Myosin Vb and rab11a regulate ezrin phosphorylation in enterocytes

Abstract

Microvilli at the apical surface of enterocytes allow the efficient absorption of nutrients in the intestine. Ezrin activation by its phosphorylation at T567 is important for microvilli development, but how such ezrin phosphorylation is controlled is not well understood. We demonstrate that a subset of kinases that phosphorylate ezrin closely co-distributes with apical recycling endosome marker Rab11a in the subapical domain. Expression of dominant-negative Rab11a mutant or depletion of the Rab11a-binding motor protein myosin Vb prevents the subapical enrichment of Rab11a and these kinases and inhibits ezrin phosphorylation and microvilli development, without affecting the polarized distribution of ezrin itself. We observe a similar loss of the subapical enrichment of Rab11a and the kinases and reduced phosphorylation of ezrin in microvillus inclusion disease, which is associated with MYO5B mutations, intestinal microvilli atrophy and malabsorption. Thus, part of the machinery for ezrin activation depends on recycling endosomes controlled by myosin Vb and Rab11a which, we propose, might act as subapical signaling platforms that enterocytes use to regulate development of microvilli and maintain human intestinal function.

Control of apico–basal epithelial polarity by the microtubule minus-end-binding protein CAMSAP3 and spectraplakin ACF7

“…Apical accumulation of actin and the phosphorylated form of ezrin, an actin-binding protein important for the formation of brush border (Dhekne et al, 2014;Overeem et al, 2015;Viswanatha et al, 2012) was unaffected in CAMSAP2-knockout cells ( Fig. 4E,F; Fig.…”

“…S3A). Previous work has shown that during brush border formation, the apical actin cytoskeleton is strongly regulated by signaling and/or trafficking processes that are dependent on the apical accumulation of Rab11A-positive recycling endosomes (Dhekne et al, 2014;Knowles et al, 2015;Overeem et al, 2015;Sobajima et al, 2015). We analyzed the distribution of Rab11A endosomes by using cells that had been stably transfected with a construct for doxycycline-inducible expression of dTomato-Rab11A.…”

“…ACF7, CAMSAP2 and CAMSAP3 were not essential for the distribution of early cell polarity markers in twodimensional (2D) cultures; however, loss of ACF7 but not of the two CAMSAP proteins affected the formation of epithelial cysts in 3D cultures, indicating that ACF7 plays a more profound role in epithelial polarity than the CAMSAP proteins. Loss of CAMSAP3 led to more centrosome-centered microtubule organization, and this affected the organization of the actin cytoskeleton at the apical side of the cell, probably owing to mislocalization of recycling endosomes that are positive for Rab11A, which are an important component in apical polarity and brush border formation in intestinal cells (Bryant et al, 2010;Dhekne et al, 2014;Knowles et al, 2015;Overeem et al, 2015;Sobajima et al, 2015). Taken together, our study identifies ACF7 as an important regulator of apico-basal polarity in mammalian intestinal cells and demonstrates the importance of non-centrosomal microtubule organization for efficient epithelial cell polarization.…”

ERM proteins at a glance

“…This will require the development of unique tools that detect and interfere with the function of each ERM individually. An understanding of the molecular and cellular functions of mammalian ERMs will yield new insights into fundamental aspects of mammalian development and into established links between aberrant ERM activity and human disease processes, including pathogen-host interactions, deafness, microvillus inclusion disease, and cancer development and metastasis (Clucas and Valderrama, 2014;Dhekne et al, 2014;Hebert et al, 2012;Khan et al, 2007;Kubo et al, 2008;Skoudy et al, 1999).…”

Rab11a regulates Syntaxin 3 localization and microvillus assembly in enterocytes

“…Investigations over the past several years have established a role for elements of the apical recycling system in the regulation of apical brush border assembly and maintenance. As in the case of primary cilia formation (Nachury et al, 2007;Westlake et al, 2011;Westlake et al, 2007), the small GTPases Rab11a and Rab8a both participate in trafficking required for the assembly of the apical intestinal microvilli (Dhekne et al, 2014;Knowles et al, 2014). Rab8a and Rab11a regulate plasma membrane recycling by nucleating multi-protein complexes that participate in the processes required for targeted delivery of recycling cargoes back to membrane surfaces (Baetz and Goldenring, 2013;Bryant et al, 2010;Roland et al, 2011).…”