Our research focuses on the interaction between fibroblasts and the surrounding 3D matrix at the cell–extracellular matrix (ECM) interphase. Using confocal reflectance microscopy, we quantitatively assessed changes in collagen fiber organization at different concentrations (1.0 and 1.5 mg/mL) in 3D hydrogels and their regulation by fibroblasts. For this, we embedded cells in a 3D collagen gel and, after incubation for the required duration, fixed and fluorescently labeled them. Cells were imaged in confocal mode, and the collagen was imaged in the reflectance mode by using a Zeiss LSM 780 scanning confocal microscope.
The image shown on the cover of the January 6 issue of Biophysical Journal is a mouse embryonic fibroblast cell treated with the Rho-associated coiled coil kinase (ROCK) inhibitor (Y 27632) embedded in 1.0 mg/mL 3D collagen gel. Individual wavelength images have been deconvoluted by using the Huygens software and processed in the ImageJ software. The final image shows the maximum intensity projection of a Z-stack of deconvolved images, with the cell labeled with phalloidin for actin (pseudo-colored in purple) and the collagen fibers (pseudo-colored in yellow).
The image thus illustrates the 3D collagen matrix around a cell, allowing for its interaction and mutual regulation. Upon ROCK inhibition in this environment, fibroblasts adopt a highly protrusive morphology, extending these processes into the 3D collagen matrix. These protrusions actively modulate cell-collagen interaction and its organization.
Our study shows that the cell regulates collagen organization around itself in the presence of Caveolin-1 in a collagen concentration–dependent manner. This regulation depends on Rho-ROCK–dependent actin-mediated cell-matrix interaction. When ROCK is inhibited, cells form numerous active protrusions into the 3D collagen hydrogel, altering cell-ECM interactions and contraction. This affects the organization of collagen in 3D hydrogels by cells. We further show that dynamin plays a role in this process. Dynamin, when targeted, disrupts ROCK inhibition-mediated cell protrusion and collagen organization in 3D hydrogels. Together, these observations quantitatively demonstrate how cells respond at the cell-matrix interphase to subtle changes in collagen concentration and organization in 3D hydrogels, regulated by a Rho-ROCK-actin–dependent pathway.
Our lab is interested in understanding how the matrix microenvironment influences cell behavior. In addition to investigating cell-ECM interactions in a 3D microenvironment, our lab also focuses on the adhesion-dependent organization of organelles, such as the Golgi and mitochondria, and their implications for cell function. You can find more information on our work at https://www.adhesionlab.com/.
Debasmita Mazumdar, Sujal Kataria, Gyanendra Prasad Panda, Atharva Kulkarni, Shivprasad Patil, Mamoni Dash, and Nagaraj Balasubramanian