Abstract
SummaryThe endosomal system constitutes a highly dynamic vesicle network used to relay materials and signals between the cell and its environment.1 Once internalized, endosomes gradually mature into late acidic compartments and acquire a multivesicular body (MVB) organization through invagination of the limiting membrane (LM) to form intraluminal vesicles (ILVs).2 Cargoes sequestered into ILVs can either be delivered to lysosomes for degradation or secreted following fusion of the MVB with the plasma membrane.3 It has been speculated that commitment to ILVs is not a terminal event, and that a return pathway exists, allowing “back-fusion” or “retrofusion” of intraluminal membranes to the LM.4 The existence of retrofusion as a way to support membrane equilibrium within the MVB has been widely speculated in various cell biological contexts, including exosome uptake5 and major histocompatibility complex class II (MHC class II) antigen presentation.6, 7, 8, 9 Given the small physical scale, retrofusion of ILVs cannot be measured with conventional techniques. To circumvent this, we designed a chemically tunable cell-based system to monitor retrofusion in real time. Using this system, we demonstrate that retrofusion occurs as part of the natural MVB lifestyle, with attributes parallel to those of viral infection. Furthermore, we find that retrofusion and exocytosis coexist in an equilibrium, implying that ILVs inert to retrofusion comprise a significant fraction of exosomes destined for secretion. MVBs thus contain three types of ILVs: those committed to lysosomal degradation, those retrofusing ILVs, and those subject to secretion in the form of exosomes.Video abstract
Highlights
In the absence of dimerizer, GFP-CD63 localized to both intraluminal vesicles (ILVs) and the limiting membrane (LM) of multivesicular body (MVB) at a 2:1 ratio (Figures 1C and 1D), mirroring the distribution of endogenous CD63,11 whereas the split protease remained inactive (Figure 1B), and no nuclear GFP was observed (Figure 2A; t = 0)
To follow relocalization of membrane components from the intraluminal vesicles (ILVs) back to the limiting membrane (LM), we furnished the canonical multivesicular body (MVB) marker tetraspanin membrane protein CD63 with a GFP tag harboring an N-terminal nuclear localization signal (NLS) and a C-terminal tobacco etch virus (TEV) protease-specific cleavage site (TCS), resulting in NLS-GFP-TCS-CD63 stably expressed in MelJuSo cells, referred to as GFP-CD63 (Figures 1A and 1B)
The TEV protease was introduced into the same cells as two inactive parts, coexpressed along with NLS-DsRED from a single polycistronic vector (Figures 1A and 1B), which can be brought together on demand by a rapamycin analog termed ‘‘dimerizer.’’10 Upon addition of dimerizer, protease activity would be reconstituted and result in cleavage of available GFP-CD63 (i.e., GFP exposed to the cytosol, unlike the GFP in ILVs)
Summary
In the absence of dimerizer, GFP-CD63 localized to both ILVs and the LM of MVBs at a 2:1 ratio (Figures 1C and 1D), mirroring the distribution of endogenous CD63,11 whereas the split protease remained inactive (Figure 1B), and no nuclear GFP was observed (Figure 2A; t = 0). Upon addition of dimerizer to the cells, and consequent reconstitution of the TEV protease (Figure 1B), GFP fluorescence in acidic vesicles of the endocytic tract marked by SiR-lysosome began to diminish over time, accompanied by a concomitant rise of GFP signal in the nucleus (Figure 2A; Video S1). Because GFP in the nucleus can arise from cleaved CD63 molecules in endosomes as well as from the cell surface, we directly measured the decay of GFP fluorescence in the late endosomes. To correct for background fluorescence, cytoplasmic GFP was subtracted from the SiR-lysosome-positive GFP signal
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
