The Golgin Protein Giantin Regulates Interconnections Between Golgi Stacks

Abstract

Golgins are a family of Golgi-localized long coiled-coil proteins. The major golgin function is thought to be the tethering of vesicles, membranes, and cytoskeletal elements to the Golgi. We previously showed that knockdown of one of the longest golgins, Giantin, altered the glycosylation patterns of cell surfaces and the kinetics of cargo transport, suggesting that Giantin maintains correct glycosylation through slowing down transport within the Golgi. Giantin knockdown also altered the sizes and numbers of mini Golgi stacks generated by microtubule de-polymerization, suggesting that it maintains the independence of individual Golgi stacks. Therefore, it is presumed that Golgi stacks lose their independence following Giantin knockdown, allowing easier and possibly increased transport among stacks and abnormal glycosylation. To gain structural insights into the independence of Golgi stacks, we herein performed electron tomography and 3D modeling of Golgi stacks in Giantin knockdown cells. Compared with control cells, Giantin-knockdown cells had fewer and smaller fenestrae within each cisterna. This was supported by data showing that the diffusion rate of Golgi membrane proteins is faster in Giantin-knockdown Golgi, indicating that Giantin knockdown structurally and functionally increases connectivity among Golgi cisternae and stacks. This increased connectivity suggests that contrary to the cis-golgin tether model, Giantin instead inhibits the tether and fusion of nearby Golgi cisternae and stacks, resulting in transport difficulties between stacks that may enable the correct glycosylation of proteins and lipids passing through the Golgi.

Keywords: Golgi; electron tomography; endoplasmic reticulum; glycosylation; golgins.

FIGURE 1

Loss of Giantin elongates Golgi cisternae. (A) Typical electron micrographs of Golgi cisternae in Giantin and control siRNA-treated cells. Bar, 500 nm. (B) Cisternal lengths were measured by tracing the cisternae manually using ImageJ software (B, n = 10 micrographs, bar, SEM, P < 0.00001).

FIGURE 2

Loss of Giantin changes the intra-connectivity between Golgi cisternae/stacks. HeLa cells stably expressing ManII-GFP with Giantin or control siRNA treatment were subjected to FRAP experiments. GFP-labeled Golgi in ManII-GFP expressing cells was photobleached, and fluorescence recoveries were monitored for 200 s and graphed (n = 10, bar, SD). Representative images of fluorescence recovery at the indicated time points are shown below. Sizes 25.5 (w) × 25.5 (h) μm. The circles represent the bleached area.

FIGURE 3

Loss of Giantin fuses Golgi cisternae. (A) Typical 3D models of Golgi cisternae in Giantin and control siRNA-treated cells. One of the tomographic slices used for these 3D models is shown in top panels. Note that the Golgi cisternae visible in RNAi cells appear longer than those in control cells. Bar, 500 nm. Arrows indicate large fenestrae in cisternae. The cis-most and trans-most cisternae are labeled C1 and C5, respectively. (B) The reduction of cisternal volumes from the corresponding no-fenestra model is shown as the % volume reduction in Giantin and control siRNA-treated cells. Bar, SD (n = 4, ^∗P < 0.005).