Fig. 9: The ß_H-Spectrin cytoskeleton is required for glial polarity.

Representative images are shown. a Orthogonal view of the dorsal aspect of a third instar control ventral nerve cord stained for ensheathing glial (EG) morphology [83E12-Gal4, UAS-CD8::GFP]. b Third instar larval brain of a zygotic karst null mutant larvae with the genotype [kst^MiMIC13613 / kst^MiMIC13613, 83E12-Gal4, UAS-CD8::GFP]. The absence of all ß_H-Spectrin protein affects the morphology of the dorsally located ensheathing glial cells. c–e Third instar larval ventral nerve cord with reduced kst expression in ensheathing glia [83E12-Gal4, UAS-kst^dsRNA, UAS-PH-AKT-GFP, UAS-PH-PLCδ-Cherry]. d PH-PLCδ-Cherry binds to PIP₂ and shows an even distribution between apical and basolateral plasma membrane domains. For quantification see (i). Quantification was performed as described in Fig. 6. e PH-AKT-GFP binds to PIP₃ and is distributed in a polarized manner. For quantification see (i). f–h Third instar larval ventral nerve cord with increased kst expression in ensheathing glia [83E12-Gal4, kst^{P{EPgy2}EY01010}, UAS-PH-AKT-GFP, UAS-PH-PLCδ-Cherry]. Note, the variable phenotype noted upon karst overexpression. Ensheathing glia with almost normal morphology (open arrowhead) is next to hyperconvoluted ensheathing glia (asterisk). Quantification of PIP₂ and PIP₃ localization is shown in (j). i, j Quantification was performed as described in Fig. 6. The mean and the standard deviation is shown. k Quantification of the distribution of Nrv2::GFP using an unpaired t-test, two-tailed. *p_{control-kstdsRNA} = 0.0474; ****p_{control-kstEP} < 0.001. The mean and the standard deviation is shown. l–n In karst mutant larvae, Nrv2 is found in ensheathing glia cell processes (open arrowhead) that face the neuropil. The dashed line indicates the neuropil ensheathing glia boundary. o–q Upon ensheathing glial specific kst knockdown, Nrv2 localizes to the basolateral cell domains, for quantification see (k). Filled arrowhead indicates the blood–brain barrier. r–t Upon overexpression of Karst, polarized localization of Nrv2 is less apparent in ensheathing glial cell processes (normal morphology: open arrowheads, hyperconvoluted morphology: asterisk), for quantification see (k). Scale bars are 50 µm. Source data are provided as a Source Data file.