Abstract
The endo-lysosomal pathway is essential for intracellular transport and the degradation of extracellular cargo. The relationship between three populations of endo-lysosomal vesicles—Rab7-positive, LAMP1-positive, and both Rab7- and LAMP1-postive—was probed with fluorescence microscopy and single particle tracking. Of specific interest was determining if these vesicles were intermediate or terminal vesicles in the transport of extracellular cargo. We find that the major organelle in the endo-lysosomal pathway, both in terms of population and cargo transport, is positive for Rab7 and LAMP1. Dextran, a fluid phase cargo, shifts from localization within all three populations of vesicles at 30 minutes and 1 hour to primarily LAMP1- and Rab7/LAMP1-vesicles at longer times. This demonstrates that LAMP1- and Rab7/LAMP1-vesicles are terminal vesicles in the endo-lysosomal pathway. We tested two possible mechanisms for this distribution of cargo, delivery to mannose 6-phosphate receptor (M6PR)-negative vesicles and the fusion dynamics of individual vesicles. We find no correlation with M6PR but do find that Rab7-vesicles undergo significantly fewer fusion events than LAMP1- or Rab7/LAMP1-vesicles suggesting that the distribution of fluid phase cargo is driven by vesicle dynamics.
Highlights
The endo-lysosomal pathway is of fundamental importance in cell biology, responsible for the transport and degradation of extracellular cargo [1,2,3,4]
We find no correlation with mannose 6-phosphate receptor (M6PR), but do observe fusion dynamics that support the observed partitioning of dextran
Majority of endo-lysosomal vesicles are positive for Rab7 and lysosomal-associated membrane protein-1 (LAMP1)
Summary
The endo-lysosomal pathway is of fundamental importance in cell biology, responsible for the transport and degradation of extracellular cargo [1,2,3,4]. A more complex picture of lysosomal degradation has emerged that demonstrates degradation can occur upstream of lysosomes and that key lysosomal proteins are not necessary for the degradation of extracellular cargo [8,9,10,11,12,13] Reconciling these results with the conventional picture of the endo-lysosomal pathway has taken on increasing importance with the advent of gene delivery and nanobiotechnology, fields in which delivery of DNA or nanoparticles to enzyme-rich, degradative vesicles is either targeted for triggered release or avoided to prevent degradation [14,15]. Are Rab7/LAMP1-vesicles intermediates between late endosomes and lysosomes or are they terminal vesicles in which cargo accumulates?
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