Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.

Durotaxis, migration of cells directed by stiffness gradient, is critical in development and disease. To study the molecular determinants of single cell durotaxis, we developed an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with different slopes, along with uniform stiffness (soft and stiff) regions. We find that fibroblasts rely on non-muscle myosin II (NMII) activity and the LIM-domain protein zyxin for durotaxis on both steep and shallow stiffness gradients. Importantly, unlike haptotaxis, the Arp2/3 complex is dispensable for durotaxis on both stiffness gradients. Lack of Arp2/3 results in a filopodia-based durotactic migration that is equally efficient as that of lamellipodia-based durotactic migration. Finally, we reveal an essential role for the actin-bundler fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis in shallow, but not steep, stiffness gradient. Together, our all-in-one hydrogel system can serve as a platform to identify, discriminate, and characterize stiffness gradient specific molecular mechanisms that cells employ to efficiently durotax.

The Systemic Capillary Leak Syndrome (SCLS) is a rare, orphan disease characterized by bouts of acute, reversible plasma extravasation into tissues, which leads to hemoconcentration, hypoalbuminemia, hypotension, and vascular collapse. Although the cause of SCLS is unknown, 80–95 % of SCLS patients have a monoclonal gammopathy of unknown significance (MGUS), in which clonal plasma cells secretes large amounts of monoclonal immunoglobulins or “paraproteins”. MGUS is a premalignant precursor to multiple myeloma (MM) and is notably detected in most SCLS patient sera. Another marked distinction is that acute, but not basal SCLS sera contain elevated concentrations of inflammatory mediators known to promote endothelial permeability, notably Vascular Endothelial Growth Factor (VEGF) and Angiopoietin 2 (Angpt2). To address these findings experimentally, we purified whole IgG from patient and control sera to examine whether these immunoglobulins have an impact on endothelial cell barrier function and performed functional assays to characterize the observed changes in morphology. Preliminary data suggest that patient IgG binds to endothelial cells and elicits significant F-actin stress fiber formation and VE-cadherin internalization. SCLS IgG seems to be critical in triggering endothelial dysfunction and dissecting the underlying signaling mechanisms involved will help us better understand disease pathogenesis and potentially pave the way for therapeutic target development.