Membrane adhesion dictates Golgi stacking and cisternal morphology

Simultaneous Depletion of GRASP65/55 or GM130/Golgin45 Disrupts Cisternal Flatness of Golgi Stacks but Does Not Result in Golgi Unstacking.

A recent study reported that double knockdown of GRASP65/55 results in complete unstacking of Golgi in HeLa cells (15). To better understand the relative contribution of Golgins and GRASPs in Golgi stack assembly and to find out whether GRASPs are both necessary and sufficient for Golgi stacking in vivo, we decided to simultaneously deplete both GRASPs or both Golgins and study the effects on Golgi stacking, using electron microscopy. In contrast to the earlier report, we observed that simultaneous depletion of GRASP65/55 or their Golgin binding partners, GM130/Golgin45, results in disruption of Golgi cisternal flatness (cf. EM photos in Fig. 1 A–C) without significant Golgi unstacking. As shown in Fig. S1, double depletion of GRASPs resulted in a significant reduction of Golgins, and vice versa. These phenomena suggest that Golgins and GRASPs are likely to affect the stability of each other to a certain extent (GM130–GRASP65 and Golgin45–GRASP55), mainly because of the role they play in the membrane association of their binding partners. Importantly, however, even with this additional reduction in the level of other adhesion proteins, we did not observe any significant Golgi unstacking in either GRASP or Golgin double-depleted HeLa cells. To exclude the possibility that this difference is a result of siRNA oligos used for GRASP65/55 depletion, we designed an additional set of siRNA oligos against GRASP65 and GRASP55, respectively. The efficiency of GRASP65/55 double knockdown routinely reached above 98% for both sets of siRNAs against GRASP65/55 (Figs. S1 and S2). Neither siRNA combinations led to significant unstacking of the Golgi when examined under EM. We have no explanation for this discrepancy, although we also observed a significant unstacking of the Golgi in a small percentage (<1%) of the knockdown cells we analyzed.

Morphologically, we noticed that Golgi membranes appeared to have lost the characteristic flattened cisternae. As a way to quantify the change in Golgi cisternal morphology, we measured the maximum luminal width of the Golgi cisternae (see Fig. S1 for an example). In control siRNA-transfected cells, the average maximum luminal width of Golgi cisternae was around 59 ± 3 nm (Fig. 1F). In single-knockdown cells, we observed a small but consistent increase (∼10–15 nm) in the average luminal width, whereas GRASP65/55 or GM130/Golgin45 double depletion led to a two- to threefold increase in the luminal width (Fig. 1 D and E for Kolmogorov–Smirnov (K-S) plots; see Fig. S1 for frequency distribution graphs).

We reasoned that increased cisternal width in these knockdown cells might be a result of a block in anterograde cargo transport through the Golgi. To test this, we used CD8 fused to conditional aggregation domain (24) to assess the efficiency of bulk cargo transport under these knockdown conditions. Surprisingly, we found that GRASP65/55 double knockdown consistently led to a ∼30–40% increase in CD8 transport to the plasma membrane compared with control cells and GM130/Golgin45 depleted cells (Fig. S2; see Fig. S3 for raw data), suggesting that the increased cisternal width in these cells is not likely to be caused by accumulation of anterograde cargo within the lumen of the Golgi cisterna.

Additional Knockdown of Golgin45 in GRASP65/55-Depleted Cells Results in Golgi Disassembly.

These findings naturally led us to the question of whether the changes in cisternal flatness in the GRASP- or Golgin-depleted cells are associated with Golgi stacking. In other words, the disrupted cisternal flatness may or may not be indicative of the progression toward Golgi disassembly in these cells. To find an answer to this seemingly elusive question, we tested whether additional depletion of Golgin45 or GM130 leads to Golgi disassembly in GRASP65/55-depleted HeLa cells. If so, the result would suggest that both cisternal flatness and cisternal stacking are determined by the collective cisternal adhesive process from these GRASPs and Golgins. In support of this idea, we found that triple knockdown of GRASP65/55 and Golgin45 (and, to a lesser extent, GM130) in HeLa cells results in significant Golgi unstacking (Fig. S4 A and B). Qualitatively, depletion of Golgin45/GRASP65/55 led to almost complete Golgi disassembly, but only partial Golgi unstacking was observed in GM130/GRASP65/55-depleted cells (compare EM photos in Fig. S4 A and B).

Is the Disruption in Golgi Cisternal Flatness Reversible?

We then asked whether the disruption in cisternal flatness can be restored by rescue transfection. To do this, we expressed an RNAi-resistant form of each adhesion protein in double-knockdown cells that eliminate both GRASP proteins or both Golgins. As expected, Golgi stack morphology was restored by either exogenously expressing GRASP65 or GRASP55 in GRASPs double-depleted cells (Fig. 2 A and B) or by expressing either Golgin GM130 or Golgin45 in Golgin double-depleted cells (Fig. 2 C and D; see Fig. 2E for summary). The average maximum luminal width for rescued Golgi cisternae was very close to the values we saw in single-knockdown cells. The knockdown and rescue expressions were confirmed by Western blots, as shown in Fig. S5. Altogether, this is consistent with the idea that GRASP55 and GRASP65 play a complementary role in Golgi stacking and cisternal morphology, as can Golgin45 and GM130, despite their differing physiological cis-trans distributions.

Are GRASP Proteins Dispensable for Golgi Stacking?

As shown in the triple-knockdown experiments (Fig. S4), there was a strong indication that GRASPs and Golgins may be playing functionally complementary roles in Golgi stacking and cisternal morphology. If this is true, is it possible that Golgins may be able to substitute for GRASPs either entirely or partially in maintaining Golgi stacking and cisternal morphology? To test this, we exogenously expressed Golgins in GRASP65/55-depleted HeLa cells to see whether Golgins can restore cisternal flatness in GRASP-depleted HeLa cells. Remarkably, crossing the functional barrier between GRASPs and Golgins was equally effective. Expressing either Golgin protein in GRASPs double-depleted cells restored the Golgi stack morphology, as judged qualitatively by EM (Fig. 3 A and B, Right). Golgi cisternal flatness was not restored by expressing trans-Golgi network localized Golgin97 (Fig. 3C). As shown in Fig. S6, we also performed reverse experiments, in which we depleted GM130/Golgin45, followed by exogenous expression of GRASP55-GFP. Although the degree of restoration is smaller compared with the effect of Golgin overexpression in GRASP-depleted cells, we still found a significant restoration in cisternal flatness in these cells (P < 0.05).

We measured maximum cisternal luminal width to quantify the degree of restoration (see K-S plots in Fig. 3 A–D). Exogenous expression of Golgin45 in GRASP65/55-depleted cells resulted in Golgi with a 61 ± 2 nm average maximum cisternal luminal width (from 155 ± 7 nm in GRASP65/55-depleted cells). Compared with control siRNA-transfected cells (59 ± 3 nm), there was essentially no significant difference. Thus, our results strongly indicate that the GRASPs and Golgin45 (and, to a lesser extent, GM130) are likely to play a functionally redundant and complementary role for Golgi stacking under physiological conditions. We measured the anterograde cargo transport in these cells (Fig. S6D) and found that the amount of CD8 transported to the plasma membrane in these cells is slightly lower compared with GRASP65/55-depleted cells.

Golgin45-Dependent Restoration of Golgi Cisternal Flatness and Morphology Is Not Cell Type-Specific.

To see whether these observations are restricted to HeLa cells, we tested human fibrosarcoma HT1080 cells depleted of either GRASP65/55 or GM130/Golgin45 for 96 h and processed these knockdown cells for EM. We found that the morphological changes in HT1080 cells depleted of GRASPs or Golgins were essentially identical (Fig. S7A). No significant unstacking of the Golgi was observed in either knockdown cell. Exogenous expression of Golgin45 in GRASP65/55-depleted HT-1080 for 18 h resulted in similar restoration of Golgi cisternal flatness as in HeLa cells (Fig. S7 A–C). Because HT-1080 cells are known to secrete matrix metalloproteinase MMP-2 abundantly, the knockdown cells were tested for the secretion of endogenous MMP-2. Strikingly, we found that both GRASP and Golgin double-depleted HT1080 cells secreted significantly increased amounts of endogenous MMP-2 (∼1.5-fold increase; Fig. S7 E and F) during the 4-h secretion assays compared with the control transfected cells. These results suggest that flattening of Golgi cisternae might have a role in determining the rate of anterograde cargo transport through the Golgi.

Can an Ectopic Adhesion Process Introduce Another Organelle into the Golgi Stack?

Our results so far have suggested that cisternal adhesion, irrespective of its molecular source, is likely to be the overriding principle of Golgi stack assembly. If so, then adding adhesive bonds between Golgi cisternae and another organelle (mitochondria) should lead to the formation of hybrid stacks within a few hours, in which mitochondria can be incorporated into preexisting interphase Golgi stacks as if they were Golgi cisternae, provided the ectopic binding energy exceeds that normally stabilizing the Golgi stack.

To engineer ectopic adhesion between Golgi cisternae and mitochondria that can be controlled pharmacologically, we used a “knock-sideways” approach (13, 25) involving the ligand-induced formation of heterodimers between the FK506-binding protein (FKBP) and a rapamycin-binding domain (FRB). These two proteins bind to each other when a rapamycin analog (AP21967, which simultaneously binds to the FRB and FKBP domains) is added to cells. The Golgi adhesion protein GRASP55 was expressed as a hybrid protein with FKBP (and a fluorescent protein [tag blue fluorescent protein (tagBFP)]) to enable visualization (Fig. S8A). FRB was expressed as a hybrid with a protein targeted to the mitochondrial outer membrane (OMP25) also tagged with the myc epitope. In this way, the expressed GRASP55 hybrid protein should relocalize from the Golgi (and cytosol if present in excess) to the mitochondria when the ligand AP21967 is added to the cells (Fig. S8B). More specifically, endogenous GRASPs were replaced by the GRASP55 hybrid protein by using GRASP double-depleted cells that also express an RNAi-resistant form of the GRASP55-FKBP-tagBFP hybrid. To simplify the analysis and focus on the effects of ectopic adhesion on the stack (compared with the ribbon), these cells were treated with nocodazole to produce ministacks before initiating knock-sideways with AP21967.

Electron microscopy was then used to follow the fate of preexisting Golgi stacks and mitochondria (Fig. 4 A–D). The result was a dramatic reorganization of the Golgi stacks within 30–60 min after addition of ligand (2 μM), in which mitochondria cluster around ministacks at earlier times (Fig. 4B) and then invade them at later times, often forming hybrid stacks (Fig. 4 C and D). In some cases, Golgi cisternae appeared to wrap around mitochondria similar to a hot dog bun wrapping around a sausage. The diversity of the resulting hybrid organelles makes it impossible to quantify the results. However, it is nonetheless clear that these results support the idea that established Golgi stacks can be dissected by ectopic adhesion to mitochondria within a few hours. This implies that the stacks had been stabilized by reversible adhesion that could then be overcome at equilibrium by an engineered alternative.

Reagents and Antibodies.

All common reagents were purchased from Sigma-Aldrich unless otherwise mentioned. The following antibodies were used: mouse monoclonal GM130 (BD Transduction Laboratories), goat polyclonal GRASP65 (Santa Cruz Biotechnology), rabbit polyclonal GRASP55 (Proteintech Inc.), rabbit polyclonal p115 (Santa Cruz Biotechnology), HRP-conjugated mouse β-actin antibody (GenScript). Rabbit polyclonal Golgin97 (AbCAM). Rabbit polyclonal Golgin45 antibody was made by injecting synthetic Golgin45 peptide (AA40-53) conjugated to KLH (GenScript). pcDNA-hGRASP55-GFP was kindly provided by Adam Linstedt (Carnegie Mellon University). Rat GM130 cDNA was obtained from Nobuhiro Nakamura (Kyoto Sangyo University). Human Golgin45 cDNA was purchased from Addgene. Human GRASP65 cDNA was from Yanzhuang Wang (University of Michigan, Ann Arbor, MI). Human Golgin97 cDNA was obtained from Sean Munro (MRC Laboratories, Cambridge, United Kingdom).

See SI Materials and Methods for further information.