Drp1-induced mitochondrial fission is required for fine astrocyte process formation and mitochondrial localization in vitro. (A) Schematic of Drp1 in mitochondrial fission. (B) Drp1 domain structure, noting the K38E mutation in the GTPase domain. (C) Representative images of rat astrocytes transfected with shControl or shDrp1 (green) and MitoDsRed (magenta) (top panels) co-cultured on neurons (unlabeled), with a MitoDsRed mask (lower panels), noting hyperfused/elongated mitochondria in shDrp1 astrocytes (red arrowheads). Scale bars: 40 μm. (D) Representative images of rat astrocytes transfected with shRNA targeting Drp1 (shDrp1) or a scrambled control (shControl) (first column) co-cultured on neurons (unlabeled) and their mitochondria (second column) across primary, secondary, fine, and terminal astrocyte process types. Scale bar: 20 μm. (E) Super-plotted quantification of average mitochondrial number in secondary, fine, and terminal branches from shControl vs. shDrp1 astrocytes. (F) Quantification of primary, secondary, fine, and terminal branch numbers from shControl and shDrp1 astrocytes. Data are mean ± SEM n = 4 independent experiments (large circles), 10 cells/condition/experiment (small gray dots). Nested t test. (G) Representative images of rat astrocytes transfected with shControl (green), shDrp1 (green), shDrp1 (green)+ hDrp1-YFP (magenta), or shDrp1 (green)+ hDrp1-K38E-CFP (magenta). Scale bar: 40 μm. (H–K) Quantification of total primary, (I) secondary, (J) fine, and (K) terminal branch number in astrocytes across the 4 conditions from G. Data are mean ± SEM n = 3 independent experiments (large circles), 10 cells/condition/experiment (small gray dots). Nested one-way ANOVA with Tukey post hoc test.