Opposing Microtubule Motors Control Motility, Morphology, and Cargo Segregation During Er-to-Golgi Transport.

Abstract

BSTRACTWe 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.

Assembly and Activation of Dynein-Dynactin by the Cargo Adaptor Protein Hook3

“…The mammalian light intermediate chains, encoded by two closely related gene products LIC1 and LIC2 (Hughes et al, 1995;Tynan et al, 2000), are involved in several different types of cargo interactions and dynein-based movements, including endosomal and lysosomal transport, ER export, Golgi transport, and axonal vesicle trafficking (Palmer et al, 2009;Horgan et al, 2010;Kong et al, 2013;Tan et al, 2011;Brown et al, 2014;Koushika et al, 2004). The domain structure of the LIC allows it to interact with cargo adaptors while integrated into the dynein holoenzyme.…”

Interaction of early secretory pathway and Golgi membranes with microtubules and microtubule motors

“…When a non motor chain of kinesin 2 KAP3 is depleted, the anterograde transport ERES -ERGICcis Golgi is not affected, whereas the reverse COPI dependent return of KDEL R marker protein to ER is inhibited [33]. Detailed analysis of movements of COPI coated ts045 VSV G containing vesicles in the cell showed that the depletion of dynein, as expected, leads to dramatically decreased number of moving particles [6]. The effect of depletion of kinesin 1 was unexpected: the number of moving particles was also reduced, but the remaining moving vesicles strongly increased the length of tracks.…”

“…The effect of depletion of kinesin 1 was unexpected: the number of moving particles was also reduced, but the remaining moving vesicles strongly increased the length of tracks. Depletion of kinesin 2 did not affect the num ber of moving vesicles, but also increased the length of the tracks [6]. From these data it can be concluded that kinesins inhibit dynein dependent anterograde transport from the ER to Golgi, and kinesin 1 is involved in retro grade transport [6].…”

“…A very convenient model system for detailed description of the kinetics of the secretory pathway is based on use of the protein of vesic ular stomatitis virus VSV G. A peak of works using this protein came in the 1990s. Thermo sensitive mutant ts045 VSV G at 40°C does not leave the ER [5], which enables the synchronization of secretion of VSV G. Cells are incubated 9 16 h at 40°C, and then secretion is induced by decreasing the temperature to 32°C [5,6] or even to 15°C [7]. At 32°C VSV G passes the entire secre tory pathway and remains on the plasma membrane, but at 15°C it is delayed in ERGIC [7].…”

Trypanosomes have divergent kinesin-2 proteins that function differentially in IFT, cell division, and motility

“…Kinesin-2 proteins perform an important and conserved function in IFT and flagellar biogenesis of primary and motile cilia (Lechtreck, 2015; Scholey, 2013). Kinesin-2 proteins have also been implicated in bidirectional endoplasmic reticulum (ER) to Golgi transport (Brown et al, 2014; Stauber et al, 2006), endosomal trafficking (Granger et al, 2014), transport in neurons (Hirokawa et al, 2010), cilium-based signaling (Goetz and Anderson, 2010), chromosome segregation (Haraguchi et al, 2006; Miller et al, 2005), and cytokinesis completion (Brown et al, 1999; Fan and Beck, 2004). Here, we report that the two kinesin-2 proteins TbKin2a and TbKin2b in BSF T. brucei function in flagellar biogenesis, but only TbKin2a appears to be crucial for cell proliferation.…”