Biology subjects

Pavlov, E.

Publications and source records attributed to Pavlov, E..

3 recordsLinked to original sources

bioRxiv2026

Label-free real-time imaging of mitochondrial matrix volume changes and permeability transition in living cells

Along with the membrane potential and respiration, mitochondrial matrix volume is a critical parameter that determines mitochondrial function. Mitochondria undergo constant changes in matrix volume and cristae dynamics, and in processes that are critical for normal metabolic rates and pathophysiological responses. Changes in matrix volume cannot be easily measured by conventional fluorescence imaging techniques due to the size of the sub-organellar structures, which are below resolution. This challenge was successfully resolved in studies of isolated mitochondria with the use of scattered light. Here we use dark-field imaging, which relies on scattered light contrast, to measure matrix volume dynamics in living cells. We demonstrate that mitochondrial volume changes can be easily detected as changes in intensity of the scattered light following matrix volume modulation with K+ ionophores or by onset of the permeability transition. Specifically, we found that stimulation of K+ influx leads to increase of mitochondrial matrix volume while stimulation of K+ efflux leads to matrix shrinkage, and that activation of the permeability transition leads to high-amplitude mitochondrial swelling in wild-type but not in cells lacking subunit c of ATP synthase. These results directly demonstrate the dynamic nature of mitochondrial matrix volume and its link to physiological and pathological ion transport.

cell biology↗

bioRxiv2026

Fabrication of the high-resistance patch-clamp pipettes for mitochondrial electrophysiological studies using optimized two step method

The patch-clamp experimental technique is widely used to study the electrical properties of ion channels in biological and artificial lipid membranes. The key to the high quality of the experiments is the manufacturing of glass pipettes that provide highly electrically resistant contact between the edge of the pipette tip and the lipid bilayer. Preparation of the pipettes is particularly challenging for studies of the mitochondrial membranes due to the need for very small pipette tip sizes. Here, we present a robust procedure for producing pipettes suitable for experiments with native mitochondrial membranes. This procedure involves a two-step approach: initial fabrication of relatively large glass micropipettes using a standard micropipette puller, followed by tip refinement using a microforger to achieve smooth glass surface and reduced opening size. Pipette tip diameters and surface structure were examined using field emission - scanning electron microscopy (FE-SEM) imaging to assess the effects of variable parameters on pipette geometry and size. The resulting pipettes were validated in patch-clamp recording of the mitochondrial inner membranes. This approach enables the reproducible production of optimized pipettes for mitochondrial patch-clamp experiments, improving the quality and throughput of electrophysiological recordings of the mitochondrial ion channels.

biophysics↗

bioRxiv2026

A robust workflow for 3D imaging of human mitochondria using cryo-electron tomography

Mitochondria are dynamic signaling organelles that transduce metabolic and biochemical cues to facilitate cellular adaptation. Their complex structure and dynamics are essential for integrating metabolic pathways, responding to stressors, and communicating inter- and intra-cellular signals. While optimal mitochondrial activity is frequently linked to cellular and organismal health--influencing processes ranging from metabolism and regulated cell death to differentiation and growth--the mechanistic links between mitochondrial dysfunction and cellular defects leading to human disease remain incompletely understood. Understanding how mitochondrial shape and function are linked is crucial for deciphering the regulatory mechanisms of cell survival and fate. Here, we present a molecular resolution cryo-electron tomography (cryo-ET) imaging and image analysis platform to investigate the structure of isolated human mitochondria under different conditions. We describe optimized protocols for isolating mitochondria from human cells, vitrifying these samples with high-pressure freezing (HPF) using the waffle method, cryo-focused ion beam (cryo-FIB) milling to generate thin sections (lamellae), and imaging with cryo-transmission electron microscopy (cryo-TEM). This is complemented by a robust downstream processing pipeline for tilt-series alignment, tomogram reconstruction, and three-dimensional (3D) segmentation of tomograms using the latest state-of-the-art algorithms. With some variations, this versatile workflow is adaptable to other subcellular compartments for structural studies in isolation or within intact cells. Furthermore, our protocols provide a critical foundation for investigating the in-situ structure of protein machineries that govern key cellular processes.

biophysics↗