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. 2023 Jul 25;13:12003. doi: 10.1038/s41598-023-38588-7

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© The Author(s) 2023

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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INF2 subcellular localization and distribution of mitochondria and actin in HeLa cells expressing wild-type or INF2 variants. Cells were labeled with MitoTracker 24 h after transfection with cMyc-tagged INF2 constructs, and then fixed with 4% paraformaldehyde. Cells were triple-stained with c-Myc antibody (Alexa 488), phalloidin (Alexa 647) and MitoTracker (Alexa 555). Cells expressing the wild-type and FSGS variant (T161N, N202S) showed a diffuse, fine reticular ER pattern with Golgi clusters, whereas those expressing CMT variant (G73D, V108D) exhibited a coarse punctate pattern with peripheral accumulation (arrows). The reduction in the number of actin stress fibers was more pronounced in cell expressing CMT/FSGS variants compared to those expressing FSGS variants. In cells expressing wild-type INF2, mitochondria were evenly distributed in the perinuclear region. In contrast, all pathogenic variants caused mitochondria to mislocalize to the cell periphery (arrowheads). The extent of mislocalization was more severe in cells expressing CMT/FSGS variants compared to those expressing FSGS variants. Cells expressing the T161N variant displayed an intermediate configuration, with an almost equal distribution of perinuclear and peripheral patterns. Scale bars: 10 μm.