Biology subjects

Kitko, K. E.

Publications and source records attributed to Kitko, K. E..

3 recordsLinked to original sources

bioRxiv2022

A Multifunctional Anchor for Multimodal Expansion Microscopy

In situ imaging of biomolecular location with nanoscale resolution enables mapping of the building blocks of life throughout biological systems in normal and disease states. Expansion microscopy (ExM), by physically enlarging specimens in an isotropic fashion, enables nanoimaging on standard light microscopes. Key to ExM is the equipping of different kinds of molecule, with different kinds of anchoring moiety, so they can all be pulled apart by polymer swelling. Here we present a multifunctional anchor, an acrylate epoxide, that enables multiple kinds of molecules (e.g., proteins and RNAs) to be equipped with anchors in a single experimental step. This reagent simplifies ExM protocols and greatly reduces cost (by 2-10 fold for a typical multiplexed ExM experiment) compared to previous strategies for equipping RNAs with anchors. We show that this unified ExM (uniExM) protocol can be used to preserve and visualize RNA transcripts, proteins in biologically relevant ultrastructure, and sets of RNA transcripts in patient-derived xenograft (PDX) cancer tissues, and can support the visualization of other kinds of biomolecular species as well. Thus, uniExM may find many uses in the simple, multimodal nanoscale analysis of cells and tissues.

bioengineering↗

bioRxiv2019

Graphene nanoflakes for acute manipulation of membrane cholesterol and transmembrane signaling

Cholesterol is one of the most essential lipids in eukaryotic cell membranes. However, acute and selective manipulation of membrane cholesterol remains challenging. Here, we report that graphene nanoflakes (GNFs) insert into the plasma membrane and directly interact with cholesterol, resulting in acute cholesterol enrichment - and thus structural and functional changes. Using two representative cell preparations, we explore the utility of GNFs in modifying cell communication pathways sensitive to membrane cholesterol. In fibroblasts, GNFs enhance ATP-induced intracellular Ca2+-release by allosteric facilitation of P2Y receptors, a subtype of G protein-coupled receptors, in a cholesterol-dependent manner. In neurons, which possess higher membrane cholesterol levels than most cell types, GNFs further increase cholesterol. Consequently, GNFs change membrane fluidity, especially at synaptic boutons, and potentiate neurotransmitter release by accelerating synaptic vesicle turnover. Together, our results provide a molecular explanation for graphenes cellular impacts and demonstrate its potential for membrane-oriented engineering of cell signaling.

cell biology↗

bioRxiv2017

RNA editing of CAPS1 regulates synaptic vesicle organization, release and retrieval.

Calcium-dependent activator protein for secretion 1 (CAPS1) facilitates the docking and priming of synaptic and dense core vesicles. A conserved hairpin structure in the CAPS1 pre-mRNA allows an post-transcriptional adenosine-to-inosine RNA editing event to alter a genomically-encoded glutamate to a glycine codon. Functional comparisons of CAPS1 protein isoforms in primary hippocampal neurons show that elevation of edited CAPS1 isoforms facilitates presynaptic vesicle clustering and turnover. Conversely, non-edited CAPS1 isoforms slow evoked release, increase spontaneous fusion, and loosen the clustering of synaptic vesicles. Therefore, CAPS1 editing promotes organization of the vesicle pool in a way that is beneficial for evoked release, while non-edited isoforms promote more lax vesicle organization that widens distribution, attenuates evoked release and eases the control of spontaneous fusion. Overall, RNA editing of CAPS1 is a mechanism to fine tune neurotransmitter release.\n\nIMPACT STATEMENTPost-transcriptional RNA editing of CAPS1 is a mechanism to regulate neurotransmitter release from synaptic vesicles.

biochemistry↗