title: Time-dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes.
abstract: Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa488) and fluorescent dextran (Dextran546), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa488from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa488. Initially, S100B-Alexa488and Dextran546internalized with distinct rates into different endocytotic vesicles; S100B-Alexa488internalized into smaller vesicles than Dextran546. At a later stage, S100B-Alexa488-positive vesicles substantially co-localized with Dextran546-positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa488, but not of Dextran546. The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa488-positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca2+]i). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products.
authors.affiliation: Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Eva
authors.initials: E
authors.lastname: Lasič
authors.affiliation: Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.|Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Galland
authors.affiliation: Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.|Celica Biomedical, Ljubljana, Slovenia.
authors.forename: Nina
authors.initials: N
authors.lastname: Vardjan
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Šribar
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Ljubljana, Slovenia.|Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Igor
authors.initials: I
authors.lastname: Križaj
authors.affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
authors.forename: Marina Concli
authors.initials: MC
authors.lastname: Leite
authors.affiliation: Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.|Celica Biomedical, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
authors.affiliation: Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia. matjaz.stenovec@mf.uni-lj.si.|Celica Biomedical, Ljubljana, Slovenia. matjaz.stenovec@mf.uni-lj.si.
authors.forename: Matjaž
authors.initials: M
authors.lastname: Stenovec
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003487:Cyanoacrylates / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D034281:Dynamins / Q000037:antagonists & inhibitors; D004705:Endocytosis; D005110:Extracellular Space / Q000378:metabolism*; D005260:Female; D007211:Indoles / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000067759:Receptor for Advanced Glycation End Products / Q000037:antagonists & inhibitors / Q000276:immunology; D064568:S100 Calcium Binding Protein beta Subunit / Q000378:metabolism*
chemical_list: C000593874:Ager protein, rat; D019138:Antibodies, Blocking; D003487:Cyanoacrylates; D007211:Indoles; D000067759:Receptor for Advanced Glycation End Products; D064568:S100 Calcium Binding Protein beta Subunit; C579158:S100b protein, rat; C000597838:dynole 34-2; D000255:Adenosine Triphosphate; D034281:Dynamins; D002118:Calcium
keywords: S100 proteins; astrocyte; intracellular trafficking; lysosome; vesicles; waste removal