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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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GlialCAM is a diffusional phase barrier for the MLC1 signalling cluster at the PM. (A) The indicated HA-MLC1 variants (wt, P92S (PS), C326R CR) interacting with GlialCAM (GCAM) were isolated from the PM using cs-IP with anti-GCAM Ab. The precipitates were analyzed using Western blot analysis with anti-HA or anti-Flag Abs. MLC1 was measured as fold change to wt (left panel) from co-IP-ed samples and the amount of GCAM pulldown was normalized to sample lacking MLC1 (right panel) in HeLa cells. (B) MLC1 lateral diffusion at the PM was measured by FLIP. Indicated areas (white circles) were photobleached and the fluorescence loss was monitored in the adjacent red squares. The normalized intensity traces show fluorescence decay in cells +/− GCAM overexpression. (C,D) Summary of decay rate constants are expressed as 1/s for MLC1-wt (C) and P92S (D) in cells +/− GCAM overexpression. GCAM substantially restricts the diffusion of MLC1-wt at the free PM (PM) and cell–cell junctions (JC), in contrast to P92S showing restriction only at the free PM. Statistical analysis against mock (J-D) or wt in the junction (D) are indicated. Means ± SEM, n ≥ 3, **p < 0.01, ***p < 0.001, ****p < 0.0001.