PE17 alters Golgi stack and trans-Golgi network (TGN) morphology and mass, and inhibits protein secretion
Our findings indicated that PE17 does not localize completely with mitochondria, but rather shows a distinct distribution that does not appear to overlap with major intracellular compartments. As PE17 has been reported to decrease protein secretion in transfected cells (Stammet al. , 2019), however, we hypothesized that PE17 might localize to, or disrupt, an organelle involved in the secretory pathway. To determine if PE17 localized to (or perturbed) the morphology of the endoplasmic reticulum (ER) we performed colocalization experiments with a marker for the ER, calnexin. We found that PE17 positive structures did not strongly colocalize with the ER marker calnexin (Figure 2A). We found that the overall morphology of the ER network was unaltered by expression of PE17 (Figure 2A). To assess any possible changes of ER mass, we analyzed the percent area of the ER in cells expressing PE17 and cells that were not expressing PE17 and found no significant difference (Figure 2B, p>0.05).
As secretory cargo is transported from the ER to the Golgi stack, we also investigated whether PE17 may affect this part of the secretory pathway. Immunolocalisation of the Golgi stack-associated protein, Gpp130, in PE17-transfected cells indicated that, while PE17 did not localize to the Golgi stack, expression of PE17 dramatically altered Golgi stack morphology (Figure 2C). In cells expressing PE17, compact Golgi cisternae were no longer observed, but rather Gpp130 labelling identified a series of short, disconnected tubules. While the Golgi stack is typically located in a perinuclear domain, these short tubules were found throughout the cytoplasm. Additionally, we noticed an overall decrease in the amount of Gpp130 staining in cells expressing PE17 as compared to cells not expressing PE17. Upon evaluating the percent area of Gpp130 staining in both conditions, we found that cells expressing PE17 had significantly less Golgi stack mass (Figure 2D, p<0.0001).
As the TGN receives modified proteins from the Golgi stack, we postulated that the TGN might also be impacted by PE17 expression. As predicted, when PE17 was expressed, we observed similar morphological changes to those observed with the Golgi stack (Figure 2E). While typically compact, the TGN in PE17-expressing cells is fragmented and dispersed throughout the cell with some cells appearing to have lost most of this structure. Notably, the mass of the TGN was significantly decreased in cells expressing PE17 (Figure 2F, p<0.0001).
Although loss of both Golgi stack and TGN would be sufficient to account for a loss of secretion, we also took advantage of a novel characteristic of the type II A549 alveolar epithelial cell line to further analyze this finding. Surfactant protein C is expressed in the lung and is secreted predominantly from type II alveolar cells after undergoing a proteolytic maturation in multivesicular bodies and subsequent delivery to the plasma membrane via exocytosis (Conkrightet al. , 2001). Therefore, we transiently transfected A549 AECs with PE17 and probed for total prosurfactant-C. Analysis of fluorescence intensity in PE17 expressing versus non-expressing cells demonstrated that total intracellular prosurfactant-C staining was significantly brighter in cells expressing PE17 than in cells that were not expressing PE17 (Figure 2G,H)(p<0.0001), suggesting a disruption in secretory traffic from the ER to later compartments along the secretory pathway.
One mechanism of organelle fragmentation is through the disruption of cytoskeletal elements, such as microtubules and actin filaments. Since PE17 expression causes severe changes in mitochondria, Golgi stack, and TGN morphology and distribution, we next sought to determine if these changes were due to disruption in F-actin or microtubule dynamics. Upon PE17-myc expression and labelling with phalloidin to image F-actin and anti-tubulin antibodies to visualize microtubules, we found that there was no difference in the morphology of F-actin or microtubules in cells expressing PE17 and cells that are not expressing PE17 (Figure 3A,B), indicating that the changes in organelle morphology previously observed in PE17-expressing strains are not due to large-scale cytoskeletal disruptions.