Naguib, N. | bioRxiv Science

bioRxiv2026

Intravital Cholesterol Depletion Reduces Membrane Dynamics and Increases Mechanosensitivity in Osteocytes In Vivo

Osteocytes detect mechanical forces within bone through signaling processes organized at the plasma membrane. Membrane cholesterol regulates membrane organization, dynamics, and mechanical properties, yet its role in osteocyte mechanotransduction in vivo remains unknown. Here, we developed an intravital multiphoton imaging approach to quantify membrane-associated uptake, retention, and clearance alongside load-induced Ca2+ signaling in osteocytes within the intact metatarsal. Using fluorescent nanoparticles and a membrane-labeling probe, we tracked these processes before and after pharmacological cholesterol depletion. Cholesterol depletion reduced osteocyte membrane uptake and retention and altered clearance in both sexes, while affecting load-induced Ca2+ signaling in a sex-dependent manner. In females, cholesterol depletion increased both the proportion of osteocytes responding to mechanical loading and the magnitude of their responses, whereas neither outcome changed in males. These findings identify plasma membrane cholesterol as a regulator of osteocyte membrane dynamics and mechanical responsiveness in vivo. More broadly, this work establishes an approach for directly examining how membrane composition and turnover regulate mechanotransduction in cells embedded within their native tissue environment.

cell biology↗

bioRxiv2023

Intravital imaging of osteocyte αvβ3 integrin dynamics with locally injectable fluorescent nanoparticles

Osteocytes are the resident mechanosensory cells in bone. They are responsible for skeletal homeostasis and adaptation to mechanical cues. Integrin proteins play an prominent role in osteocyte mechanotransduction, however the details are not well stratified in vivo. Intravital imaging with multiphoton microscopy presents an opportunity to study molecular level mechanobiological events in vivo, and could be used to study integrin dynamics in osteocytes. However, fluorescent imaging limitations with respect to excessive optical scattering and low signal to noise ratio caused by mineralized bone matrix make such investigations non-trivial. Here we demonstrate that ultra-small and bright fluorescent core-shell silica nanoparticles (<7nm diameter), known as Cornell Prime Dots (CDots), are well-suited for the in vivo bone microenvironment and can improve intravital imaging capabilities. We report validation studies for CDots as a novel, locally injected in vivo osteocyte imaging tool for both non-specific cellular uptake and for targeting integrins. The pharmacokinetics of CDots reveal distinct sex differences in nanoparticle cycling and clearance in osteocytes, which represents a novel topic of study in bone biology. Integrin-targeted CDots were used to study osteocyte integrin dynamics. To the best of our knowledge, we report here the first evidence of osteocyte integrin endocytosis and recycling in vivo. Our results provide novel insights in osteocyte biology and will open up new lines of investigation that were previously unavailable in vivo.

bioengineering↗