near-simultaneous-laser-scanning-confocal-atomic-force-microscopy

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

University of Kentucky

The cytoskeletal architecture directly affects the morphology, motility, and tensional homeostasis of the cell. In addition, the cytoskeleton is important for mitosis, intracellular traffic, organelle motility, and even cellular respiration. The organelle responsible for a majority of the energy conversion for the cell, the mitochondrion, has a dependence on the cytoskeleton for mobility and function. In previous studies, we established that cytoskeletal inhibitors altered the movement of the mitochondria, their morphology, and their respiration in human dermal fibroblasts. Here, we use this protocol to investigate applicability of power law diffusion to describe mitochondrial locomotion, assessment of rates of fission and fusion in healthy and diseased cells, and differences in mitochondria locomotion in more open networks either in response to cytoskeletal destabilizers or by cell line. We found that mitochondria within fibrosarcoma cells and within fibroblast cells treated with an actin-destabilizing toxin resulted in increased net travel, increased average velocity, and increased diffusion of mitochondria when compared to control fibroblasts. Although the mitochondria within the fibrosarcoma travel further than mitochondria within their healthy counterparts, fibroblasts, the dependence on mitochondria for respiration is much lower with higher rates ofhydrogen peroxide production and was confirmed using the OROBOROS O2K. We also found that rates of fission and fusion of the mitochondria equilibrate despite significant alteration of the cytoskeleton. Rates ranged from 15% to 25%, where the highest rates were observed within the fibrosarcoma cell line. This result is interesting because the fibrosarcoma cell line does not have increased respiration metrics including when compared to fibroblast. Mitochondria travel further, faster, and have an increase in percent mitochondria splitting or joining while not dependent on the mitochondria for a majority of its energy production. This study illustrates the complex interaction between mitochondrial movement and respiration through the disruption of the cytoskeleton.

Mitochondrial dynamics and respiration within cells with increased open pore cytoskeletal meshes.

Functionalization of planar and curved glass surfaces with spiropyran (SP) molecules and localized UV-induced activation of the mechanophore are demonstrated. Fluorescence spectra of UV-irradiated SP-functionalized surfaces reveal that increases in surface roughness or curvature produce more efficient conversion of the mechanophore to the open merocyanine (MC) form. Further, force-induced activation of the mechanophore is achieved at curved glass-polymer interfaces and not planar interfaces. Minimal fluorescence signal from UV-irradiated SP-functionalized planar glass surfaces precluded mechanical activation testing. Curved glass-polymer interfaces are prepared by SP functionalization of E-glass fibers, which are subsequently embedded in a poly(methyl methacrylate) (PMMA) matrix. Mechanical activation is induced through shear loading by a single fiber microbond testing protocol.  detection of SP activation at the interface is monitored by fluorescence spectroscopy. The fluorescence increase during interfacial testing suggests that attachment of the interfacial SP molecule to both fiber surface and polymer matrix is present and able to achieve significant activation of SP at the fiber-polymer matrix interface. Unlike previous studies for bulk polymers, SP activation is detected at relatively low levels of applied shear stress. By linking SP at the glass-polymer interface and transferring load directly to that interface, a more efficient mechanism for eliciting the SP response is achieved.

Martha E. Grady

Department of Mechanical Engineering

University of Kentucky

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Martha E. Grady

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Mechanical Engineering

University of Kentucky

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Department of Mechanical Engineering