Their co-trafficking may result from SBD's affinity for glycosphingolipids, or may reflect a phenomenon we have documented in a separate study, termed "hijacking", whereby one label influences the trafficking behavior of the other.
Strikingly, this co-trafficking of SBD and lac-Cer was only observed when the two labels were present at the membrane simultaneously, but not when they were added sequentially (fig. 8B, "simultaneous" vs. "sequential"; see Methods).
When cells were pretreated with LPA, they displayed perinuclear accumulation of E-cadherin (green) along with β-catenin (red), consistent with co-trafficking to the same compartment (colocalization in yellow in merged image) (Figure 2).
Filled arrowhead indicates a CAPS-1-EYFP punctum co-trafficking with a mobile DCV.
The reviewers state that documentation of CAPS-1-EYFP and DCV co-trafficking is too anecdotal and deserves better documentation.
The best fungal model system to study co-trafficking of endosomes and mRNAs is the corn pathogen Ustilago maydis (Jansen et al., 2014).
(C ) Percentage of mobile DCVs co-trafficking with CAPS-1-EYFP (mobile DCVs not co-trafficking with CAPS-1-EYFP (green bar): 99 or 93.4%; mobile DCVs co-trafficking with CAPS-1-EYFP: 7 or 6.3%, total number of cells = 20, moving DCVs analyzed = 106).
Furthermore, the number of mobile CAPS-1-EYFP puncta that co-trafficked with dynamic NPY-mCherry labeled DCVs was very low (seven out of 106 mobile DCVs showed co-trafficking of CAPS-1-EYFP, Figure 8C).
Although the mechanism whereby endogenous CD44 might accordingly reduce its affiliation with rafts is unknown, one speculation is that it reflects hetero-dimerisation between endogenous and overexpressed CD44 and their consequent co-trafficking in/out of rafts.
We agree and added a new, quantitative analysis of DCV and CAPS-1-EYFP dynamics to Figure 7 (new Figure 7 B and C) to support our conclusion that co-trafficking of CAPS-1 with mobile DCVs is very rare.
It remains to be determined whether Dsh co-traffics with Fmi, and whether this is the normal mode of removal of Dsh from junctions; however, it is interesting to note that in vertebrates Dsh has been reported to act as an endocytic adapter (Chen et al., 2003; Yu et al., 2007), and in some contexts such adaptors are marked for internalisation by ubiquitylation (reviewed by Traub and Lukacs, 2007).
