Loss of the Essential Autophagy Regulators FIP200 or Atg5 Leads to Distinct Effects on Focal Adhesion Composition and Organization.

Emelia A Assar,David A Tumbarello

doi:10.3389/fcell.2020.00733

Emelia A Assar, David A Tumbarello

Open Access

https://doi.org/10.3389/fcell.2020.00733

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Abstract

Autophagy is an essential catabolic intracellular pathway that maintains homeostasis by degrading long-lived proteins, damaged organelles, and provides an energy source during nutrient starvation. It is now understood that autophagy has discrete functions as a selective lysosomal degradation pathway targeting large cytosolic structural and signaling complexes to influence cell motility and adhesion. We provide evidence suggesting the primary autophagy regulators Atg5 and FIP200 both play a role in cell motility and extracellular matrix adhesion. However, their loss of function has a differential impact on focal adhesion composition and organization, as well as signaling in response to fibronectin induced cell spreading. This differential impact on focal adhesions is illustrated by smaller focal adhesion complexes and a decrease in FAK, paxillin, and vinculin expression associated with FIP200 loss of function. In contrast, Atg5 loss of function results in production of large and stable focal adhesions, characterized by their retention of phosphorylated FAK and Src, which correlates with increased vinculin and FAK protein expression. Importantly, autophagy is upregulated during processes associated with focal adhesion reorganization and their exhibits colocalization of autophagosomes with focal adhesion cargo. Interestingly, FIP200 localizes to vinculin-rich focal adhesions and its loss negatively regulates FAK phosphorylation. These data collectively suggest FIP200 and Atg5 may have both autophagy-dependent and -independent functions that provide distinct mechanisms and impacts on focal adhesion dynamics associated with cell motility.

Highlights

The autophagy pathway is essential to maintain cellular homeostasis by facilitating the removal of aggregated or long-lived proteins, damaged organelles, and invading pathogens, as well as acting as an energy source during nutrient deprivation
Our results indicate that loss of Atg5 and family Interacting Protein of 200 kDa (FIP200) both negatively impact on cell motility and enhance fibronectininduced adhesion, but have differential impacts on focal adhesion composition, organization, and dynamics
It should be noted that FIP200 KO cells did retain a partial level of LC3 lipidation, illustrated by an accumulation of LC3-II expression in response to Bafilomycin A1 (BfnA1) by western blot (Figure 1B and Supplementary Figure S1), this was characterized by an atypical LC3 localization shown by microscopy (Figure 1A), which appears as large, swollen aggregates

Summary

Introduction

The autophagy pathway is essential to maintain cellular homeostasis by facilitating the removal of aggregated or long-lived proteins, damaged organelles, and invading pathogens, as well as acting as an energy source during nutrient deprivation. It has been suggested that autophagy directly regulates focal adhesion dynamics by facilitating the targeted degradation of key signaling and adaptor proteins (Sandilands et al, 2012a; Kenific et al, 2016a; Sharifi et al, 2016) This selective form of autophagy is mediated by the ubiquitylation of discrete cargo that subsequently recruits autophagy receptors, such as NBR1, optineurin, NDP52, tax1bp, or p62, which contain conserved ubiquitin binding domains that recognize distinct ubiquitin species and specified LC3-interacting regions (LIRs) that interact with autophagosome membrane (Pankiv et al, 2007; Kirkin et al, 2009; Von Muhlinen et al, 2010; Wild et al, 2011; Tumbarello et al, 2015). It is thought that this selective process for the targeted turnover of focal adhesion components helps facilitate coordinated cell movement (Kenific et al, 2016b; Sharifi et al, 2017)

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