The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

Sauvanet, Cécile; Garbett, Damien; Bretscher, Anthony

doi:10.1091/mbc.e15-07-0498

Cited by 6 publications

(10 citation statements)

References 55 publications

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“…The presence or absence of microvilli was scored as described previously ( Garbett et al, 2010 ; Hanono et al, 2006 ; LaLonde et al, 2010 ; Pelaseyed et al, 2017 ; Sauvanet et al, 2015 ). More than 50 cells per replicate were stained using the WGA, ezrin, and phalloidin and binned into two categories: microvilli and no microvilli.…”

Section: Methodsmentioning

confidence: 99%

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

Zaman

Lombardo

Sauvanet

et al. 2021

Journal of Cell Biology

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Activated ezrin-radixin-moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate apical structures, including microvilli. Among many kinases implicated in ERM activation are the homologues LOK and SLK. CRISPR/Cas9 was used to knock out all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM-activating phosphorylation. The apical domains of cells lacking LOK/SLK or ERMs are strikingly similar and selectively altered, with loss of microvilli and with junctional actin replaced by ectopic myosin-II–containing apical contractile structures. Constitutively active ezrin can reverse the phenotypes of either ERM or LOK/SLK knockouts, indicating that a central function of LOK/SLK is to activate ERMs. Both knockout lines have elevated active RhoA with concomitant enhanced myosin light chain phosphorylation, revealing that active ERMs are negative regulators of RhoA. As RhoA-GTP activates LOK/SLK to activate ERM proteins, the ability of active ERMs to negatively regulate RhoA-GTP represents a novel local feedback loop necessary for the proper apical morphology of epithelial cells.

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Section: Methodsmentioning

confidence: 99%

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

Zaman

Lombardo

Sauvanet

et al. 2021

Journal of Cell Biology

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“…, 2010 ), loss of EBP50 results in a variable reduction in the number of surface microvilli ( Figure 2D ). Jeg-3 cells do not express the closely related PDZ-containing scaffolding protein E3KARP ( Sauvanet et al. , 2015 ).…”

Section: Resultsmentioning

confidence: 99%

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells^†

Miller

McDermitt

Sauvanet

et al. 2022

MBoC

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Here we report on the related TBC/RabGAPs EPI64A and EPI64B and show that they function to organize the apical aspect of epithelial cells. EPI64A binds the scaffolding protein EBP50/NHERF1, which itself binds active ezrin in epithelial cell microvilli. Epithelial cells additionally express EPI64B that also localizes to microvilli. However, EPI64B does not bind EBP50 and both proteins are shown to have a microvillar localization domain that spans the RabGAP domains. CRISPR/Cas9 was used to inactivate expression of each protein individually or both in Jeg-3 and Caco2 cells. In Jeg-3 cells, loss of EPI64B resulted in a reduction of apical microvilli, and a further reduction was seen in the double knockout, mostly likely due to misregulation of Rab8 and Rab35. In addition, apical junctions were partially disrupted in cells lacking EPI64A, and accentuated in the double knock out. In Caco2 loss of EPI64B resulted in wavy junctions, whereas loss of both EPI64A and EPI64B had a severe phenotype often resulting in cells with a stellate apical morphology. In the knockout cells, the basal region of the cell remained unchanged, so EPI64A and EPI64B specifically localize to and regulate the morphology of the apical domain of polarized epithelial cells.

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“…The closest homologue of E3KARP is EBP50 (or NHERF1) (Reczek et al ., 1997; Sauvanet et al , 2015a). Similar to E3KARP, EBP50 consists of two PDZ domains and a C-terminal ezrin-binding domain (EBD), is localized at microvilli, and has been implicated in microvilli formation (Garbett & Bretscher, 2012; Garbett et al , 2010; Garbett et al , 2013; LaLonde et al , 2010; Morales et al , 2004).…”

Section: Resultsmentioning

confidence: 99%

“…S3). JEG-3 cells, a choriocarcinoma-derived cell line, has widely been used as a model to study microvilli formation and dynamics (Garbett & Bretscher, 2012; Garbett et al ., 2010; Garbett et al ., 2013; Hanono et al ., 2006; Sauvanet et al ., 2015a; Viswanatha et al ., 2012). The recovery rates for ezrin did not significantly differ in the absence and presence of TMIGD1 expression (Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel adhesive complex at the base of intestinal microvilli

Hartmann

Thuering

Michels³

et al. 2021

Preprint

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Intestinal epithelial cells form dense arrays of microvilli at the apical membrane to enhance their functional capacity. Microvilli contain a protocadherin-based intermicrovillar adhesion complex localized at their tips which regulates microvillar length and packaging. Here, we identify a second adhesive complex in microvilli of intestinal epithelial cells. This complex is localized at the basal region of microvilli and consists of the adhesion molecule TMIGD1, the phosphoprotein EBP50 and the F-actin – plasma membrane cross-linking protein ezrin. Ternary complex formation requires unmasking of the EBP50 PDZ domains by ezrin binding and is strongly enhanced upon mutating Ser162 located in PDZ domain 2 of EBP50. Dephosphorylation of EBP50 at S162 is mediated by PP1α, a serine/threonine phosphatase localized at the microvillar base and involved in ezrin phosphocycling. Importantly, the binding of EBP50 to TMIGD1 enhances the dynamic turnover of EBP50 at microvilli in a Ser162 phosphorylation-dependent manner. We identify an adhesive complex at the microvillar base and propose a potential mechanism that regulates microvillar dynamics in enterocytes.

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The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

Cited by 6 publications

References 55 publications

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells^†

A novel adhesive complex at the base of intestinal microvilli

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The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation

Cited by 6 publications

References 55 publications

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

Effector-mediated ERM activation locally inhibits RhoA activity to shape the apical cell domain

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells†

A novel adhesive complex at the base of intestinal microvilli

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Product

Resources

About

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells^†