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. 2021 Sep 16;11:18435. doi: 10.1038/s41598-021-97777-4

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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A misaligned GlialCAM signalling cluster causes late endosomal enlargement and increases the early endosomal resident time of ClC-2. (A) MLC1 expression effect on late endosomes-lysosomes was monitored using siMLC1 and siNT (non-targeted) mediated depletion in astrocytic U251N cells. Lamp2 marker is for lysosomes and EEA1 for early endosomes. Bar: 5 µm. (B) High-content image analysis of immunostaining and treatments in panel (A). EAA1/Lamp2 + colocalization was analyzed using Pearson’s correlation coefficient. siNT was used as a control in comparison to siMLC1 and siGCAM depleted U251N cells (~ 300/repeat, n = 3). (C) As in (B), but the mean diameter of individual Lamp2⁺ lysosomes was measured (n = 21,656–47,877/repeat of lysosomes). (D) Representative histograms of the endo-lysosomal pathway vesicular pH (pHv) measurement in siNT, siMLC1 and siGCAM astrocytic U251N cells. Live cells were loaded with pH-sensitive dextran for 5 min and chased for 20 min before single vesicle analysis. (E) Mean vesicular pH (pHv) of cells in (D) chased for indicated times. Characteristic endosomal pH in control siNT cells is indicated. (F) Mean vesicular pH (pHv) of cells in (D) labelled for transferrin receptor (TfR) containing recycling or misfolded (CD4tl-L57C) and constitutively ubiquitinated (CD4tl-Ub) model cargo endosomes. (G) Strongly recycling transferrin receptor (TfR) amount at the PM was measured using cs-ELISA. (H) Immunofluorescence of internalized ClC2 and colocalization with EEA1 in cells +/− GCAM and +/− MLC1 overexpression in HeLa cells. Bar: 5 µm. (I) The impact of GCAM-wt, GCAM-R92W, and MLC1 expression on chloride channel HA-ClC-2 internalization was measured for 5 min at 37 °C using cs-ELISA in HeLa cells. (J) Manders' overlap coefficient of ClC2 with EEA1 + early endosomes. Means ± SEM, n ≥ 3. p-value: ns non-significant, *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.