Live cell tracking of macrophage efferocytosis during Drosophila embryo development in vivo.

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.

The vSNAREs VAMP2 and VAMP4 control recycling and intracellular sorting of post-synaptic receptors in neuronal dendrites.

The endosomal recycling system dynamically tunes synaptic strength, which underlies synaptic plasticity. Exocytosis is involved in the expression of long-term potentiation (LTP), as postsynaptic cleavage of the SNARE (soluble NSF-attachment protein receptor) protein VAMP2 by tetanus toxin blocks LTP. Moreover, induction of LTP increases the exocytosis of transferrin receptors (TfRs) and markers of recycling endosomes (REs), as well as post-synaptic AMPA type receptors (AMPARs). However, the interplay between AMPAR and TfR exocytosis remains unclear. Here, we identify VAMP4 as the vesicular SNARE that mediates most dendritic RE exocytosis. In contrast, VAMP2 plays a minor role in RE exocytosis. LTP induction increases the exocytosis of both VAMP2- and VAMP4-labeled organelles. Knock down (KD) of VAMP4 decreases TfR recycling but increases AMPAR recycling. Moreover, VAMP4 KD increases AMPAR-mediated synaptic transmission, which consequently occludes LTP expression. The opposing changes in AMPAR and TfR recycling upon VAMP4 KD reveal their sorting into separate endosomal populations.