Cell type-specific adaptation of cellular and nuclear volume in micro-engineered 3D environments.

Bio-functionalized three-dimensional (3D) structures fabricated by direct laser writing (DLW) are structurally and mechanically well-defined and ideal for systematically investigating the influence of three-dimensionality and substrate stiffness on cell behavior. Here, we show that different fibroblast-like and epithelial cell lines maintain normal proliferation rates and form functional cell-matrix contacts in DLW-fabricated 3D scaffolds of different mechanics and geometry. Furthermore, the molecular composition of cell-matrix contacts forming in these 3D micro-environments and under conventional 2D culture conditions is identical, based on the analysis of several marker proteins (paxillin, phospho-paxillin, phospho-focal adhesion kinase, vinculin, ß1-integrin). However, fibroblast-like and epithelial cells differ markedly in the way they adapt their total cell and nuclear volumes in 3D environments. While fibroblast-like cell lines display significantly increased cell and nuclear volumes in 3D substrates compared to 2D substrates, epithelial cells retain similar cell and nuclear volumes in 2D and 3D environments. Despite differential cell volume regulation between fibroblasts and epithelial cells in 3D environments, the nucleus-to-cell (N/C) volume ratios remain constant for all cell types and culture conditions. Thus, changes in cell and nuclear volume during the transition from 2D to 3D environments are strongly cell type-dependent, but independent of scaffold stiffness, while cells maintain the N/C ratio regardless of culture conditions.

Multifunctional polymer scaffolds with adjustable pore size and chemoattractant gradients for studying cell matrix invasion.

Transmigrating cells often need to deform cell body and nucleus to pass through micrometer-sized pores in extracellular matrix scaffolds. Furthermore, chemoattractive signals typically guide transmigration, but the precise interplay between mechanical constraints and signaling mechanisms during 3D matrix invasion is incompletely understood and may differ between cell types. Here, we used Direct Laser Writing to fabricate 3D cell culture scaffolds with adjustable pore sizes (2-10 µm) on a microporous carrier membrane for applying diffusible chemical gradients. Mouse embryonic fibroblasts invade 10 µm pore scaffolds even in absence of chemoattractant, but invasion is significantly enhanced by knockout of lamin A/C, a known regulator of cell nucleus stiffness. Nuclear stiffness thus constitutes a major obstacle to matrix invasion for fibroblasts, but chemotaxis signals are not essential. In contrast, epithelial A549 cells do not enter 10 µm pores even when lamin A/C levels are reduced, but readily enter scaffolds with pores down to 7 µm in presence of chemoattractant (serum). Nuclear stiffness is therefore not a prime regulator of matrix invasion in epithelial cells, which instead require chemoattractive signals. Microstructured scaffolds with adjustable pore size and diffusible chemical gradients are thus a valuable tool to dissect cell-type specific mechanical and signaling aspects during matrix invasion.

Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner.

BACKGROUND: Cells sense the extracellular environment using adhesion receptors (integrins) linked to the intracellular actin cytoskeleton through a complex network of regulatory proteins that, all together, form focal adhesions (FAs). The molecular basis of how these sensing units are regulated, how they are implicated in transducing mechanical stimuli, and how this leads to a spatiotemporal coordination of FAs is unclear. RESULTS: Here we show that vinculin, through its links to the talin-integrin complex and F-actin, regulates the transmission of mechanical signals from the extracellular matrix to the actomyosin machinery. We demonstrate that the vinculin interaction with the talin-integrin complex drives the recruitment and release of core FA components. The activation state of vinculin is itself regulated by force, as underscored by our observation that vinculin localization to FAs is dependent on actomyosin contraction. Using a variety of vinculin mutants, we establish which components of the cell-matrix adhesion network are coordinated through direct and indirect associations with vinculin. Moreover, using cyclic stretching, we demonstrate that vinculin plays a key role in the transmission of extracellular mechanical stimuli leading to the reorganization of cell polarity. Of particular importance is the actin-binding tail region of vinculin, without which the cell's ability to repolarize in response to cyclic stretching is perturbed. CONCLUSIONS: Overall our data promote a model whereby vinculin controls the transmission of intracellular and extracellular mechanical cues that are important for the spatiotemporal assembly, disassembly, and reorganization of FAs to coordinate polarized cell motility.