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

Abstract

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 networking in human blood cells with application in acute care illnesses

“…We have advanced our previous work in which we published whole cell mitochondrial tracking based on fluorescence live imaging. 17,18 We have expanded our method to now track mitochondria networking in whole cells using PBMCs freshly obtained from subjects and easily compare networking between PBMCs obtained from healthy subjects and PBMCs obtained from subjects with sepsis, an acute care illness. We have demonstrated that the methodology is accessible within a clinically meaningful time frame, thus illustrating the translational strength of our method.…”

“…We preprocessed raw image files in ImageJ using an approach previously presented. 17,18 Briefly, time-lapse images were first convolved using the 5×5 edge-detection filter given in. Next, images were converted to the frequency domain using a Fast Fourier Transform (FFT) in ImageJ, and subjected to a bandpass filter ranging from 2 pixels (~0.3 μm with our resolution) to 100 pixels (~16 μm).…”

“…16 We advanced this approach by demonstrating “whole-cell” mitochondrial analysis, tracking all mitochondria present in the fluorescence microscopy focal plane. 17,18 Our technique enables the evaluation of whole-cell mitochondrial networking that includes measurement of motility as well as rates of fission and fusion events in PBMCs.…”

Parvalbumin alters mitochondrial dynamics and affects cell morphology

“…Besides the obviously visible rounding of the cells, whole cell volumes in PV-MDCK cells (− 30%) were decreased approximately proportional to the decrease in mitochondrial mass (− 40%). This effect might be linked to the mitochondrial Ca 2+ buffering capacity, shown to be implicated in cytoskeleton dynamics [ 66 ] and also to the intracellular distribution of mitochondria, which strongly depends on mitochondria–cytoskeleton interactions [ 45 , 50 ]. In line with our findings, blocking of mitochondrial Ca 2+ uptake in MCU-knockdown cells results in an increased circularity coefficient [ 66 ].…”

Translational Application of Measuring Mitochondrial Functions in Blood Cells Obtained from Patients with Acute Poisoning

“…We performed a specialized protocol to establish the respiratory capacities at various complexes providing more detailed respiratory activity at complex (C) I, II, III, IV. Our published manuscripts describe these methods in further detail in regard to injections with specific concentrations [12,25,28].…”