Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

BOXCAR IMAGING FLUORESCENCE CORRELATION SPECTROSCOPY REVEALS KINETICS OF RAFT STABILIZATION IN ANTIGEN-ACTIVATED MAST CELLS Nirmalya Bag 1,2 ; Gil-Suk Yang 2 ; Alice Wagenknecht-Wiesner 2 ; Barbara A Baird 2 ; 1 Indian Institute of Technology Kharagpur, Department of Chemistry, Kharagpur, India 2 Cornell University, Department of Chemistry and Chemical Biology, Ithaca, NY, USA Antigen (Ag) crosslinking of immunoglobulin E (IgE) receptor, FcεRI, leads to the clustering of the receptors followed by their phosphorylation by Lyn kinase at the plasma membrane (PM). Recently, we exploited unprecedented statistical robustness of camera-based imaging fluorescence correlation spectroscopy (ImFCS) to show reorganization of lipidic interactions in the inner leaflet of the PM corresponding to stabilization of ordered regions (colloquially known as rafts) around Ag-clustered IgE- FcεRI in liv e RBL mast cells (Bag et al, PNAS, 2021). We have now developed a time-resolved diffusion analysis in which the raw image stack is divided in overlapped segments (boxcar), and ImFCS is performed on each segment. In this manner, we obtained diffusion coefficients of membrane components at every 35 sec from live cells. Our data reveals membrane organization, as sensed by order- and disorder-preferring lipid probes, occurring within 500 sec of Ag- stimulation corresponding to about 50 FcεRI per cluster. These results represent one of the first direct demonstrations of raft stabilization in live cells in real time. We anticipate widespread application of this method in future studies of membrane dynamics.

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