Biology subjects

Sorokina, E. M.

Publications and source records attributed to Sorokina, E. M..

4 recordsLinked to original sources

bioRxiv2024

Multiplexed DNA-PAINT Imaging of the Heterogeneity of Late Endosome/Lysosome Protein Composition

Late endosomes/lysosomes (LELs) are crucial for numerous physiological processes and their dysfunction is linked to many diseases. Proteomic analyses have identified hundreds of LEL proteins, however, whether these proteins are uniformly present on each LEL, or if there are cell-type dependent LEL sub-populations with unique protein compositions is unclear. We employed a quantitative, multiplexed DNA-PAINT super-resolution approach to examine the distribution of six key LEL proteins (LAMP1, LAMP2, CD63, TMEM192, NPC1 and LAMTOR4) on individual LELs. While LAMP1 and LAMP2 were abundant across LELs, marking a common population, most analyzed proteins were associated with specific LEL subpopulations. Our multiplexed imaging approach identified up to eight different LEL subpopulations based on their unique membrane protein composition. Additionally, our analysis of the spatial relationships between these subpopulations and mitochondria revealed a cell-type specific tendency for NPC1-positive LELs to be closely positioned to mitochondria. Our approach will be broadly applicable to determining organelle heterogeneity with single organelle resolution in many biological contexts. SummaryThis study develops a multiplexed and quantitative DNA-PAINT super-resolution imaging pipeline to investigate the distribution of late endosomal/lysosomal (LEL) proteins across individual LELs, revealing cell-type specific LEL sub-populations with unique protein compositions, offering insights into organelle heterogeneity at single-organelle resolution.

cell biology↗

bioRxiv2024

Piggybacking functionalized DNA nanostructures into live cell nuclei

DNA origami (DO) are promising tools for in vitro or in vivo applications including drug delivery; biosensing, detecting biomolecules; and probing chromatin sub-structures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing visualizing and controlling important biological processes in live cells. Here we present an approach to deliver DO strucures into live cell nuclei. We show that labelled DOs do not undergo detectable structural degradation in cell culture media or human cell extracts for 24 hr. To deliver DO platforms into the nuclei of human U2OS cells, we conjugated 30 nm long DO nanorods with an antibody raised against the largest subunit of RNA Polymerase II (Pol II), a key enzyme involved in gene transcription. We find that DOs remain structurally intact in cells for 24hr, including within the nucleus. Using fluorescence microscopy we demonstrate that the electroporated anti-Pol II antibody conjugated DOs are efficiently piggybacked into nuclei and exihibit sub-diffusive motion inside the nucleus. Our results reveal that functionalizing DOs with an antibody raised against a nuclear factor is a highly effective method for the delivery of nanodevices into live cell nuclei.

bioengineering↗

bioRxiv2020

ORP1L regulates dynein clustering on endolysosmal membranes in response to cholesterol levels

The sub-cellular positioning of endolysosomes is crucial for regulating their function. Particularly, the positioning of endolysosomes between the cell periphery versus the peri-nuclear region impacts autophagy, mTOR (mechanistic target of rapamycin) signaling and other processes. The mechanisms that regulate the positioning of endolysosomes at these two locations are still being uncovered. Here, using super-resolution microscopy, we show that the retrograde motor dynein forms nano-clusters on endolysosomal membranes containing 1-2 dyneins, with an average of ~3 nanoclusters per endolysosome. These data suggest that a very small number of dynein motors (1-6) drive endolysosome motility. Surprisingly, dynein nano-clusters are slightly larger on peripheral endolysosomes having higher cholesterol levels compared to peri-nuclear ones. By perturbing endolysosomal membrane cholesterol levels, we show that dynein copy number within nano-clusters is influenced by the amount of endolysosomal cholesterol while the total number of nano-clusters per endolysosome is independent of cholesterol. Finally, we show that the dynein adapter protein ORP1L (Oxysterol Binding Protein Homologue) regulates the number of dynein motors within nano-clusters in response to cholesterol levels. We propose a new model by which endolysosomal transport and positioning is influenced by the cholesterol sensing adapter protein ORP1L, which influences dyneins copy number within nano-clusters.

cell biology↗

bioRxiv2020

Tau forms oligomeric complexes on microtubules that are distinct from pathological oligomers in disease

Tau is a microtubule-associated protein, which promotes neuronal microtubule assembly and stability. Accumulation of tau into insoluble aggregates known as neurofibrillary tangles (NFTs) is a pathological hallmark of several neurodegenerative diseases. The current hypothesis is that small, soluble oligomeric tau species preceding NFT formation cause toxicity. However, thus far visualizing the spatial distribution of tau monomers and oligomers inside cells under physiological or pathological conditions has not been possible. Here, using single molecule localization microscopy (SMLM), we show that, in vivo, tau forms small oligomers on microtubules under physiological conditions. These physiological oligomers are distinct from those found in cells exhibiting tau aggregation and could be pre-cursors of aggregated tau in pathology. Further, using an unsupervised shape classification algorithm that we developed, we show that different tau phosphorylation states are associated with distinct tau aggregate species. Our work elucidates taus nanoscale composition under physiological and pathological conditions in vivo.

cell biology↗