ATG9A is the only essential integral membrane protein involved in autophagy. ATG9A contains six transmembrane domains and initiates the assembly of autophagosomes. The autophagosome is a double-membrane structure that engulfs and eventually degrades cytoplasmic materials such as organelles or macromolecules. Assembly of the autophagosome requires the delivery of lipids and membrane components to initiate and expand the double-membrane pre-autophagosome structure called the isolation membrane. ATG9A localizes to highly mobile cytoplasmic vesicles which are thought to play a key role in recruiting and delivering membrane and lipids to the assembling autophagosome. Interestingly ATG9A localizes only to the outer membrane layer of the double-membrane pre-autophagosome structure. This essential role in autophagosome assembly makes ATG9A an excellent marker for autophagy induction and for examining the membrane dynamics of early autophagosome assembly. ATG9A is only transiently associated with the autophagosome membrane suggesting ATG9A is recycled back to the cytoplasm for subsequent rounds of autophagosome initiation. Autophagy is a constant process happening at basal levels within the cell and is further induced in response to nutrient starvation, cellular damage, or intracellular pathogens. Upon induction of autophagy by starvation cells generate increased levels of ATG9A containing vesicles that are derived from the Golgi apparatus and also contain LC3, a known marker of autophagosomes.
Immunocytochemistry/Immunofluorescence: ATG9A Antibody (EPR2450(2)) [NBP1-95342] HepG2 cells.
The Schekman group from UC Berkeley has designed a cell-free small vesicle generation assay to examine the steps involved in starvation induced autophagy (1). They shared their findings in eLife where, using the ATG9A antibody, the group identified PI3K (consisting of VPS34, VPS15, Beclin 1, and ATG14) and COPII as essential components needed to generate autophagosome precursors containing ATG9A and LC3. Chen et al. identified CD133, a marker for cancer stem cells, as an important protein involved in cell survival through the regulation of autophagy and energy metabolism (2). They used the ATG9A antibody to monitor protein expression levels in response to CD133 depletion along with the autophagy associated genes Beclin 1, ATG5, and LC3. The Heazell group used the ATG9A antibody for immunohistochemistry in combination with the autophagy markers LC3, ATG5, Beclin 1, and LAMP-2 to monitor autophagy in normal and dysfunctional placenta tissue (3). Their results indicated a potential role for increased levels of autophagy in Fetal Growth Restriction, a serious pregnancy complication. Bejarano et al. identified a novel mechanism of inhibiting autophagosome assembly (4). In their Nature Cell Biology article they showed connexins, the protein components of gap junctions, sequester the autophagy initiation complex containing PI3K and ATG16 at the plasma membrane. They used the ATG9A antibody for immunocytochemistry and showed upon cell starvation ATG9A and ATG14 localize to the plasma membrane where they trigger the internalization of Connexin 43 to release its inhibitory effect. The Sada group characterized the role of NOD-1 induction of autophagy during Mycobacterium tuberculosis infection (5). NOD-1 is a cytoplasmic receptor present in alveolar macrophages. Upon recognition of an infection NOD-1 triggers autophagy, which the authors monitored through immunoblotting with theATG9A antibody.
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