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Motoneuron Endocytosis and Intracellular Trafficking of Cholera Toxin B Modified Protocells
Author(s) -
Porras Maria Alejandra Gonzalez,
Durfee Paul,
Brinker Jeffrey,
Sieck Gary,
Mantilla Carlos Bernardo
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.873.3
Subject(s) - protocell , microbiology and biotechnology , endocytosis , endocytic cycle , biology , colocalization , intracellular , nocodazole , biophysics , biochemistry , cell , membrane , cytoskeleton
Cholera toxin B modified protocells (CTB‐protocells) are a promising, customizable approach for targeting and delivering drugs to motoneurons. Understanding the endocytic mechanism and intracellular fate of CTB‐protocells is essential for safe therapeutic application and cargo delivery efficiency, and also to elucidate characteristics of motoneuron physiology that are not well understood. To assess the energy dependent uptake of CTB‐protocells by motoneurons, intracellular protocell fluorescence was measured at 4°C and 37°C. Confocal analysis demonstrated that after 3 h at 4°C, there is a 40% reduction in CTB‐protocell uptake compared to the uptake observed at 37°C (p<0.001). Pharmacological endocytic pathway inhibitors were used to elucidate the preferential endocytotic mechanism. Results showed that amiloride (macropinocytosis inhibitor) and methyl‐β‐cyclodextrin (inhibitor of cholesterol dependent endocytic processes) significantly reduced the uptake of CTB‐protocells (p<0.001), consistent with endocytosis being dependent on the integrity of lipid rafts. To track intracellular fate, immunofluorescence techniques and live confocal microscopy were used to demonstrate colocalization of CTB‐protocells with lysosomes, endoplasmic reticulum (ER) and Golgi apparatus at 1 h and 3 h. Colocalization of CTB‐protocells with lysosomes was less than 20%. Both, ER and Golgi colocalization increased from ~20 to ~40% in a 2‐h period (from 1 h to 3 h). To evaluate retrograde transport in motoneurons in vivo , CTB‐protocells were intrapleurally injected in rats. Evidence of protocells in phrenic nerve axons measured by protocell fluorescence intensity supported retrograde transport. In conclusion, uptake of CTB‐protocells is energy‐dependent and mediated principally by macropinocytosis. Additionally, CTB‐protocells are able to escape lysosomal degradation, and are trafficked to relevant subcellular compartments after endocytosis.