A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte

Frolikova, M.; Blazikova, M.; Capek, M.; Chmelova, H.; Valecka, J.; Kolackova, V.; Valaskova, E.; Gregor, M.; Komrskova, K.; Horvath, O.; Novotny, I.

doi:10.1101/2023.03.07.531472

bioRxiv · 10.1101/2023.03.07.531472

A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte

Frolikova, M.·Blazikova, M.·Capek, M.·Chmelova, H.·Valecka, J.·Kolackova, V.·Valaskova, E.·Gregor, M.·Komrskova, K.·Horvath, O.·Novotny, I.

View original source ↗Export metadata ↓

Abstract

Super-resolution (SR) microscopy is a cutting-edge method that can provide detailed structural information with high resolution. However, the thickness of the specimen has been a major limitation for SR methods, and larger structures have posed a challenge. To overcome this, the key step is to optimize sample preparation to ensure optical homogeneity and clarity, which can enhance the capabilities of SR methods for the acquisition of thicker structures. Oocytes are the largest cells in the mammalian body and are crucial objects in reproductive biology. They are especially useful for studying membrane proteins. However, oocytes are extremely fragile and sensitive to mechanical manipulation and osmotic shocks, making sample preparation a critical and challenging step. We present an innovative, simple, and sensitive approach to oocyte sample preparation for 3D STED acquisition. This involves alcohol dehydration and mounting into a high refractive index medium. This extended preparation procedure allowed us to successfully obtain a unique 2-channel 3D STED super-resolution image of an entire mouse oocyte. By optimizing sample preparation, we can overcome the limitations of SR methods and obtain high-resolution images of larger structures, such as oocytes, Knowledge of which are important for understanding fundamental biological processes. RESEARCH HIGHLIGHTSO_LIThis study aimed to develop a successful sample preparation protocol for imaging mouse oocytes using 3D STED super-resolution microscopy. C_LIO_LIThe results showed that the oocyte sample was optically homogenous, enhancing the capability of the 3D STED method to capture high-resolution images throughout the full depth of the sample, resulting in highly similar SR images. C_LIO_LIThe 3D STED point spread function (PSF) pattern of the depletion laser was successfully secured throughout the entire volume of the sample, including the top and bottom. C_LIO_LIThis study presents the first-ever full volume 3D image of the mouse oocyte, which was acquired using a 2-channel 3D STED method. C_LI GRAPHICAL ABSTRACTIntroducing an extended sample preparation procedure resulted in an outstanding optical quality sample environment pronounced by high refractive index, high transparency, and minimal spherical aberration. This procedure allowed 3D STED within the entire oocyte. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Explore related subjects

cell biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Frolikova, M., Blazikova, M., Capek, M., Chmelova, H., Valecka, J., Kolackova, V., Valaskova, E., Gregor, M., Komrskova, K., Horvath, O., Novotny, I.. 2023-03-07. A sample preparation procedure enables acquisition of 2-channel super-resolution 3D STED image of an entire oocyte. https://doi.org/10.1101/2023.03.07.531472

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Functional characterization of Rho GTPase activating proteins SYDE1 and SYDE2

The human genome encodes more than 60 proteins containing Rho GTPase activating protein (RhoGAP) domains, many of which remain understudied with respect to their target specificity and biological roles. SYDE1 and SYDE2 are two such orphan RhoGAPs, for which there are few studies characterizing their biochemical and cellular functions and conflicting reports identifying their cognate GTPases. We previously identified SYDE1 and SYDE2 in a screen for substrates of the c-Jun N-terminal kinases. Here, we show that SYDE1 and SYDE2 are preferentially phosphorylated by JNK1 relative to other mitogen-activated protein kinases (MAPKs) at sites proximal to a kinase docking region. Purified SYDE1 and SYDE2 are shown to have significant catalytic GAP activity toward RhoA, Rac1, and Cdc42. However, neither up- nor down-regulation of SYDE1/2 expression leads to detectable changes in bulk GTP loading of any of these GTPases. Nevertheless, we demonstrate that SYDE1 and SYDE2, in a partially GAP-dependent manner, increase cell spreading and number of focal adhesions, and promote more directionally persistent migration in HEK293 cells. Together, these findings establish SYDE1 and SYDE2 as robust JNK substrates with catalytic activity toward a set of Rho GTPases and reveal basic functions of SYDE1 and SYDE2 in regulating cell morphology, adhesion, and migration.

cell biology↗

bioRxiv2026

The filopodial scaffold polyphosphate dictates cell adhesion-versus-invasion decisions

Inorganic polyphosphate (polyP) is an ancient polymer conserved across all life, serving cell type and location specific functions in every major compartment. Yet its role at the plasma membrane, where it accumulates to peak levels in many primary cells, is largely unknown. Here we identify polyP as a stabilizing component of filopodia, actin based membrane protrusions that govern cell adhesion, contact inhibition, and chemotaxis. Elevating cellular polyP increases filopodial stability and enhances cell adhesion, whereas reducing polyP accelerates filopodial disassembly and promotes cell migration. Mechanistically, we find that polyP acts as a structural filopodial scaffold, recruiting and organizing IRSp53, a membrane curvature inducing protein. We show that metastatic fibroblasts and breast cancer organoids carry markedly reduced and intracellularly reorganized polyP levels relative to their non transformed counterparts. Restoring endogenous polyP via lipid nanoparticle delivery suppresses their invasive phenotypes and reverses prometastatic gene expression signatures, implicating polyP as a primordial tumor suppressor.

cell biology↗

bioRxiv2026

Mitochondrial transfer mediates metabolic communication between beta cells and islet macrophages

Pancreatic islet macrophages support islet homeostasis and adapt their metabolic program in response to environmental cues, including beta cell released factors. Intercellular mitochondrial transfer is a biological process that modulates cellular responses. To test whether beta cells, which are strongly secretory, transfer mitochondria to islet macrophages, we generated mice with beta cell-specific expression of mitochondrial GFP (PhAMfloxIns1Cre). We demonstrate that beta cells transfer mitochondria to islet macrophages in vivo and in vitro. Diabetogenic stressors did not alter the frequency of mitochondrial transfer and macrophages containing beta cell-derived GFP exhibit increased protein synthesis rates. RNA-seq identified upregulation of activity-regulated cytoskeleton associated protein (Arc) in macrophages receiving beta cell-derived mitochondria, while disruption of actin cytoskeleton dynamics prevented mitochondrial transfer. Together, these findings identify mitochondrial transfer as a previously unrecognized mechanism of beta cell-macrophage communication that may contribute to islet homeostasis and immune regulation.

cell biology↗