Partial colocalization was observed among VAC and lysosomes loaded with dextran , suggesting that some VAC is also localized on lysosomes. Interestingly, the limiting membrane of vacuoles in Vac fibroblasts is positive for LAMP, but damaging for LBPA , implying that the large vacuoles derive solely from lysosomes. In metazoans, the PIKfyve VAC FIG pathway is thought to play a function in autophagy, either during fusion of autophagosomes with endosomes lysosomes, or recycling of lysosomes from autolysosomes . LC is usually a normal marker of autophagosomes. We transfected wildtype or Vac fibroblasts with LC RFP and colabelled transfected cells with anti VAC. VAC partially colocalized with LC , suggesting that autophagosomes might possibly include PI P and or PI P. Alternatively, these PI P and or PI P containing regions may represent the interface among autophagosomes and endosomes lysosomes.
Localization of VAC in neurons To ascertain the localization of VAC in neurons, we 1st examined its distribution within the soma. Within this case, neurons had been not permeabilized with saponin before fixation; as a result, the images indicate each membrane bound and cytosolic pools of VAC. A substantial portion on the from this source VAC localized to punctate structures . As in fibroblasts, VAC puncta colocalized both with the early endosome marker, EEA , and together with the late endosome lysosome marker, LAMP . To test irrespective of whether VAC is present in dendrites, hippocampal neurons were labelled with antibodies against VAC and against MAP, a microtubule linked protein that may be highly expressed in dendrites but not in axons. Notably, discrete VAC puncta were located in MAP positive dendrites .
In addition, one more pool of VAC puncta was evident in MAP negative neurites, implying an axonal localization. To test this further, we labelled neurons with anti VAC and anti TAU , which Tie-2 inhibitor preferentially labels axons in younger cultures . Once more, VAC puncta have been present in TAU labelled axons , even though this axonal VAC pool was significantly less prominent than the dendritic pool. In neurites, VAC puncta partially colocalized with both EEA and LAMP , suggesting that VAC in neuronal processes functions in pathways that involve early and late endosomes at the same time as lysosomes. Endogenous VAC localizes to synapses Interestingly, probably the most striking colocalization was observed amongst VAC and synaptic markers. A substantial variety of VAC puncta colocalized together with the presynaptic terminal markers synapsin, synaptotagmin and VAMP synaptobrevin .
To test whether or not VAC colocalizes with excitatory synapses, we performed triple labelling against VAC, vGlut , plus the postsynaptic scaffolding protein PSD. VAC puncta colocalized extensively with vGlut PSD double good puncta , suggesting a role for VAC in excitatory synapse function.