Biology subjects

Agasti, S. S.

Publications and source records attributed to Agasti, S. S..

4 recordsLinked to original sources

bioRxiv2026

An optopharmacological tool for on-demand perturbation of branched actin polymerization

Regulated actin cytoskeletal organization and dynamics are essential for life-sustaining processes in eukaryotic cells. The spatiotemporal regulation of filamentous actin can generate a staggering variety of higher-order architectures to meet diverse cellular needs. However, connecting a specific actin substructure to the selective cellular function remains challenging because tools to specifically inhibit F-actin in a spatiotemporally controlled manner are currently limited. To address this crucial technical gap, we have synthesized and characterized a photocrosslinkable small molecule inhibitor of a major and evolutionarily conserved actin nucleation factor, Arp2/3 complex. The new inhibitor "AZ" combines the specificity of a preexisting inhibitor CK666 with photosensitive crosslinking by an Azido group, such that a light-induced, instantaneous, on-demand, irreversible inhibition of Arp2/3 complex-dependent actin nucleation is achieved in a spatiotemporally defined manner at a subcellular, supracellular, and organismal scale. Whole cell treatment with AZ, followed by subcellular photoirradiation, revealed a divergent actin nucleation homeostasis in the cortex of different cells. Thus, AZ could be a powerful tool to infer actin polymerization homeostasis and network properties in live cells-processes which otherwise remain obscure.

cell biology↗

bioRxiv2023

Supramolecular catch-and-release strategy for bioorthogonal fluorogenic imaging across the visible spectrum

Fluorogenic probes that unmask fluorescence signals in response to a bioorthogonal reaction are a powerful new addition to biological imaging. They can provide significantly reduced background fluorescence and minimize non-specific signals, potentially allowing real-time high-contrast imaging without washing out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are readily available now, their integration into a bioorthogonal fluorogenic scheme still necessitates another level of extensive design efforts and customized structural alterations to optimize quenching mechanisms for each given fluorophore scaffold. Herein, we present an easy-to-implement and highly generalizable supramolecular catch-and-release strategy for generating an efficient bioorthogonal fluorogenic response from essentially any readily available fluorophores without further structural alterations. We designed this distinct strategy based on the macrocyclic cucurbit[7]uril (CB7) host, where a fluorogenic response is achieved by programming a guest displacement reaction from the macrocycle cavity. We used this strategy to rapidly generate fluorogenic probes across the visible spectrum from structurally diverse classes of fluorophore scaffolds, including coumarin, bodipy, rhodamine, and cyanine. These probes were applied to no-wash fluorogenic imaging of various target molecules in live cells and tissue with minimal background and no appreciable non-specific signal. Notably, the orthogonal reactivity profile of the system allowed us to pair this host-guest fluorogenic probe with the covalently clickable fluorogenic probe to achieve high-contrast super-resolution and multiplexed fluorogenic imaging in cells and tissue.

bioengineering↗

bioRxiv2019

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. In this work, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and movement of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA motion in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of LTP in culture revealed GluR-dependent biphasic up-regulation of mRNA translation with a similar effect in dendrites and somata. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

neuroscience↗

bioRxiv2017

Rapid Sequential In Situ Multiplexing With DNA-Exchange-Imaging

To decipher the molecular mechanism of biological function, it is critical to map the molecular composition of individual cells in the context of their biological environment in situ. Immunofluorescence (IF) provides specific labeling for molecular profiling. However, conventional IF methods have finite multiplexing capabilities due to spectral overlap of the fluorophores. Various sequential imaging methods have been developed to circumvent this spectral limit, but are not widely adopted due to the common limitation of requiring multi-rounds of slow (typically over 2 hours at room temperature to overnight at 4 {degrees}C in practice) immunostaining. DNA-Exchange-Imaging is a practical platform for rapid in situ spectrally-unlimited multiplexing. This technique overcomes speed restrictions by allowing for single-step immunostaining with DNA-barcoded antibodies, followed by rapid (less than 10 minutes) buffer exchange of fluorophore-bearing DNA imager strands. By eliminating the need for multiple rounds of immunostaining, DEI enables rapid spectrally unlimited sequential imaging. The programmability of DNA-Exchange-Imaging allows us to further adapt it to diverse microscopy platforms (with Exchange-Confocal, Exchange-SIM, Exchange-STED, and Exchange-PAINT demonstrated here), achieving highly multiplexed in situ protein visualization in diverse samples (including neuronal and tumor cells as well as fresh-frozen or paraffin-embedded tissue sections) and at multiple desired resolution scales (from ~300 nm down to sub-20-nm). Validation highlights include 8-target imaging using single-channel Exchange-Confocal in tens of micron thick retina tissue sections in 2-3 hours (as compared to days required in principle by previous methods using comparable equipment), and 8-target super-resolution imaging with ~20 nm resolution using Exchange-PAINT in primary neurons. These results collectively suggest DNA-Exchange as a versatile, practical platform for rapid, highly multiplexed in situ imaging, potentially enabling new applications ranging from basic science, to drug discovery, and to clinical pathology.

bioengineering↗