IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules

Abstract

It is unclear whether the establishment of apical–basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.

A mechanosensing mechanism mediated by IRSp53 controls plasma membrane shape homeostasis at the nanoscale

“…Fig. 3A-G), indicating that the presence of IRSp53 in the PM is not mediated exclusively by the I-BAR domain and rather occurs as an interplay of all different domains, as already suggested in previous studies (63, 64). We next analyzed the dynamics of EGFP-IRSp53-FL at the resorbing evaginations.…”

“…I403P and W413G mutations of the SH3 domain also led to long lags and inefficient resorption. Although in this case the I-BAR domain is not impaired, lack of interaction of IRSp53 with NPFs, which could be already bound to the PM and target IRSp53 there (56, 64), could delay both recruitment and the subsequent resorption process. Similarly, the I268N-CRIB mutant is probably delayed due to the lack of interaction with active Cdc42 already bound to the PM.…”

The biosynthetic-secretory pathway, supplemented by recycling routes, specifies epithelial membrane polarity

“…Routing of such a trajectory from the ER to the apical membrane might require components of: (1) pre-Golgi secretory carriers (this study); (2) Golgi-/post-Golgi endomembranes/carriers (GSLs, clathrin/AP-1; prior studies by us and others) (7,10,11,(50)(51)(52)(53)77); (3) apical recycling carriers and the apical-vesicle/membrane interface (e.g. RAB-11 (19), V-ATPases (38), EPS-8 (40); this study and other studies; Fig. 9).…”

“…3B), were enriched for biosynthetic-secretory pathway components of membrane-directed (anterograde) vesicle trajectories (7/12), implicating this pathway in the regulation of membrane polarity and suggesting that GSLs position the apical domain on biosynthetic-secretory or parallel trafficking routes. Validating this finding, enhancers also identified molecules previously implicated in apical domain positioning via apical recycling or by functions at the apical-membrane-vesicle interface (RAB-11, VHA-1/4, EPS-8; (19,33,(38)(39)(40) note that clathrin/AP-1, previously characterized as GSL enhancers (11), are not included in this analysis). In contrast, suppressors, whose strong depletion reduces apicobasal polarity conversion in a let-767(-/-) background (Fig.…”

Role of Lipids in Morphogenesis of T-Cell Microvilli

“…It supports membrane ruffling and protrusions in T cells ( 65 ). In epithelial cells, it is expressed at the microvilli-containing apical membrane and functionally associates with podocalyxin-1 ( 66 ). As a protein of countless functions, it will be important to characterize its specific role in microvilli of T cells.…”