Three-dimensional (3D) visualization of vitrified cells can uncover structures of subcellular complexes without chemical fixation or staining. Here, we present a pipeline integrating three imaging modalities to visualize the same specimen at cryogenic temperature at different scales: cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be applied to other types of cells, it could be used for large-scale phenotypic studies of frozenhydrated specimens in a variety of healthy and diseased conditions with and without treatments.
Mechanical stimulation of osteoblasts activates many cellular mechanisms including the release of ATP. Binding of ATP to purinergic receptors is key to load-induced osteogenesis. Osteoblasts also respond to fluid shear stress (FSS) with increased actin stress fiber formation (ASFF) that we postulate is in response to activation of the P2Y2 receptor (P2Y2R). Furthermore, we predict that ASFF increases cell stiffness and reduces the sensitivity to further mechanical stimulation. We found that small interfering RNA (siRNA) suppression of P2Y2R attenuated ASFF in response to FSS and ATP treatment. In addition, RhoA GTPase was activated within 15 min after the onset of FSS or ATP treatment and mediated ASFF following P2Y2R activation via the Rho kinase (ROCK)1/LIM kinase 2/cofilin pathway. We also observed that ASFF in response to FSS or ATP treatment increased the cell stiffness and was prevented by knocking down P2Y2R. Finally, we confirmed that the enhanced cell stiffness and ASFF in response to RhoA GTPase activation during FSS drastically reduced the mechanosensitivity of the osteoblasts based on the intracellular Ca2+ concentration ([Ca2+]i) response to consecutive bouts of FSS. These data suggest that osteoblasts can regulate their mechanosensitivity to continued load through P2Y2R activation of the RhoA GTPase signaling cascade, leading to ASFF and increased cell stiffness.
During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12dyn/cm2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis.
dimensional (3D) visualization of vitrified cells can uncover structures of subcellular 27 complexes without chemical fixation or staining. Here, we present a pipeline integrating three 28 imaging modalities to visualize the same specimen at cryogenic temperature at different scales: 29 cryo-fluorescence confocal microscopy, volume cryo-focused ion beam scanning electron 30 microscopy, and transmission cryo-electron tomography. Our proof-of-concept benchmark 31 revealed the 3D distribution of organelles and subcellular structures in whole heat-shocked yeast 32 cells, including the ultrastructure of protein inclusions that recruit fluorescently-labelled chaperone 33 Hsp104. Since our workflow efficiently integrates imaging at three different scales and can be 34 applied to other types of cells, it could be used for large-scale phenotypic studies of frozen-35 hydrated specimens in a variety of healthy and diseased conditions with and without treatments. 36 37 KEYWORDS 38 Airyscan microscopy; cryo correlative-light and electron microscopy, cryoCLEM; volume 39 cryo-focused ion bean scanning electron microscopy, cryoFIB-SEM; cryo-electron tomography, 40 cryoET; Hsp104 chaperone; protein aggregation 41 42 46 determination 1 . However, several limitations preclude its wider application, including the thickness 47 of some samples and difficulties in locating and identifying features of interest within them. To48 visualize molecular details in thicker samples by cryoET, such as regions in eukaryotic cells away 49 from the thin cell periphery, cryogenic focused ion beam scanning electron microscopy (cryoFIB-50 SEM) has been used to generate thin lamellae from vitrified cells 2-5 (i.e. thin layers through the 51 3 bulky cell) with a process called "ion beam milling", enabling many exciting biological observations 52 inside the cell 6 . However, technical challenges remain in ensuring that the milled lamellae contain 53 the features of interest. Correlative light and electron microscopy (CLEM) can overcome this 54 challenge by fluorescently labelling targets 7,8 , thereby guiding cryoFIB-SEM milling 9,10 and the 55 selection of optimal imaging areas for cryoET experiments, as well as aiding the interpretation of 56 observed features. However, even the smallest eukaryotic cells are typically several microns thick 57 and the desirable lamella thickness range is ~100-500 nm; thus, targeting nanoscale features 58 along the z direction remains extremely challenging. Here, as a proof-of-principle, we present a 59 pipeline to address this challenge by using high-resolution CryoAiryscan Confocal Microscopy 60 (CACM) to determine the z position of fluorescent targets within cells that were vitrified on electron 61 microscopy (EM) grids, followed by cryoFIB-SEM "mill and view" (MAV) imaging, which provides 62 a 3D view of whole cells with resolvable organelles as they are being milled to produce a lamella 63 containing the target of interest, and ending with visualization of molecular details of regions of 64 interest by cryoET. 65 66...
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