Scanning ion conductance microscopy of live human glomerulus

Bohovyk, Ruslan; Fedoriuk, Mykhailo; Isaeva, Elena; Shevchuk, Andrew; Palygin, Oleg; Staruschenko, Alexander

doi:10.1111/jcmm.16475

Cited by 2 publications

(2 citation statements)

References 19 publications

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“…Samples were manually positioned in the x – y direction under the inverted optical microscope, Nikon TE2000-U (Nikon Instruments, Tokyo, Japan). For the hopping probe, SICM imaging borosilicate glass nanopipettes with a resistance of approximately 100 MΩ, which corresponds to an estimated tip diameter of around 120 nm, were used as described previously [ 56 , 57 ]. The nanopipettes were filled with the same PSS solution used for the bath and were positioned in the z-direction with a piezoelectric actuator.…”

Section: Methodsmentioning

confidence: 99%

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes

Shalygin

Shuyskiy

Bohovyk

et al. 2021

IJMS

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The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.

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Section: Methodsmentioning

confidence: 99%

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes

Shalygin

Shuyskiy

Bohovyk

et al. 2021

IJMS

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“…Scanning ion conductance microscopy (SICM) is a technique that enables high-resolution, non-optical 3D-imaging of living cell surfaces with complex morphology. So far, SICM was only performed on whole glomeruli [6,7], but not yet on cultured human podocytes.…”

Section: Introductionmentioning

confidence: 99%

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

Kraus¹,

Rose

Krüger

et al. 2023

Cells

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Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future.

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Scanning ion conductance microscopy of live human glomerulus

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 2 publications

References 19 publications

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes

Cytoskeleton Rearrangements Modulate TRPC6 Channel Activity in Podocytes

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques

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