Time‐dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes

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‐Alexa 488 ) and fluorescent dextran (Dextran 546 ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B‐Alexa 488 from extracellular space and monitored trafficking of vesicles that internalized S100B‐Alexa 488 . Initially, S100B‐Alexa 488 and Dextran 546 internalized with distinct rates into different endocytotic vesicles; S100B‐Alexa 488 internalized into smaller vesicles than Dextran 546 . At a later stage, S100B‐Alexa 488 ‐positive vesicles substantially co‐localized with Dextran 546 ‐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‐Alexa 488 , but not of Dextran 546 . The dynamin inhibitor dynole 34‐2 inhibited internalization of both fluorescent probes. Directional mobility of S100B‐Alexa 488 ‐positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca 2+ ] 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.