Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites.

Abstract

Microtubule disruption has dramatic effects on the normal centrosomal localization of the Golgi complex, with Golgi elements remaining as competent functional units but undergoing a reversible "fragmentation" and dispersal throughout the cytoplasm. In this study we have analyzed this process using digital fluorescence image processing microscopy combined with biochemical and ultrastructural approaches. After microtubule depolymerization, Golgi membrane components were found to redistribute to a distinct number of peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed rapidly (within 15 minutes) to these sites after first moving through the ER. Prior to this redistribution, Golgi enzyme processing of proteins exported from the ER was inhibited and only returned to normal levels after Golgi enzymes redistributed to peripheral ER exit sites where Golgi stacks were regenerated. Experiments examining the effects of microtubule disruption on the membrane pathways connecting the ER and Golgi suggested their potential role in the dispersal process. Whereas clustering of peripheral pre-Golgi elements into the centrosomal region failed to occur after microtubule disruption, Golgi-to-ER membrane recycling was only slightly inhibited. Moreover, conditions that impeded Golgi-to-ER recycling completely blocked Golgi fragmentation. Based on these findings we propose that a slow but constitutive flux of Golgi resident proteins through the same ER/Golgi cycling pathways as ERGIC-53 underlies Golgi Dispersal upon microtubule depolymerization. Both ERGIC-53 and Golgi proteins would accumulate at peripheral ER exit sites due to failure of membranes at these sites to cluster into the centrosomal region. Regeneration of Golgi stacks at these peripheral sites would re-establish secretory flow from the ER into the Golgi complex and result in Golgi dispersal.

The Golgi Complex Is a Microtubule-organizing Organelle

“…As shown for WIF-B cells (Figure 4B), the Golgi remained essentially in a centrosomal location, although very small Golgi fragments also occurred throughout the cytoplasm and were probably rebuilt from the ER (Cole et al, 1996;Storrie et al, 1998). In these conditions, there was a dramatic drop in the amount of noncentrosomal MTs ( Figure 4B) compared with cells in which nocodazole treatment was performed at 37°C ( Figure 4A).…”

“…This means that our in vivo observations were likely not to result from the severing and release of centrosomal MTs (Baas and Joshi, 1992;Yu et al, 1993;Keating et al, 1997;Vorobjev et al, 1997) or even spontaneous cytoplasmic assembly, followed by treadmilling or migration en bloc Vorobjev et al, 1997;Yvon and Wadsworth, 1997;Tucker et al, 1998) until they were captured and stabilized by scattered Golgi elements. Because the reconstitution of mini-Golgi stacks at ER exit sites actively participates in the dispersion of the Golgi complex after nocodazole-mediated MT depolymerization (Cole et al, 1996;Storrie et al, 1998), the ER would also have been a likely candidate for stimulating noncentrosomal MT assembly. This possibility was clearly ruled out because the vast majority of newly assembled MTs followed the Golgi when the organelle was kept in a central location during MT depolymerization.…”

“…Conversely, the central localization of the Golgi apparatus involves cytoplasmic dynein (Corthésy-Theulaz et al, 1992;Fath et al, 1994;Burkhardt et al, 1997;Harada et al, 1998). It is also worth noting that, in addition to the kinesin-mediated disruption of the central Golgi complex during MT depolymerization, the dispersion of Golgi elements also relies on the reconstitution of mini Golgi stacks at endoplasmic reticulum (ER) exit sites (Cole et al, 1996;Storrie et al, 1998). Such data on the scattering and the reclustering of the Golgi complex have led to the view that Golgi membranes undergo an intimate relationship with MTs and especially with a population of nocodazole-resistant, stable MTs.…”

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

“…Microtubules are not an absolute requirement for Golgito-ER transport, because Golgi enzymes have been proposed to recycle through the ER to form scattered Golgi ministacks in the absence of microtubules by treatment of cells with noccodazole. However, completion of Golgi recycling under these conditions is inefficient and takes many hours (Cole et al, 1996).…”

“…Enzymes seemingly enter the ER close to the microtubule organizing center (MTOC), diffuse through the ER, and then reemerge at peripheral ER exit sites. Here, Golgi stacks are then reassembled into discrete, yet fully functional Golgi stacks that remain in proximity to the ER exit site (Cole et al, 1996). On repolymerization of microtubules, the 100 or so Golgi stacks move inwards toward the MTOC, reforming the Golgi ribbon.…”