Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

6-POS Board 6 ASTROGLIAL ACTIVATION BY INTERFERON GAMMA AUGMENTS CELL SURFACE EXPRESSION OF ANTIGEN PRESENTING MHCII MOLECULES VIA INCREASING EXOCYTOTIC BUT DECREASING ENDOCYTOTIC VESICLE INTERACTIONS WITH THE PLASMALEMMA Mico Božic 2 ; Robert Zorec 1,2 ; Matjaž Stenovec 1,2 ; 1 Celica BIOMEDICAL, Ljubljana, Ljubljana, Slovenia 2 University of Ljubljana, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, Ljubljana, Slovenia Astrocytes are homeostatic cells in the central nervous system that partake in neuroinflammation upon activation; a process also termed reactive astrocytosis. It was shown previously that interferon gamma (IFNgamma), an inflammatory cytokine, induces expression of major histocompatibility type II molecules (MHCII) engaged in antigen presentation at the cell surface. To elucidate the vesicular pathway involved in MHCII delivery and segregation at the plasmalemma of cultured rat astrocytes, we investigated the subcellular localization of MHCII by confocal and structured illumination (SIM) microscopies and examined the dynamics of single vesicle interactions with the plasmalemma by high-resolution cell-attached membrane capacitance measurements. Astrocyte activation with IFNgamma increased the expression of MHCII, which upon immunolabelling co-localized highly with LAMP1-EGFP, a lysosomal marker, and minutely with immunolabelled Rab4A, EEA1 and TPC1, markers of various endosomes. As revealed by SIM measurements, numerous MHCII-positive vesicles in IFNgamma-activated astrocytes had larger diameter, consistent with their lysosomal identity. Contrastively, these were scarce in controls. MHCII localized to the cell surface only in IFNgamma-activated astrocytes. Membrane capacitance measurements revealed distinct types of reversible (kiss-and-run) and irreversible (full) exo-/endocytotic events in both groups. Following IFNgamma treatment reversible fusion of vesicles with larger diameter that exhibited a prolonged fusion pore dwell time and an increased fusion pore conductance was observed. Furthermore, IFNgamma activation reduced the frequency of full endocytotic events. ATP stimulation, which increases intracellular calcium activity, increased the frequency of exocytotic and decreased the frequency of full endocytotic events in both groups. Therefore, in IFNgamma- activated astrocytes, the altered balance between exo- and endocytosis is favoring exocytosis, predominantly of larger lysosomes, which contributes to prolonged residence of MHCII molecules at the plasmalemma, promoting antigen presentation in reactive astrocytes.

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