The intra-Golgi transport kinetics for RUSH Reporters in Golgi ministacks. HeLa cells transiently co-expressing individual RUSH reporter and GalT-mCherry were incubated with 33 µM nocodazole for 3 hours. This was followed by a chase with 33 µM nocodazole, 10 µg/ml cycloheximide, and 40 µM biotin for variable time durations (t) before fixation and GM130 immunostaining. Except for TNFα-SBP-GFP, which uses Ii-Streptavidin as the ER hook, all RUSH reporters employ signal sequence fused streptavidin-KDEL as their ER hook. Using GLIM, LQs for the RUSH reporters were then calculated and plotted. Each panel has an LQ vs. time plot on the left, with n (the number of analyzable Golgi ministacks), adj. R2, y0, and tintra indicated. On the right side of each panel, the dLQ/dt vs. time plot represents the first-order derivative of its corresponding LQ vs. time plot on the left. ERES/ERGIC (LQ < −0.25), cis (−0.25 ≤ LQ < 0.25), medial (0.25 ≤ LQ < 0.75), trans-Golgi (0.75 ≤ LQ < 1.25), and TGN (1.25 ≤ LQ) zones were color-shaded. Aside from the one in panel C, all LQ vs. time plots are from our previous reports (see Table 1).

Intra-Golgi transport kinetics of secretory RUSH reporters in Golgi ministacks. See the legend of Figure 1 for details. #1-3 indicate independent replicates. Except for TNFα-SBP-GFP, which uses Ii-Streptavidin as the ER hook, all RUSH reporters employ signal sequence fused streptavidin-KDEL as their ER hook. Superscripts 1 and 2 indicate data were re-analyzed from previous publications (Tie et al., 2016) and (Sun et al., 2020). WT, wild type. tintra (intra-Golgi transport time), y0, and adj. R2 were calculated by fitting measured LQ vs. time kinetics data to Equation 1. dLQ/dt (at LQ = 0.4) was calculated by Equation 3, and converted to nm/min by multiplying 274 nm per LQ unit. For furin cytosolic tail RUSH reporters, YA is YKGL, a tyrosine-based motif, to AKGL mutation, while AC is SDSEEDE, an acidic cluster sequence, to ADAAAAA mutation(Tie et al., 2016). YA + AC indicates mutations in both sites (Tie et al., 2016).

Different secretory cargos exhibit distinct intra-Golgi transport velocities within the same cisternae of Golgi ministacks. (A) Plots of dLQ/dt vs. LQ for selected RUSH reporters reveal diverse intra-Golgi transport velocities at the same cisternae. These plots were constructed based on Equation 3, using parameters from Table 1. Different regions within the Golgi — cis (−0.25 ≤ LQ < 0.25), medial (0.25 ≤ LQ < 0.75), trans-Golgi (0.75 ≤ LQ < 1.25), and TGN (1.25 ≤ LQ) — are color-shaded for easier identification. To convert dLQ/dt to nm/min, we multiplied dLQ/dt by 274 nm per LQ unit. A dotted vertical line marks the LQ value of 0.40. (B) Modifying the classic cisternal progression model to explain the distinct intra-Golgi transport velocities at the same cisternae. According to this model, Golgi cisternae progress at a constant velocity (v). Direct-continuity-based anterograde transport could accelerate the intra-Golgi transport velocity (v + Δv), while the COPI-mediated retrograde transport could reduce it (v - Δv).

Golgi residence times of transmembrane secretory cargos and Golgi glycosyltransferases in native Golgi (without nocodazole). Superscripts 1 and 2 indicate data were from previous publications (Sun et al., 2020) and (Sun et al., 2021), respectively. The RUSH reporter, TNFα-SBP-GFP, employs signal sequence fused streptavidin-KDEL as the ER hook. n, the number of quantified cells; SEM, standard error of the mean.

GFP-Tac-TC and GFP-CD8a-TC might not have a Golgi retrieval mechanism once exiting the Golgi. (A) Domain organization of GFP-tagged Tac, Tac-TC, CD8a, and CD8a-TC. ss, signal sequence. Mem., membrane. t1/2 values are from Table 2 and shown as mean ± SEM. n, the number of cells analyzed. (B) The plasma membrane-localized GFP-Tac-TC and GFP-CD8a-TC are not retrieved to the Golgi. HeLa cells transiently expressing indicated GFP-tagged protein were incubated continuously with VHH-anti-GFP-mCherry for 8 h before fixation and immunostaining for the endogenous Golgi marker, GM130. Images were acquired under a wide-field microscope. The internalized VHH-anti-GFP-mCherry has negative Golgi localization for all GFP-tagged proteins except for MGAT2 (arrows), a positive control. The percentage of cells showing the Golgi localization of VHH-anti-GFP-mCherry is labeled at right. n, the number of cells analyzed. Scale bar, 10 µm.

The organization of Golgi ministacks under the BFA treatment. HeLa cells transiently co-expressing GalT-mCherry and indicated GFP or CD8a-tagged Golgi proteins were incubated with 33 µM nocodazole for 3 hours. This was followed by additional treatment with 33 µM nocodazole and 1 µM BFA for variable durations before fixation. Endogenous GS15 and TGN46 were immunostained. LQs of different Golgi proteins were then determined using GLIM. The dotted lines represent LQs for GM130 (LQ = 0.00) and GalT-mCherry (LQ = 1.00). The total number of analyzed Golgi ministacks is denoted by ‘n’.

Additional intra-Golgi transport kinetics of RUSH reporters. All LQ vs. time plots, except those in panels (A-C), are from our previous reports (see Table I). See Figure 1’s legend for details.

Acquiring Golgi residence times of RUSH SBP-GFP-CD59 and GFP-CD8a-TC. (A) HeLa cells were transiently transfected to co-express RUSH SBP-GFP-CD59 and GalT-mCherry. Cells were incubated with 40 µM biotin and 10 µg/ml cycloheximide at 20 °C to accumulate the RUSH reporter in the Golgi before live-cell imaging under a wide-field microscope at 37 °C. Only RUSH SBP-GFP-CD59 images are shown. (B) HeLa cells transiently co-expressing GFP-CD8a-TC and GalT-mCherry were treated with cycloheximide and imaged live under a wide-field microscope. Only GFP-CD8a-TC images are shown. In both (A) and (B), the total fluorescence intensity within the Golgi was quantified, normalized, and plotted against the time. Each intensity series was fitted to the first-order exponential function to calculate t1/2. Grey and red indicate individual and averaged time series, respectively. CHX, cycloheximide. Error bar, mean ± stand error of the mean. n, the number of quantified cells. Scale bar, 10 µm.

The localization of Arf1 and golgin-84 under the treatment of nocodazole and BFA. (A) HeLa cells transiently expressing Arf1-GFP and ST6GAL1-DMyc were treated with 33 µM nocodazole for 3 hours. This was followed by additional treatment with 33 µM nocodazole and 1 µM BFA for 5 min before fixation and immunostaining of Myc-tag and giantin. Images were acquired by Airyscan microscopy. (B) HeLa cells transiently expressing GalT-mCherry and GFP-golgin-84 were treated with 33 µM nocodazole for 3 hours. This was followed by additional treatment with 33 µM nocodazole and 1 µM BFA for 30 min before fixation and immunostaining for GM130. Images were acquired by wide-field microscopy. Scale bar, 10 µm.

LQ data employed in plots presented in Figure 1, Figure 4, and Supplementary Figure 1. n, the number of quantified cells. SEM, standard error of the mean.