Biology subjects

Shevchuk, A.

Publications and source records attributed to Shevchuk, A..

6 recordsLinked to original sources

bioRxiv2026

Investigating nanostructure and -mechanics of contracting actin stress fibers by scanning ion conductance microscopy

Scanning ion conductance microscopy (SICM) provides gentle, non-contact cell surface imaging, but it has not been used to investigate intracellular structures because the plasma membrane restricts nanopipette access. Here, we combined SICM with microsonication-based cell de-roofing to expose intracellular actin stress fibers (SFs) in U2OS cells for nanotopographical and -mechanical characterization. Importantly, the de-roofing conditions preserved actomyosin contractility, allowing analysis of SF structural and biomechanical changes during ATP-induced contraction. Resting SFs displayed an average height of 203{+/-}38 nm and width of 357{+/-}73 nm, and a complex surface architecture characterized by regularly spaced long-range height modulations ([~]500 nm periodicity; Wq [~]25 nm) and smaller irregular corrugations (Ra [~]19.2 nm). ATP stimulation reduced SF height and width by [~]39% and [~]15%, respectively, while largely preserving surface corrugation patterns. During contraction, some SFs separated into two longitudinal strands. High-resolution SICM imaging also revealed filamentous crosslinks mechanically coupling neighboring SFs, and nanomechanical measurements demonstrated local stiffening during contraction. These findings provide new insight into the structural and mechanical regulation of SF contraction and highlight the potential of SICM combined with cell de-roofing as a powerful platform for studying dynamic intracellular processes at nanometer resolution.

biophysics↗

bioRxiv2026

Constructing a single-objective oblique plane microscope (OPM) for fast, multi-colour, high-resolution volumetric fluorescence imaging

Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture (NA) objective for both illuminating the sample and collecting emission fluorescence from a tilted plane within the specimen. OPM has become indispensable in biological and biomedical research, providing rapid, high-resolution volumetric fluorescence imaging of live cells and tissues while minimising phototoxicity and photobleaching. It also overcomes the sample mounting challenges associated with conventional light sheet microscopes that require two orthogonally placed objectives. However, the application of OPM has been limited by the complex design and the intricate optical alignment and characterisation needed, particularly with the remote-refocusing system (RFS) in the emission path. This protocol offers a detailed, step-by-step guide for constructing an OPM setup using commercially available components and for characterising its performance to ensure optimal imaging quality. We aim to deliver the unique merits of OPM to researchers in life science and medicine, enabling them to visualise the spatiotemporal organisation of key biomolecules, structures, and cells in 3D at high resolutions.

biophysics↗

bioRxiv2026

Multimodal mechano-SICM and FRET with stretch for probing cardiomyocyte function.

Cardiac function is dependent upon the ability of cardiomyocytes to adapt their contractions to meet the demands of the body. Increased preload lengthens the sarcomere, altering the efficiency of contraction. The surface topography of cardiomyocytes is distinct from other cell types. T-tubules are key membrane structures which protrude into the cell body, aligned with the edges of the sarcomere. These are key signalling domains which ensure efficient and adaptable excitation-contraction coupling. It has been shown that T-tubules are dynamic structures which deform during the contraction cycle however, how the T-tubule structure adapts to increased preload has not been realised. Here we demonstrate a methodology for the measurement of the surface topography and sub-cellular signalling of isolated adult cardiomyocytes under diastolic stretch. We track individual T-tubule openings, showing that increased load causes them to shift, increase diameter and become stiffer. Future applications of this system include experimental modelling of preload-reducing therapies, for the treatment of acute and chronic heart failure.

physiology↗

bioRxiv2025

Multiparametric Correlative Topographical and Volumetric Fluorescence Microscopy

Live-cell imaging of cell surface topography and intracellular architecture is essential for understanding cellular function. However, conventional approaches often involve trade-offs between resolution, invasiveness, and volumetric coverage. Here, we present an integrated Scanning Ion Conductance Microscope and single-objective Oblique Plane Microscope (SICM-OPM) system that enables simultaneous non-contact topographical imaging and volumetric fluorescence imaging within the same live cell. Beyond correlative live imaging, the platform supports nanomechanical mapping with tens-of-nanometres resolution, fluorescence-guided localised molecular delivery via the SICM, and benefits from reduced photobleaching due to light-sheet excitation. We demonstrate this platforms capabilities by visualising imipramine-induced T-tubule remodelling in live cardiomyocytes, revealing subsurface detubulation while surface morphology remains preserved. Additionally, we show precision delivery of fluorescent cargos--including dextrans and -synuclein--into diatom and mammalian cells, alongside localised stiffness mapping to evaluate mechanical responses. We believe this technique opens new avenues for correlative structural, functional, and biophysical studies in live cells, with broad relevance to cell biology, neurodegeneration, and mechanobiology.

biophysics↗

bioRxiv2024

Depleting trafficking regulator CASK promotes intercalated disc organization and ventricular function

BackgroundThe intercalated disc (ID) electromechanically couples adjacent cardiomyocytes. Alteration of this structure plays a central role in cardiac arrhythmias, notably arrhythmogenic cardiomyopathy (ACM), an inherited genetic disorder of desmosomes. Calcium/Calmodulin-Dependent Serine Protein Kinase (CASK), a costameric component of the lateral membrane, regulates cardiomyocyte protein trafficking. Here, we investigated CASK regulation of the organization of ID components in normal and pathological contexts. Here, we investigated CASK regulation of the organization of ID components in normal and pathological contexts. MethodsWe studied the outcomes of CASK depletion in neonatal rat hearts using a cardiac-specific adeno-associated virus strategy. We used conventional and strain echocardiography, hemodynamics, electrocardiography, histology, and gene and protein expression studies to characterize adult rat hearts. We also studied the effects of CASK depletion in neonatal rat ventricular cardiomyocytes (NRVM), and control and ACM (PKP2+/-) IPS cell-derived CM (hIPS-CM) using proteomics, electron microscopy, high-resolution imaging, mechano-scanning ion conductance microscopy (mechano-SICM), and stress resistance tests. We studied cardiac CASK expression and localization in human ACM and non-transplantable control hearts. ResultsDepletion of CASK in our multiple experimental models revealed that CASK regulates cardiomyocyte IDs. In rats, CASK depletion improved contractile reserve and compliance. In cultured rat cardiomyocytes, CASK knockdown increased localization of connexin 43 (Cx43) and PKP2 at IDs, resulting in increased contact stiffness. In PKP2+/- hIPS-CMs, CASK expression was increased. CASK depletion in these cells promoted PKP2 accumulation at cell contacts, formation of desmosome-like structures, and stress resistance. In the right ventricles of ACM patients, CASK protein level was also increased and CASK abnormally localized at the ID. ConclusionCASK functions as a repressor of ID organization and tissue cohesion, suggesting novel mechanisms for regulating ID structure and function. These observations, along with CASK upregulation and mislocalization in ACM, open up new perspectives on understanding the pathophysiology of ACM and suggest innovative strategies for its treatment.

physiology↗

bioRxiv2020

Release of insulin granules by simultaneous, high-speed correlative SICM-FCM

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, non-adherent, surface of pancreatic {beta}-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically-induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

cell biology↗