar collagen matrices and cultured for 1–2 days in serum‐free media. Time‐lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y‐27632 or blebbistatin were used to inhibit Rho‐kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac‐induced cell spreading within 3‐D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II‐based tractional forces during cell spreading within 3‐D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II‐independent. J. Cell. Physiol. 217: 162–171, 2008.

Local thermal injury elicits immediate dynamic behavioural responses by corneal Langerhans cells.

Langerhans cells (LCs) represent a special subset of immature dendritic cells (DCs) that reside in epithelial tissues at the environmental interfaces. Although dynamic interactions of mature DCs with T cells have been visualized in lymph nodes, the cellular behaviours linked with the surveillance of tissues for pathogenic signals, an important function of immature DCs, remain unknown. To visualize LCs in situ, bone marrow cells from C57BL/6 mice expressing the enhanced green fluorescent protein (EGFP) transgene were transplanted into syngeneic wild‐type recipients. Motile activities of EGFP+ corneal LCs in intact organ cultures were then recorded by time lapse two‐photon microscopy. At baseline, corneal LCs exhibited a unique motion, termed dendrite surveillance extension and retraction cycling habitude (dSEARCH), characterized by rhythmic extension and retraction of their dendritic processes through intercellular spaces between epithelial cells. Upon pinpoint injury produced by infrared laser, LCs showed augmented dSEARCH and amoeba‐like lateral movement. Interleukin (IL)‐1 receptor antagonist completely abrogated both injury‐associated changes, suggesting roles for IL‐1. In the absence of injury, exogenous IL‐1 caused a transient increase in dSEARCH without provoking lateral migration, whereas tumour necrosis factor‐α induced both changes. Our results demonstrate rapid cytokine‐mediated behavioural responses by LCs to local tissue injury, providing new insights into the biology of LCs.

Analysis of the pattern of subcellular force generation by corneal fibroblasts after Rho activation

Purpose. To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. Methods. Human corneal fibroblasts were plated at low density in 100-&mgr;m thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 &mgr;mol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. Results. Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (−12.1% ± 7.0%; P<0.05) and cell area (−13.1% ± 13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. Conclusions. The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.

1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor

AIM Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.