Chondrocyte-specific response to stiffness-mediated primary cilia formation and centriole positioning

Mechanical stress and the stiffness of the extracellular matrix are key drivers of tissue development and homeostasis. Aberrant mechanosensation is associated with a wide range of pathologies, including osteoarthritis. Matrix (or substrate) stiffness plays a major role in cell spreading, adhesion, proliferation, and differentiation. However, how specific cells sense substrate stiffness still remains unclear. The primary cilium is an essential cellular organelle that senses and integrates mechanical and chemical signals from the extracellular environment. We hypothesized that the primary cilium dynamically alters its length and position to fine-tune cell mecha-nosignaling based on substrate stiffness alone. We used a hydrogel system of varying substrate stiffness to examine the role of stiff-ness on cilia frequency, length, and centriole position as well as cell and nuclei area over time. Contrary to other cell types, we show that chondrocyte primary cilia shorten on softer substrates, demonstrating tissue-specific mechanosensing that is aligned with the tis-sue stiffness the cells originate from. We further show that stiffness determines centriole positioning to either the basal or apical mem-brane during attachment and spreading, with centrioles positioned toward the basal membrane on stiffer substrates. These phenomena are mediated by force generation actin-myosin stress fibers in a time-dependent manner. Finally, we show on stiff sub-strates that primary cilia are involved in tension-mediated cell spreading. We propose that substrate stiffness plays a role in cilia posi-tioning, regulating cellular responses to external forces, and maybe a key driver of mechanosignaling-associated diseases.