Quasi-3D Cytoskeletal Dynamics of Osteocytes under Fluid Flow

Abstract

Osteocytes respond to dynamic fluid shear loading by activating various biochemical pathways, mediating a dynamic process of bone formation and resorption. Whole-cell deformation and regional deformation of the cytoskeleton may be able to directly regulate this process. Attempts to image cellular deformation by conventional microscopy techniques have been hindered by low temporal or spatial resolution. In this study, we developed a quasi-three-dimensional microscopy technique that enabled us to simultaneously visualize an osteocyte's traditional bottom-view profile and a side-view profile at high temporal resolution. Quantitative analysis of the plasma membrane and either the intracellular actin or microtubule (MT) cytoskeletal networks provided characterization of their deformations over time. Although no volumetric dilatation of the whole cell was observed under flow, both the actin and MT networks experienced primarily tensile strains in all measured strain components. Regional heterogeneity in the strain field of normal strains was observed in the actin networks, especially in the leading edge to flow, but not in the MT networks. In contrast, side-view shear strains exhibited similar subcellular distribution patterns in both networks. Disruption of MT networks caused actin normal strains to decrease, whereas actin disruption had little effect on the MT network strains, highlighting the networks' mechanical interactions in osteocytes.

A noninvasive approach to determine viscoelastic properties of an individual adherent cell under fluid flow

“…Each image of the plasma membrane over time is further processed by means of a digital image correlation technique to measure the displacement of the cell contour. The accuracy of the deformation of the cell acquired by the quasi-3D microscopy system was verified in our previous work, with the precision for displacement ±0.2 pixel and strain ±0.00287 (%), respectively (Baik et al, 2010; Qiu et al, 2012). …”

“…Fluorescent images of individual osteocytes are captured by two objectives simultaneously at 12 Hz along the loading history, using the custom-built quasi-3D microscope as described in our previous study (Baik et al, 2010). Each image of the plasma membrane over time is further processed by means of a digital image correlation technique to measure the displacement of the cell contour.…”

“…In our previous studies, the 3D cell geometry of an osteocyte under fluid flow was recorded by a quasi-3D microscopy (Baik et al, 2010). The current study combines quasi-3D microcopy and a fluid-structure interaction (FSI) finite element method to simulate the viscoelastic creep behavior of osteocytes under steady flow.…”

T-Type voltage-sensitive calcium channels mediate mechanically-induced intracellular calcium oscillations in osteocytes by regulating endoplasmic reticulum calcium dynamics

“…The FRET biosensor was excited at 430nm, and fluorescence emissions of YFP (530nm) and CFP (470nm) were captured simultaneously using a quadview beamsplitter and custom quad-band polychroic 27 . To monitor ER depletion, cells were imaged for 20 minutes following the addition of thapsigargin (1µM).…”

Experimental studies of bone mechanoadaptation: bridging in vitro and in vivo studies with multiscale systems

“…Our laboratory developed a technique to observe the deformation of cytoskeletal elements in rounded osteocytes under fluid flow at high temporal resolution (less than 1 s) in both bottom-and side-view [99] (figure 4a). Using this technique, we can reconstruct a quasi-three-dimensional image of the cell under fluid flow (figure 4b).…”