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. 2023 Jan 10;24(2):1375. doi: 10.3390/ijms24021375

Figure 5.

Figure 5

There is no equilibration of cargos across Golgi cisternae during the outgoing transport wave. (A–D; quantified in [E]) HeLa cells transfected with VFP were incubated at 40 °C for 3 h. Next, cells were placed at 32 ° for 30 min, returned at 40 ° (transport block), and examined after 4, 6, 8, and 12 min. VFP was detected using antibody against luminal portion of VSVG and biochemical reaction based on the application of the secondary antibody conjugated with HRP and their subsequent incubation with DAB. Time points are indicated on images. White arrows show the empty Golgi cisternae. (E) Dynamics of the emptying of the GC from VFP. After 7 and 12 min, the number of the DAB-negative Golgi cisternae is significantly (p < 0.05) higher than before the outgoing wave (StSt). (F–H) Emptying of the GC filled with AFP at steady state (quantified in Q). The delivery of AFP was blocked with CHM. AFP was detected using antibody against GFP and biochemical reaction based on the application of the secondary antibody conjugated with HRP and their subsequent incubation with DAB. Time points are indicated on images. (I,J) Human fibroblasts transfected with PFP were subjected to the ER accumulation–chase protocol (quantified in [P]). After 30 min, the GC was completely filled with PFP-positive distensions. Then the ascorbic acid was eliminated, and cells were placed at 40 °C in order to block the delivery of new portions of PFP at the Golgi complex. (I) Before this shift (0 min), all Golgi cisternae contained PFP distensions. (J) After 15 min, PFP distension was observed only within TGN, and no distensions were visible within the cisternae. Arrow indicates the post-Golgi carrier filled with PFP. (K–O) The outgoing wave for VFP examined on the basis of cryosections (quantified in [R]). (K) Starting point when VFP is present on all cisternae. (L) Already after 4 min, a few Golgi cisternae at the cis-side of the stack did not contain VFP. Arrows indicate labelling for ERGIC53. (M) After 6 min, a significant portion of Golgi cisternae do not contain VFP. After 8 (N) and 12 min (O), VFP was present only at the trans-side of the stack or in TMC labeled for TGN46 (arrows in [O]). No equilibration of VFP within different Golgi cisternae. (P,Q) Dynamics of the emptying of Golgi cisternae after synchronization of PFP (P) and AFP (Q) correspondingly according to the outgoing wave protocol. After 7 min and 12 min, the number of the DAB-negative (see [F–H]) Golgi cisternae is significantly (p < 0.05) higher than before the outgoing wave (0’). Similarly, the number of cisternae not containing PFP-positive distensions is significantly (p < 0.05) higher than before the outgoing wave (0’). (R) Initially, the GC was filled. Then the delivery of VFP was blocked, and the labeling density of VFP was measured on Golgi cisternae after different times (see graph). Initially all cisternae were filled with VFP. After 6 min, the medial cisternae at the cis-side of the stack became empty: their labeling density became significantly (p < 0.05) lower than at times 0’ and 2’. Scale bars: 200 nm (A–D,F–H); 420 nm (K); 320 nm (L–O).