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The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

ABSTRACT: Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of β1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.

SUBMITTER: Campisi D

PROVIDER: S-EPMC8932524 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

Campisi Daniele D Desrues Laurence L Dembélé Kléouforo-Paul KP Mutel Alexandre A Parment Renaud R Gandolfo Pierrick P Castel Hélène H Morin Fabrice F

The Journal of cell biology 20220218 3

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)-rich lamellipodia that normally drive directional migration. Using live-cell TIRF mi ...[more]

PMID: 35180289

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Project description:BACKGROUND:Nef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways. RESULTS:To gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~?fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein. CONCLUSIONS:These studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

Project description:Invasion of the colon wall by Entamoeba histolytica during amoebic dysentery entails migration of trophozoites through tissue layers that are rich in extracellular matrix. Transcriptional silencing of the E. histolytica surface metalloprotease EhMSP-1 produces hyperadherent less-motile trophozoites that are deficient in forming invadosomes. Reversible protein phosphorylation is often implicated in regulation of cell motility and invadosome formation. To identify such intermediaries of the EhMSP-1-silenced phenotype, here we compared the phosphoproteomes of EhMSP-1-silenced and vector control trophozoites by using quantitative tandem mass spectrometry-based proteomics. Six proteins were found to be differentially phosphorylated in EhMSP-1-silenced and control cells, including EhCoactosin, a member of the ADF/cofilin family of actin-binding proteins, which was more frequently phosphorylated at serine 147. Regulated overexpression of wild-type, phosphomimetic, and nonphosphorylatable EhCoactosin variants was used to test if phosphorylation functions in control of E. histolytica actin dynamics. Each of the overexpressed proteins colocalized with F-actin during E. histolytica phagocytosis. Nonetheless, trophozoites overexpressing an EhCoactosin phosphomimetic mutant formed more and poorly coordinated cell membrane protrusions compared to those in control or cells expressing a nonphosphorylatable mutant, while trophozoites overexpressing nonphosphorylatable EhCoactosin were significantly more motile within a model of mammalian extracellular matrix. Therefore, although EhCoactosin's actin-binding ability appeared unaffected by phosphorylation, EhCoactosin phosphorylation helps to regulate amoebic motility. These data help to understand the mechanisms underlying altered adherence and motility in EhMSP-1-silenced trophozoites and lay the groundwork for identifying kinases and phosphatases critical for control of amoebic invasiveness.IMPORTANCE Invasive amoebiasis, caused by the intestinal parasite Entamoeba histolytica, causes life-threatening diarrhea and liver abscesses, but, for unknown reasons, only approximately 10% of E. histolytica infections become symptomatic. A key requirement of invasion is the ability of the parasite to migrate through tissue layers. Here, we systematically looked for differences in protein phosphorylation between control parasites and a previously identified hyperadherent E. histolytica cell line that has reduced motility. We identified EhCoactosin, an actin-binding protein not previously known to be phosphoregulated, as one of the differentially phosphorylated proteins in E. histolytica and demonstrated that EhCoactosin phosphorylation functions in control of cell membrane dynamics and amoebic motility. This and the additional differentially phosphorylated proteins reported lay the groundwork for identifying kinases and phosphatases that regulate tissue invasiveness.

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The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

Publications

The core autophagy protein ATG9A controls dynamics of cell protrusions and directed migration.

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