Biology subjects

Klevanski, M.

Publications and source records attributed to Klevanski, M..

4 recordsLinked to original sources

bioRxiv2025

Glyco-STORM: Nanoscale Mapping of the Cellular Glycosylation Landscape

Glycosylation is a crucial biochemical modification of proteins and other biomolecules in cells that generates an exceptional structural and functional diversity. Aberrant glycosylation is implicated in numerous diseases, including neurodegenerative disorders and cancer. Despite its significance, methodological constraints to date have limited the exploration of the nanometer scale spatial arrangement of glycans across entire cells. We developed Glyco-STORM, a super-resolution imaging approach that generates nano-structural maps of cellular glycosylation. Glyco-STORM employs fluorophore-labeled lectins and multiplexed single-molecule super-resolution microscopy, in combination with nanoscale spatial pattern analysis. For example, Glyco-STORM unraveled nanodomains within the endoplasmic reticulum, subdomains along the Golgi axes, and a polarized lysosomal clathrin coat. At synaptic contact sites, mature glycans delineate the synaptic cleft and subsynaptic tubules adjacent to the postsynaptic density. In summary, Glyco-STORM elucidates the spatial arrangement of glycosylation sites from subcellular to molecular levels, revealing the previously obscured glycosylation landscape at nanoscale and establishing a spatial glycosylation code that provides a unique perspective on cellular organization distinct from traditional protein-centric views.

cell biology↗

bioRxiv2024

Super-resolved protein imaging using bifunctional light-up aptamers

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single-molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light-up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super-resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein-binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein-binding DNA or RNA aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP-binding aptamer AP3 facilitates high- and super-resolution imaging of GFP-tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody-based immunofluorescence protocols, as they are 7-fold smaller than antibody conjugates and exhibit higher bleaching-resistance. We demonstrate the effectiveness of our approach in super-resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST-PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST.

biophysics↗

bioRxiv2021

Visualizing multi-protein patterns at the synapse of neuronal tissue with DNA-assisted single-molecule localization microscopy

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.

biophysics↗

bioRxiv2019

Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues

Understanding the nano-architecture of protein machines in diverse sub-cellular compartments remains a challenge despite rapid progress in super-resolution microscopy. While singlemolecule localization microscopy techniques allow the visualization and identification of cellular structures with near-molecular resolution, multiplex-labeling of tens of target proteins within the same sample has not yet been achieved routinely. However, single sample multiplexing is essential to detect patterns that threaten to get lost in multi-sample averaging. Here, we report maS3TORM (multiplexed automated serial staining stochastic optical reconstruction microscopy), a microscopy approach capable of fully automated 3D dSTORM imaging and solution exchange employing a re-staining protocol to achieve highly multiplexed protein localization within individual biological samples. We demonstrate 3D super-resolution images of 15 target proteins in single cultured cells and 16 targets in individual neuronal tissue samples with <10 nm localization precision. This allowed us to define novel nano-architectural features of protein distribution within the presynaptic nerve terminal.

neuroscience↗