Land-locked mammalian Golgi reveals cargo transport between stable cisternae

Abstract

The Golgi is composed of a stack of cis, medial, trans cisternae that are biochemically distinct. The stable compartments model postulates that permanent cisternae communicate through bi-directional vesicles, while the cisternal maturation model postulates that transient cisternae biochemically mature to ensure anterograde transport. Testing either model has been constrained by the diffraction limit of light microscopy, as the cisternae are only 10-20 nm thick and closely stacked in mammalian cells. We previously described the unstacking of Golgi by the ectopic adhesion of Golgi cisternae to mitochondria. Here, we show that cargo processing and transport continue-even when individual Golgi cisternae are separated and "land-locked" between mitochondria. With the increased spatial separation of cisternae, we show using three-dimensional live imaging that cis-Golgi and trans-Golgi remain stable in their composition and size. Hence, we provide new evidence in support of the stable compartments model in mammalian cells.The different composition of Golgi cisternae gave rise to two different models for intra-Golgi traffic: one where stable cisternae communicate via vesicles and another one where cisternae biochemically mature to ensure anterograde transport. Here, the authors provide evidence in support of the stable compartments model.

Fig. 1

Re-targeting GRASP55 from Golgi to mitochondria generates “land-locked” Golgi cisternae. a Schematic of GRASP55 re-targeting assay, adapted from Supplementary Fig. 8a . GRASP55 is localized to the cytoplasmic face of the Golgi by N-terminal myristoylation. The C-terminal end of GRASP55 was fused to FKBP and a FP. OMP25 is anchored to the cytoplasmic face of the outer mitochondrial membrane by a C-terminal transmembrane domain. FRB and FP were fused to the N-terminal end of OMP25. Addition of the dimerizer induces FKBP and FRB heterodimerization. b Cartoon showing expected outcome of GRASP55 re-targeting to mitochondria. Addition of dimerizer induces heterodimerization of GRASP55-FKBP-BFP and FRB-FP-OMP25 leading to the adhesion of mitochondria to individual Golgi cisterna. c Confocal images of HeLa cells transfected with GRASP55-FKBP-GFP and FRB-Myc-OMP25 and immunostained for Myc and gpp130. Where indicated, cells were treated with dimerizer (2 μM) for 3 h. Scale bar is 10 μm. d Quantification of Golgi areas (gpp130 fluorescence) with and without dimerizer from c. e STED microscopy images of thin section (70 nm) HeLa cells that were transfected with GRASP55-FKBP-HA and FRB-Myc-OMP25 and stained for mitochondria and immunostained for gpp130. Where indicated, cells were treated with dimerizer (2 μM) for 3 h. Scale bar is 500 nm. f Live confocal imaging of Golgi in HeLa cells co-transfected with GalT-RFP, GRASP55-FKBP-BFP and FRB-GFP-OMP25, and pretreated with nocodazole, or dimerizer, or both. Overlay of frames from time 0 to time 0 and time 0 to time 120 min are shown. Scale bar is 10 μm. g PCC over time t of overlay images of GalT-RFP at time 0 to time t based on live image movies from f. Error bars = s.d. For statistical analysis, t-tests were used

Fig. 2

Land-locked Golgi cisternae are unstacked. a STED microscopy images of serial thin sections of HeLa cells cotransfected with GRASP55-FKBP-HA and FRB-Myc-OMP25 and stained for mitochondria (CMXRos) and immunostained for cis-Golgi (gpp130) and trans-Golgi (p230). Complete separation of cis-cisterna and trans-cisterna is evident only in the presence of the dimerizer. Scale bar is 500 nm. b TEM of HeLa cells transfected with GRASP55-FKBP-BFP and FRB-Myc-OMP25. Blue M indicates mitochondria. Red G indicates Golgi. Scale bar is 1 μm. c TEM of ManII-HRP HeLa cells transfected with GRASP55-FKBP-BFP and FRB-Myc-OMP25, then treated with DAB and H₂O₂ post-fixation to turn Golgi-specific membranes black. Scale bar is 1 μm. d Quantification of the number of cisternae/stack in GRASP55 re-targeted HeLa cells from b. e Optical section of an EM tomogram of HeLa cells transfected with GRASP55-FKBP-BFP and FRB-Myc-OMP25, then treated with dimerizer. Scale bar is 200 nm. f A surface-rendered 3D model of EM tomogram from e was generated to visualize the unstacked cisternae of land-locked Golgi. Error bars = s.d. For statistical analysis, t-tests were used

Fig. 3

Land-locked cisternae contain coatomer, coated buds and vesicles. a Confocal images of HeLa cells transfected with GRASP55-FKBP-HA and FRB-Myc-OMP25, then stained for mitochondria and immunostained for GM130 and COPI. Where indicated, cells were treated with dimerizer (2 μM) for 3 h. Scale bar is 5 μm. b TEM images of vesicles and budded regions on land-locked Golgi in HeLa cells transfected with GRASP55-FKBP-BFP and FRB-Myc-OMP25 and treated with dimerizer. Scale bar is 200 nm

Fig. 4

Live imaging a wave of anterograde cargo through individual normal or land-locked Golgi areas. a Cartoon schematic of the anterograde cargo ss-GFP-FM4-FCS-hGH. This cargo is aggregated and trapped in the ER until solubilizer is added. Then, the monomeric cargo is released from the ER for secretion. b Cartoon of ss-GFP-FM4-FCS-hGH cargo traffic and effect of temperature block. A wave of anterograde traffic is initiated from the ER by the addition of solubilizer at 37 °C, and the cargo is secreted out of the Golgi. However, at 20 °C, traffic is slowed so that the cargo accumulates at the TGN instead of getting secreted. c Quantification of a wave of cargo released at 37 °C from normal (n = 9) or land-locked (n = 9) Golgi in HT1080 cells that stably express ss-GFP-FM4-FCS-hGH (refer to Supplementary Fig. 6a, b for representative images and Table 2 for numbers). d Quantification of a wave of cargo after release from 20 °C block from normal (n = 9) or land-locked (n = 6) Golgi in HT1080 cells that stably express ss-GFP-FM4-FCS-hGH (refer to Supplementary Fig. 6c, d for representative images and Table 2 for numbers). The Golgi exit rate is almost indistinguishable between normal and land-locked Golgi. e Cartoon of the effect of 20 °C block combined with solubilizer washout on ss-GFP-FM4-FCS-hGH, which re-aggregates the cargo in the TGN. f, g TEM images of re-aggregated ss-GFP-FM4-FCS-hGH cargo in HT1080 cells that stably express ss-GFP-FM4-FCS-hGH. Large aggregated cargo can be seen in the lumen of f normal, stacked cisternae and g land-locked cisternae. Scale bar is 1 μm. Error bars = s.d

Fig. 5

Bulk secretion through land-locked Golgi. a HT1080 cells that stably express ss-GFP-FM4-FCS-hGH were transfected with GRASP55-FKBP-HA and FRB-GFP-OMP25, then treated with dimerizer for 3 h before initiating secretion by adding solubilizer. Secretion was carried out at 37 °C in the presence of cycloheximide. For each time point, 10% of chase media and cell lysates were loaded for analysis by SDS-PAGE and Western blot with GFP and actin antibodies. Experiments were done in triplicate. b Quantification of Western blots from a. The % of cargo (full-length and cleaved) secreted was determined by taking the ratio of the secreted cargo (medium) over total cargo (medium + cell). c Secretion after release from 20 °C block. Cells were pre-incubated in dimerizer for 3 h at 37 °C, then cooled to 20 °C for 1 h before adding solubilizer for 2 h, then warmed back to 37 °C to initiate the secretion assay. For each time point, 10% of chase media and cell lysates were loaded for analysis by SDS-PAGE and Western blot with GFP and actin antibodies. Experiments were done in triplicate. d Quantification of Western blots from c. e Normalized furin cleavage efficiency of ss-GFP-FM4-FCS-hGH for normal and land-locked Golgi was calculated for each time point from cell fractions in a by taking the ratio of cleaved/(full-length + cleaved), and plotting that as a percentage of the maximum secreted amount. The data were fit to a single exponential curve and normalized to the plateau observed for furin cleavage in land-locked Golgi. Error bars = s.d

Fig. 6

Land-locked cisternae are stable and do not mature. a Cartoons showing the two main intra-Golgi trafficking models and the land-locked Golgi. The cisternal maturation model postulates that the cisternae are mobile, maturing from cis to medial to trans as they progress through the stack. These mobile cisternae serve as anterograde cargo carriers, processing the cargo within as they uptake resident enzymes from older cisterna by retrograde COPI vesicles. The stable compartments model postulates that the Golgi exists as a static compartment that receives and exports cargo through anterograde COPI-coated vesicles. Cisternal identity is preserved by the recycling of any leaked enzymes in retrograde COPI vesicles. In land-locked Golgi, the cisterna are separated and adhered to mitochondria, increasing the spatial separation between cisternae from ~30 to ~500 nm. While a role for recycling Golgi enzymes by retrograde COPI vesicles is established, the question mark denotes how it is unclear what mediates anterograde traffic. b Live imaging land-locked Golgi. HeLa cells were transfected with GRASP55-FKBP-BFP and FRB-Myc-Omp25 to be able to re-target Golgi to the mitochondria, as well as GRASP65-GFP to mark the cis-Golgi and GalT-RFP to mark the trans-Golgi. Cells were treated with nocodazole to break the Golgi ribbon into mini-stacks, and then with dimerizer to produce land-locked Golgi. Then the cells were imaged every minute as a z-stack for 30 min. A single confocal section of a land-locked Golgi whose cis-Golgi and trans-Golgi markers flank a mitochondria is followed over time (encircled in blue). c Plot profiles were taken across land-locked Golgi shown in b for cis-Golgi (GRASP65-GFP, green), trans-Golgi (GalT-RFP, red line), and mitochondria (GRASP55-FKBP-BFP, black line) fluorescence. Cis and trans-Golgi markers remain well separated at t = 1 min and t = 30 min. d PCC was calculated for GRASP65-GFP and GalT-RFP for normal Golgi (n = 7) and land-locked Golgi (n = 20) over the course of 15 min on merged planes. Error bars = s.e.m. e Quantification of the mean distance between the peak fluorescence of cis and trans-Golgi markers for normal Golgi (n = 7) and land-locked Golgi (n = 20). Error bars = s.d. f Quantification of the integrated intensity of cis and trans-Golgi markers for land-locked Golgi over time (merged planes, n = 20). The inversed rate of hGH arrival at the trans-Golgi (Fig. 5e) is plotted for comparison (expected rate of maturation). Error bars = s.e.m. g Quantification of the average number (left y-axis, triangles) and area (right y-axis, circles) of cis-Golgi particles on merged planes from different cells and experiments (n = 3). Inset: visual correlates for thresholded cis Golgi-marker positive particles tracked at t = 1, 7, and 14 min. Error bars = s.e.m