A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Tomoki Yano,Tomoaki Mizuno,Takeshi Matsui,Hiroka Kashihara,Hatsuho Kanoh,Kazuhiro Aoki,Akira Komatsubara,Yuhei Goto,Atsushi Tamura,Hiroo Tanaka,Shogo Nakayama,Ryosuke Takahashi,Kazuto Tsukita,Sachiko Tsukita

doi:10.15252/embj.2020104712

Abstract

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di‐phosphorylated myosin light chain (ppMLC)‐driven contraction of actomyosin‐based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC‐triggered system at TJ‐associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule‐associated proteins in the AJC‐enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ‐, but not at AJ‐, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ‐associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule‐facilitated manner. Our results uncovered a hitherto unknown microtubule‐LUZP1 association at TJ‐associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Highlights

Apical constriction is critical for epithelial morphogenesis, including neural tube formation
LUZP1 is an MT-associated protein that localizes at tight junctions (TJs)-associated circumferential rings (CRs)
We generated antibodies against the N-terminal, middle, and C-terminal regions of LUZP1. The specificities of these antibodies were confirmed by the lack of their immunoblot signals in LUZP1 KO mouse mammary gland epithelial Eph4 cells, which were generated (Fig EV1F). Immunoblotting using these antibodies confirmed that LUZP1 was enriched in the apical junctional complexes (AJCs) fraction (Fig EV1G) and showed that LUZP1 was ubiquitously expressed in various tissues (Fig EV1H)

Summary

Introduction

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di-phosphorylated myosin light chain (ppMLC)-driven contraction of actomyosin-based circumferential rings (CRs), known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). We revealed a ppMLC-triggered system at TJ-associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. LUZP1 was found localized at TJ-, but not at AJ-, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubulefacilitated manner. Our results uncovered a hitherto unknown microtubule-LUZP1 association at TJ-associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction

Methods

Results

Conclusion