Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

Vera Dugina,Pavel Kopnin,Irina Alieva,Natalya Khromova,Peter W Gunning,Igor Kireev

doi:10.18632/oncotarget.12236

Abstract

Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of β- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not β-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of β-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

Highlights

Actin microfilaments are highly dynamic cellular structures with their turnover time on the order of seconds at the cell periphery [1]; they effectively regulate cell shape
Microtubule assembly is characterized by enrichment of newly polymerized portions of microtubules with GTP- tubulin, and microtubule plus-end-tracking proteins (+TIPs) which bind to these fragments [6, 7]. +TIPs represent a large group of structurally and functionally diverse microtubule regulators (End-Binding (EB) proteins; Cytoplasmic Linker Proteins (CLIP); CLIP Associated Proteins (CLASP) etc.), which are involved in microtubule interaction with the cell cortex [8]
We investigated the pattern of interaction of the γ-actin cortex and the microtubule system using 3D-SIM microscopy (Figure 2C; Figure 3B-3D and Figure 5A). 3D-SIM microscopy showed the localization of the microtubule termini within the γ-actin cortical layers in each thin (0.12 μm) z-step

Summary

Introduction

Actin microfilaments are highly dynamic cellular structures with their turnover time on the order of seconds at the cell periphery [1]; they effectively regulate cell shape. Microtubules are highly dynamic - they are continuously growing or shortening even when no visual changes can be observed in the region of the cytoplasm they occupy. The ends of individual microtubules are growing or disassembling over distances of several microns [2, 3] and the whole system is continuously exchanging with the pool of monomeric tubulin with a turnover time of 5-20 min [4,5]. +TIPs represent a large group of structurally and functionally diverse microtubule regulators (End-Binding (EB) proteins; Cytoplasmic Linker Proteins (CLIP); CLIP Associated Proteins (CLASP) etc.), which are involved in microtubule interaction with the cell cortex [8]. Microtubuleactin interaction is the basis for endothelial cell barrier function [11,12,13,14]

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