Rapid, Endoplasmic Reticulum-independent Diffusion of the Mitotic Golgi Haze

Abstract

Early in mitosis, the mammalian Golgi apparatus disassembles, and fluorescence microscopy reveals Golgi clusters and an extensive, nonresolvable haze that either represents scattered vesicles or a merged endoplasmic reticulum (ER)-Golgi compartment. To help decide between these alternatives, we have carried out a combined microscopic and pharmacological analysis, by using a BS-C-1 cell line stably coexpressing ER and Golgi markers. Video fluorescence microscopy showed that these two organelles were morphologically distinguishable at all stages of mitosis, and photobleaching experiments showed that diffusion of the Golgi marker was unaffected by the presence of the ER. Fragmentation of the ER by using filipin III completely blocked diffusion of the ER marker but had no effect on the Golgi marker, unless it was first relocated to the ER by using brefeldin A. The Golgi haze was also studied using BODIPY ceramide. Its diffusion was slower in mitotic Golgi than in mitotic ER, but similar to that of a Golgi enzyme marker in the mitotic Golgi haze or in Golgi vesicles generated by ilimaquinone. Together, these results support the idea that the Golgi and the ER remain separate during mitosis and strongly suggest that Golgi markers move by vesicle diffusion, as opposed to lateral diffusion in continuous membranes. INTRODUCTIONThe status of the Golgi apparatus as an organelle has been the subject of a long-running debate that focused initially on the route taken by transiting cargo (cisternal maturation or vesicle-mediated transport; Glick and Malhotra, 1998;Pelham and Rothman, 2000) and, more recently, on the mechanisms of duplication and partitioning that underlie its biogenesis (Marsh and Howell, 2002;Munro, 2002). If the Golgi apparatus is responsible for its biogenesis, then it can be viewed as an autonomous organelle (Pelletier et al., 2002). If, on the other hand, it depends on the endoplasmic reticulum, then it can be viewed as a dependent organelle, which exists only as a consequence of ER functioning (Zaal et al., 1999;Bevis et al., 2002;Munro, 2002).Golgi partitioning during mitosis in animal cells has been particularly controversial, with models ranging from partitioning by Golgi elements themselves (Lucocq et al., 1989;Misteli and Warren, 1995;Jesch and Linstedt, 1998;Shima et al., 1998;Jesch et al., 2001;Jokitalo et al., 2001) to the partial or even complete merger of the Golgi with the ER, which then mediates the partitioning process (Thyberg and Moskalewski, 1992;Zaal et al., 1999;Kano et al., 2000;Terasaki, 2000). Although most biochemical experiments have yielded consistent results, suggesting a separation of the ER and Golgi during mitosis (Jesch and Linstedt, 1998;Farmaki et al., 1999;Jesch et al., 2001), microscopic experiments have often yielded contradictory results (Thyberg and Moskalewski, 1992;Shima et al., 1998;Zaal et al., 1999;Jokitalo et al., 2001).A particular issue has been the Golgi haze observed by fluorescence microscopy of mitotic cells. Breakdown and fragmentation of the Golgi ribbon a...

Golgi inheritance in small buds of Saccharomyces cerevisiae is linked to endoplasmic reticulum inheritance

“…(Scale bar: 2 m.) (B) The late Golgi inheritance defect in the sec8 -599 strain was quantified as in Fig. 2. cytosol (43), and transmembrane Golgi proteins may travel in small vesicles, which diffuse through the cytosol quite rapidly (7). We favor the interpretation that ER-derived membranes fuse with Golgi-derived vesicles to form new cisternae, which recruit peripheral Golgi proteins from the cytosol (44).…”

“…It has been suggested that the Golgi fuses with the endoplasmic reticulum (ER) during mitosis and then reemerges as a separate organelle during cytokinesis (5). However, the preponderance of evidence now indicates that the mitotic Golgi consists mainly of small membrane fragments that are distinct from the ER (6)(7)(8). Yet Golgi reassembly during cytokinesis is inhibited by blocking ER export with a dominantnegative form of the Sar-1 GTPase (5, 9), possibly indicating a role for the ER in vertebrate Golgi inheritance.…”

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

“…At prometaphase, the released Golgi stacks undergo unstacking and vesiculation, resulting in the formation of mitotic Golgi clusters comprising vesicular and tubular mitotic Golgi fragments, whose fate in metaphase/anaphase is still debated. [18][19][20] At telophase, the mitotic Golgi fragments undergo a reverse series of changes leading to the reassembly of the stacks and eventually the Golgi ribbon formation in the two daughter cells. The mitotic Golgi ribbon disassembly has been considered as a stochastic mechanism for equal Golgi partitioning.…”

Mitotic regulation of SREBP and ATF6 by separation of the Golgi and ER

“…47,48 Other studies, however, did not find evidence for a merged ER and Golgi apparatus. [49][50][51][52] Furthermore, mitotic Golgi membranes are often found in close proximity to the spindle poles, suggesting an ER-independent but spindle-dependent partitioning mechanism. 16,49,53,54 Whether the spindle plays an active role in the division of the Golgi apparatus remains to be demonstrated, since it is so far not possible to uncouple the association of Golgi membranes from the spindle poles.…”

“…These approaches relied on the overexpression of chimeric proteins 47,51,52 in combination with temperature alterations to trap the molecules in the ER during mitosis. 48 There might be conditions under which Golgi enzymes enter the ER during mitosis for purposes of partitioning and/or regulation of other pathways.…”