Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA.
Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York, USA.
FASEB J. 2023 Apr;37(4):e22801. doi: 10.1096/fj.202201368RR.
Charged multivesicular body protein 4b (CHMP4B) is a core sub-unit of the endosomal sorting complex required for transport III (ESCRT-III) machinery that serves myriad remodeling and scission processes of biological membranes. Mutation of the human CHMP4B gene underlies rare forms of early-onset lens opacities or cataracts, and CHMP4B is required for lens growth and differentiation in mice. Here, we determine the sub-cellular distribution of CHMP4B in the lens and uncover a novel association with gap junction alpha-3 protein (GJA3) or connexin 46 (Cx46) and GJA8 or Cx50. Immunofluorescence confocal microscopy revealed that CHMP4B localized to cell membranes of elongated fiber cells in the outer cortex of the lens-where large gap junction plaques begin to form-particularly, on the broad faces of these flattened hexagon-like cells in cross-section. Dual immunofluorescence imaging showed that CHMP4B co-localized with gap junction plaques containing Cx46 and/or Cx50. When combined with the in situ proximity ligation assay, immunofluorescence confocal imaging indicated that CHMP4B lay in close physical proximity to Cx46 and Cx50. In Cx46-knockout (Cx46-KO) lenses, CHMP4B-membrane distribution was similar to that of wild-type, whereas, in Cx50-KO lenses, CHMP4B localization to fiber cell membranes was lost. Immunoprecipitation and immunoblotting analyses revealed that CHMP4B formed complexes with Cx46 and Cx50 in vitro. Collectively, our data suggest that CHMP4B forms plasma membrane complexes, either directly and/or indirectly, with gap junction proteins Cx46 and Cx50 that are often associated with "ball-and-socket" double-membrane junctions during lens fiber cell differentiation.
带电荷的多泡体蛋白 4b（CHMP4B）是内体分选复合物必需的核心亚基，该复合物是参与运输 III（ESCRT-III）的机器，为生物膜的多种重塑和分裂过程提供服务。人类 CHMP4B 基因突变是早期白内障或白内障的罕见形式的基础，并且 CHMP4B 是小鼠晶状体生长和分化所必需的。在这里，我们确定了 CHMP4B 在晶状体中的亚细胞分布，并发现了与间隙连接蛋白α-3（GJA3）或连接蛋白 46（Cx46）和 GJA8 或 Cx50 的新关联。免疫荧光共聚焦显微镜显示，CHMP4B 定位于晶状体外皮层伸长纤维细胞的细胞膜上，在那里大的间隙连接斑开始形成-特别是在这些扁平六边形细胞的宽阔面上，这些细胞在横截面上。双免疫荧光成像显示 CHMP4B 与含有 Cx46 和/或 Cx50 的间隙连接斑共定位。当与原位邻近连接测定法结合使用时，免疫荧光共聚焦成像表明 CHMP4B 与 Cx46 和 Cx50 紧密物理接近。在 Cx46 敲除（Cx46-KO）晶状体中，CHMP4B 膜分布与野生型相似，而在 Cx50-KO 晶状体中，CHMP4B 定位于纤维细胞膜的定位丢失。免疫沉淀和免疫印迹分析表明 CHMP4B 在体外与 Cx46 和 Cx50 形成复合物。总的来说，我们的数据表明 CHMP4B 与间隙连接蛋白 Cx46 和 Cx50 形成质膜复合物，无论是直接和/或间接的，这些蛋白通常与晶状体纤维细胞分化过程中的“球窝”双膜连接有关。
