Title: Application of a Novel Quasi-3D Microscopy Technique to Investigate Early Osteocyte Mechanotransduction Events
Description: The objective of this thesis is to observe and characterize the early mechanical and biochemical events in osteocyte mechanotransduction. Physical forces have been increasingly implicated in normal physiological and pathological cellular activities of osteocytes. The mechanotransduction process in osteocytes involves spatiotemporally complex changes in cytoskeletal organization, signal activation, and whole cell mechanical properties. Most in vitro biophysical techniques currently available sacrifice either spatial or temporal resolution and are unable to visualize 3D cellular behavior on the millisecond time scale. Here, we develop a novel multi-channel quasi-3D microscopy technique to simultaneously visualize and measure whole-cell mechanics, intracellular cytoskeletal deformation, and biochemical signal activation under fluid shear flow.The technique was applied to visualize cell dilatation and cytoskeletal deformation in osteocytes under steady fluid shear flow. Analysis of the plasma membrane and either the intracellular actin or microtubule cytoskeletal networks provided characterization of their deformations over time.

No volumetric dilatation of the whole cell was observed under flow, and both cytoskeletal networks experienced primarily tensile viscoelastic creep and recovery in all measured strain components. Intra- and inter- cellular mechanical heterogeneity was observed in both cytoskeletal networks. Cytoskeletal disruption pointed towards a unidirectional mechanical interaction where microtubule networks affected actin network strains, but not vice versa.The second study in this thesis investigated the effects of steady and oscillatory flow on the actin and microtubule networks within the same cell. Shear strain was the predominant strain in both steady and oscillatory flows, in the form of viscoelastic creep and elastic oscillations, respectively. Under oscillatory fluid shear flow, the actin networks displayed an oscillatory strain profile more often than the MT networks in all the strains tested and had a higher peak-to-trough magnitude. Taken together with the first study, the actin networks were determined to be the more responsive cytoskeletal networks in osteocytes to fluid flow and may play a bigger role in mechanotransduction.The final culminating study tracked [Ca+2]i and F-actin network strains simultaneously in a single osteocyte.

We demonstrated novel osteocyte mechano- and transduction behavior where [Ca+2]i oscillations activate phasic actomyosin contractions using a smooth muscle-like mechanism. Fluid shear, ATP, and ionomycin induced [Ca+2]i signaling with a subsequent compression and recovery in actin strains of the cell, being most apparent in the height direction strain. This contraction was reversible over the period of hundreds of seconds. ML-7, a myosin light chain kinase inhibitor, significantly slowed down the kinetics of contraction initiation, but blebbistatin, a potent skeletal and non-muscle inhibitor, had no effect on the actin contraction. Furthermore, smooth muscle contraction-related proteins were detected by Western blot. The observation of muscle-like contractility in osteocytes demonstrates a possible positive feedback mechanism of osteocytes to activate mechanotransduction pathways.