Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. However, the molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the contribution of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Our results demonstrate that optimal PSC durotaxis requires an intermediary level of ion channel activity, which we simulated via a numerically discretized mathematical model. These findings suggest that mechanosensitive Piezo1 channels detect the differential stiffness microenvironment. The resulting intracellular signals are amplified by TRPV4 and TRPC1 channels to guide efficient PSC durotaxis.

Malignant neoplasms, such as pancreatic ductal adenocarcinoma (PDAC), show pathologically increased rigidity, which results in a stiffness gradient between tumorous and healthy tissue. Cells are able to detect such differences in rigidity and to migrate into the direction of higher stiffness which is referred to as durotaxis. In PDAC, pancreatic stellate cells (PSCs) are mainly responsible for the production of the rigid tumor environment. Thereby they indirectly attract more PSCs, and ultimately, promote the invasiveness of cancer cells.

The giant muscle protein titin is a major contributor to passive force; however, its role during active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of the titin springs to quantify how titin-based forces define myocyte ultrastructure and mechanics. We conduct a series of passive and active mechanical tests on permeabilized psoas fiber bundles from wildtype, heterozygote, and homozygote TC mice. We show that under mechanical strain, as titin cleavage doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly non-linear, while passive and active forces are progressively reduced. Furthermore, interactions of elastic titin with sarcomeric actin filaments are revealed, as cleaved I-band titins only partially recoil to the Z-disk, and instead stick to the thin filaments. Strikingly, when titin-cleaved fibers contract, myosin-containing A-bands quickly stream to the point that they split and adjacent thick filaments move in opposite directions. Cleavage of I-band titin also destabilizes the myosin filaments during contraction, which leads to a shedding of individual myosin rods from the D-zone of thick filaments. Taken together, these results establish intact titin filaments as critical force-transmission networks, buffering the forces between myosin filaments during contraction, while also stabilizing the myosin rods within the thick filament. Based on our calculations, a structural change in titin that increases stiffness compared to passive muscle is critical for titin to perform these buffering tasks, unveiling its fundamental role as an activation-dependent spring in contracting muscle.

The giant muscle protein titin is a major contributor to passive force; however, its role in active force generation is unresolved. Here, we use a novel titin-cleavage (TC) mouse model that allows specific and rapid cutting of elastic titin to quantify how titin-based forces define myocyte ultrastructure and mechanics. We show that under mechanical strain, as TC doubles from heterozygous to homozygous TC muscles, Z-disks become increasingly out of register while passive and active forces are reduced. Interactions of elastic titin with sarcomeric actin filaments are revealed. Strikingly, when titin-cleaved muscles contract, myosin-containing A-bands become split and adjacent myosin filaments move in opposite directions while also shedding myosins. This establishes intact titin filaments as critical force-transmission networks, buffering the forces observed by myosin filaments during contraction. To perform this function, elastic titin must change stiffness or extensible length, unveiling its fundamental role as an activation-dependent spring in contracting muscle.