Chemotactic cell migration: the core autophagy protein ATG9A is at the leading edge

ABSTRACT

Accumulating data indicate that several components of the macroautophagy/autophagy machinery mediate additional functions, which do not depend on autophagosome biogenesis or lysosomal cargo degradation. In this context, we found that the core autophagy protein ATG9A participates in the chemotactic movement of several cell lines, including highly invasive glioblastoma cells. Accordingly, ATG9A-depleted cells are unable to form large and persistent leading-edge protrusions. By the design of an ATG9A-pHluorin construct and TIRF imaging, we established that ATG9A-positive vesicles are targeted toward the migration front, where their exocytosis is synchronized with protrusive activity. We finally demonstrated that ATG9A, through its interaction with clathrin adaptor complexes, controls the delivery of ITGB1 (integrin subunit beta 1) to the migration front and normal adhesion dynamics. Together, our work indicates that ATG9A protein has a wider role than anticipated and constitutes a critical component of vesicular trafficking allowing the expansion of cell protrusions and their anchorage to the extracellular matrix.

Since the discovery of autophagy-related (ATGs) proteins, decades of intense research have led to seminal advances in understanding the molecular mechanisms of the various stages of autophagy. At the same time, we have begun to appreciate that various ATGs mediate one or multiple cellular functions that are not directly instrumental for autophagosome formation or lysosomal cargo degradation. As the only transmembrane component of the core autophagy machinery, the ATG9A protein is present in numerous intracellular compartments, including the trans-Golgi network (TGN), endosomes and the plasma membrane. Extensive studies have demonstrated its key role in autophagosome formation, proposedly by functioning in vesicular delivery to the site of phagophore initiation, and by translocating lipids from the outer to the inner leaflet of the phagophore membrane in order to enable its expansion. In addition to this traditional view of ATG9A as an autophagy factor, recent data highlighted its critical importance for diverse functions unrelated to autophagosome biogenesis, but still depending on membrane trafficking, including protection from plasma membrane damage, mitochondrial fatty acid import, and transport of hydrolases from the TGN to the lysosomes.

Chemotactic cell migration is a fundamental process that strongly relies on the trafficking of vesicles, emanating from the TGN and endosomes, toward the cell front. Focal exocytosis of these vesicles allows the delivery of bulk membrane and specific cargo proteins that tightly control the expansion of the leading-edge protrusion and its anchorage to the extracellular matrix. In this context and by using complementary approaches, our recent study [1] identified ATG9A-positive vesicles as essential components of chemotactic cell migration.

We first demonstrated that knockdown of ATG9A abolishes or markedly reduces chemotactic movement of several human cell lines, including highly invasive glioblastoma cells. Accordingly, ATG9A-depleted cells are unable to form large and polarized F-actin-rich lamellipodia, which normally drive efficient cell migration. We also focused on the dynamics of these protrusions and found that ATG9A-depleted cells exhibit protrusions with normal velocity, but reduced distance and persistence. Live-cell TIRF microscopy revealed that most of the motile ATG9A-positive vesicles located in protrusions display an anterograde movement toward the cell edge. Do ATG9A-positive vesicles indeed perform exocytosis at the cell front? To answer this question, we designed a pH-sensitive fluorescently tagged ATG9A, and established that exocytosis of ATG9A-positive vesicles is highly polarized toward the cell front, induced by chemotactic stimuli, and synchronized with protrusive activity. It is worth noting that most of the exocytotic events are characterized by the dispersal of ATG9A into the plasma membrane, with a clear signature of full vesicle fusion, suggesting that ATG9A-positive vesicles may act as a reservoir of bulk lipids for lamellipodial expansion.

Where do the ATG9A-positive vesicles come from and what do they carry? In agreement with the critical role of TGN-to-plasma membrane trafficking of integrins during cell migration, we found that ATG9A partially colocalizes with the trans-Golgi marker TGOLN2/TGN46 and ITGB1/β1 integrin, in the perinuclear area but also in puncta located near the cell edge, likely representing bona fide post-Golgi carriers. We further demonstrated that stimulus-induced redistribution of TGOLN2 and ITGB1 from the perinuclear area to cell protrusions is dependent on ATG9A, and more specifically on its ability to interact with clathrin adaptor complexes through a canonical sorting signal (₈YXXØD/E₁₂) located in its N terminus. As additional evidence of its pro-migratory role, we finally found that ATG9A protein is necessary for normal adhesion dynamics and that exocytosis of ATG9A-positive vesicles occurs in close proximity to cell adhesions, with an evident clustering.

In-depth investigations will be necessary to clarify the molecular mechanisms underlying this new function of ATG9A. One hypothesis is that ATG9A could be an intrinsic regulator of vesicle biogenesis from the TGN. This possibility is supported by several lines of data from the literature, i.e. (i) the critical role of ATG9A for the export of hydrolases from the TGN, (ii) the ability of ATG9A to interact with PI4KB (phosphatidylinositol 4-kinase beta), a major phosphatidylinositol-4-phosphate-producing enzyme regulating the budding of vesicles from this compartment, and (iii) computer simulations showing that ATG9A protein clusters can elicit long-range membrane curvature. Another attractive hypothesis, sustained by the recently described interaction of ATG9A with IQGAP1 (a major pro-migratory protein coupling dynamic microtubules with the cell cortex), is that ATG9A can actively participate in the transport of vesicles along microtubules and/or the final steps of vesicle fusion at the migration front. In this framework, identification of the signaling pathways and the repertoire of ATG9A interactors controlling the biogenesis of particular post-Golgi carriers and their targeting to the phagophore, the lysosomes or the cell front, is a critical subject for further studies.

In summary, our findings indicate that, in addition to its well-documented role during phagophore expansion, ATG9A protein is mobilized during chemotactic stimulation and should be viewed as an essential component of vesicular trafficking driving cell migration (Figure 1). From a clinical point of view, these data pave the way for further research aimed at better understanding the protumoral functions of ATG9A already reported in glioblastoma and breast cancer.

Figure 1.

Proposed model for autophagic and non-autophagic functions of ATG9A mediated by its trafficking from the TGN. Biogenesis of ATG9A-positive vesicles from the TGN delivers lipids and/or cargo proteins to sustain the expansion of the phagophore. ATG9A also participates in the export of hydrolases from the TGN and their delivery to the lysosomal compartment. Under chemotactic stimulation, a fraction of ATG9A-positive vesicles, loaded with ITGB1/β1 integrin, are targeted to the migration front. Their exocytosis allows the maturation of adhesions and the stable anchoring of the leading-edge protrusion to the substrate. The cell can then efficiently move toward the chemotactic stimulus.

Funding Statement

This work was supported by the Institut National de la Santé et de la Recherche Médicale (Inserm), Groupement des Entreprises Françaises dans la Lutte contre le Cancer (GEFLUC), TC2N network, the Ligue contre le Cancer Normandie, the french Agence Nationale de la Recherche and the University of Rouen Normandy. D.C. is recipient of a fellowship from Normandy.

Disclosure statement

All authors have no conflicts of interest to declare.