Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ?F508 mutation is the most common. ?F508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ?F508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ?F508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ?F508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ?F508 macrophages.

Differential efficiency of the endocytic machinery in tonic and phasic synapses

Efficient synaptic vesicle membrane recycling is one of the key factors required to sustain neurotransmission. We investigated potential differences in the compensatory endocytic machineries in two glutamatergic synapses with phasic and tonic patterns of activity in the lamprey spinal cord. Post-embedding immunocytochemistry demonstrated that proteins involved in synaptic vesicle recycling, including dynamin, intersectin, and synapsin, occur at higher levels (labeling per vesicle) in tonic dorsal column synapses than in phasic reticulospinal synapses. Synaptic vesicle protein 2 occurred at similar levels in the two types of synapse. After challenging the synapses with high potassium stimulation for 30 min the vesicle pool in the tonic synapse was maintained at a normal level, while that in the phasic synapse was partly depleted along with expansion of the plasma membrane and accumulation of clathrin-coated intermediates at the periactive zone. Thus, our results indicate that an increased efficiency of the endocytic machinery in a synapse may be one of the factors underlying the ability to sustain neurotransmission at high rates.