Abstract
Purpose :
Cell shape and organization in the tissue are fundamental to cell biology, and how it is altered in many diseased state is understudied despite changes in cytoskeletal dynamics. Attachment of cells to its substrate fundamentally dictates the shape and likely affects signal transduction. Here, we determine if constraining cells to defined patterns alters cytoskeletal organization, its biomechanics, and consequently its response to pro-fibrotic cytokines.
Methods :
Primary human trabecular meshwork cells (hTM) were plated as single cells in different geometry (square, rectangle, ellipse, circle; 2500 µm2 area) on gelatin coated glass substrates in the presence or absence of 5ng/ml TGFβ2 for 7 days in serum free media. Cell adhesion area also was controlled by micropatterning techniques. Cytoskeletal elements (actin, microtubules, and intermediate filaments) were visualized by immunocytochemistry, elastic moduli, and creep/stress relaxation was determined using atomic force microscopy (AFM).
Results :
Significant differences in cell height, volume and nuclear aspect ratio were observed between cells plated on circular vs rectangular shapes. This was correlated with F-actin organization with more stress fibers seen in rectangular and square cells, while more cortical arrangement observed in circular cells. TGFβ2 treatment resulted in cell stiffening (p<0.01, t-test) although the extent to which stiffness was altered was shape dependent.
Conclusions :
Our results demonstrate that cell geometry alters fundamental characteristics such as cell mechanics, actin organization and nuclear aspect ratio in hTM cells. Response to TGFβ stimuli was dependent on geometry and the implications warrant further studies. We infer that fundamental changes in nuclear shape and cytoskeleton are likely due to altered receptor distribution, which will consequently alter signal transduction differentially.
This is a 2020 ARVO Annual Meeting abstract.