Abstract
ABSTRACTWe recently demonstrated that dynein and kinesin motors drive multiple aspects of endosomal function in mammalian cells. These functions include driving motility, maintaining morphology (notably through providing longitudinal tension to support vesicle fission), and driving cargo sorting. Microtubule motors drive bidirectional motility during traffic between the endoplasmic reticulum (ER) and Golgi. Here, we have examined the role of microtubule motors in transport carrier motility, morphology, and domain organization during ER-to-Golgi transport. We show that, consistent with our findings for endosomal dynamics, microtubule motor function during ER-to-Golgi transport of secretory cargo is required for motility, morphology, and cargo sorting within vesicular tubular carriers en route to the Golgi. Our data are consistent with previous findings that defined roles for dynein-1, kinesin-1 (KIF5B) and kinesin-2 in this trafficking step. Our high resolution tracking data identify some intriguing aspects. Depletion of kinesin-1 reduces the number of motile structures seen, which is in line with other findings relating to the role of kinesin-1 in ER export. However, those transport carriers that were produced had a much greater run length suggesting that this motor can act as a brake on anterograde motility. Kinesin-2 depletion did not significantly reduce the number of motile transport carriers but did cause a similar increase in run length. These data suggest that kinesins act as negative regulators of ER-to-Golgi transport. Depletion of dynein not only reduced the number of motile carriers formed but also caused tubulation of carriers similar to that seen for sorting nexin-coated early endosomes. Our data indicated that the previously observed anterograde–retrograde polarity of transport carriers in transit to the Golgi from the ER is maintained by microtubule motor function.
Highlights
Endoplasmic reticulum (ER)-to-Golgi transport is required for the delivery of newly synthesized secretory cargo to the Golgi apparatus
It is not known how the ER–Golgi intermediate compartment (ERGIC) is itself linked to microtubules but considerable data have shown that cytoplasmic dynein-1 is required for the movement of cargo in vesicular–tubular clusters (VTCs) to the Golgi (Burkhardt et al, 1997; Palmer et al, 2009; Presley et al, 1997)
Using GFP-tagged sorting nexins (SNXs) and total internal reflection fluorescence (TIRF) microscopy to maximize signal-to-noise ratios, we found that specific combinations of microtubule motors were coupled to individual sorting SNX-coated membrane domains
Summary
Endoplasmic reticulum (ER)-to-Golgi transport is required for the delivery of newly synthesized secretory cargo to the Golgi apparatus. The link to microtubules is established at the point of ER export through direct interaction of the Sec subunit of the COPII coat complex with the p150Glued subunit of dynactin (Watson et al, 2005). It is not known how the ERGIC is itself linked to microtubules but considerable data have shown that cytoplasmic dynein-1 is required for the movement of cargo in vesicular–tubular clusters (VTCs) to the Golgi (Burkhardt et al, 1997; Palmer et al, 2009; Presley et al, 1997). Using GFP-tagged sorting nexins (SNXs) and total internal reflection fluorescence (TIRF) microscopy to maximize signal-to-noise ratios, we found that specific combinations of microtubule motors were coupled to individual sorting SNX-coated membrane domains
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