Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

Abstract

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in brain homeostasis, astrocytes supply neurons with vital metabolites and buffer extracellular water, ions, and glutamate. An integral component of the "tri-partite" synapse, astrocytes are also critical in the formation, pruning, maintenance, and modulation of synapses. To enable these highly interactive functions, astrocytes communicate among themselves and with other glial cells, neurons, the brain vasculature, and the extracellular environment through a multitude of specialized membrane proteins that include cell adhesion molecules, aquaporins, ion channels, neurotransmitter transporters, and gap junction molecules. To support this dynamic flux, astrocytes, like neurons, rely on tightly coordinated and efficient intracellular transport. Unlike neurons, where intracellular trafficking has been extensively delineated, microtubule-based transport in astrocytes has been less studied. Nonetheless, exo-and endocytic trafficking of cell membrane proteins and intracellular organelle transport orchestrates astrocytes' normal biology, and these processes are often affected in disease or in response to injury. Here we present a straightforward protocol to culture high quality murine astrocytes, to fluorescently label astrocytic proteins and organelles of interest, and to record their intracellular transport dynamics using time-lapse confocal microscopy. We also demonstrate how to extract and quantify relevant transport parameters from the acquired movies using available image analysis software (i.e., ImageJ/FIJI) plugins.

Interactions between the Astrocytic Volume-Regulated Anion Channel and Aquaporin 4 in Hyposmotic Regulation of Vasopressin Neuronal Activity in the Supraoptic Nucleus

“…It may well be that the interruption of interactions between individual types of, or a combination of, volume-regulating channels and GFAP emerges as a fertile ground for therapeutic intervention. Furthermore, as GFAP is involved in organelle trafficking in astrocytes [ 5 , 40 ], it may be that this intermediary filament governs the trafficking/cycling of volume-regulated channel-laden organelles (i.e., the cytosolic pool) to/from the plasmalemma. If so, the modulation of cycling parameters may represent an additional site for therapeutic intervention, as the effect of preventing/enhancing the delivery of VRAC to the plasma membrane or its retrieval from the plasma membrane would affect HOC, RVD, and the modulation of VP neuronal activity in the SON.…”

Microtubule organization and dynamics in oligodendrocytes, astrocytes, and microglia

“…Astrocytes also rely on transport of diverse cargos for cell‐specific functions. Primary MD astrocytes display fast transport of lysosomes, excitatory amino acid transporter 1 (EAAT1), and the gap junction protein connexin 43 (Creighton, Ruffins, & Lorenzo, 2019). Interestingly, connexin 43 plays an important role in glioma cell invasiveness (Section 5.3) and knockdown decreases tumor area in xenografted mice (Osswald et al., 2015).…”