Endosome is an important sorting compartment, as it mediates biosynthetic and endocytic pathways, including bulk recycling, lysosomal degradation and sequence-dependent sorting. In this paper, Puthenveedu hypothesize that the sequence-dependent pathway uses distinctive features, such as local enrichment of cargo and cytoskeleton. These features allow sorting-sequence-containing receptors to escape from conventional pathways. The sequence-dependent pathway is newly emerged. As the mechanism of this novel pathway remains fragmentary, this paper is remarkable as it shed light on how some receptors are recycled in a regulated fashion.
Puthenveedu approached the hypothesis by utilizing four major tools: (i) fluorescence microscopy; (ii) fluorescene recovery after photobleaching (FRAP); (iii) latrunculin treatment; and (iv) recombinant and mutant receptors. β 2-adrenergic receptor (B2AR) was used as a model as it is recycled to cell surface via sequence-dependent sorting pathway. First, various FLAG-tagged and GFP-tagged proteins were expressed in 293 cells to identify the localization of proteins. To trace the translocation of B2AR membrane receptors from endosome to cytosol, sGFP-B2AR was used. sGFP-B2AR was sensitive to pH as it fluoresces at neutral pH in cytosol but not in acidic endosomes or transporting vesicles. Next, FRAP was used to study the diffusion rate of receptors and the turnover rate of cytoskeleton by making a “dark box” to observe the movement of fluorescent molecules of interests. To study the role of actin polymerization, latrunculin was used to inhibit actin polymerization. Then, to confirm the importance of PDZ domain in the pathway, a B2AR mutant with Ala insertion in PDZ domain was used.
By utilizing the above techniques, B2AR is found to be enriched in the tubular domain on endosomes. The majority of B2AR is delivered to the cell surface using this tubular domain that is biochemically distinct from other domain for bulk recycling pathway. In particular, actin filaments were found to be concentrated at the B2AR-containing tubules. In the absence of endosomal actin filaments, less than 20% of endosomes have B2AR-containing tubules, suggesting the importance of actin in the sequence-dependent sorting pathway. To promote actin polymerization, Arp 2/3 complex enables the initial nucleation step. Interestingly, Arp 3 is found at the base of the B2AR-containing tubular domain. While some Arp 2/3 activators, N-WASP and WAVE-2, are absent from endosomes, WASH protein complex is localized with the B2AR-containing tubules and cortactin is localized at the base of the tubules. Furthermore, the PDZ domain of B2AR is important in promoting the formation of B2AR-containing tubules. Comparing to Tfr which is dependent on bulk flow recycling pathway, B2AR has a significantly lower diffusion rate than Tfr, and extrusion of B2AR-containing tubules is lower than Tfr tubules
Puthenveedu and his group have presented some convincing results as to how sequence-dependent sorting pathway works distinctively from the other conventional pathways. However, as “24.4% of Tfr tubules contained detectable B2AR”, I am not convinced as most experiments are qualitative and observation-based. The significant amount of B2AR found in Tfr tubules suggests that sequence-dependent sorting pathway goes parallel with bulk recycling or an unknown pathway. So, how can a significant amount of B2AR escape from the sequence-dependent sorting pathway? One possibility is that a protein is required to mediate the interaction of B2AR to the actin filament. Due to the limited quantity of this mediator protein, a constant flow of B2AR can be recycled back to the cell surface via the bulk recycling pathway. In other words, this potential mediator holds the key in regulating the sequence-dependent sorting pathway in response to cellular demand and environmental stimulus. To initiate the approach to validate my hypothesis, we need to sort out B2AR-containing endosomes, then capture B2AR and its interacting partner for mass spectroscopy to identify the potential mediator protein. To confirm the interaction, mammalian or yeast two-hybrid systems and gel shift can be performed.