A schematic diagram showing the domain organizations of ER hooks and RUSH reporters used in this study.

(1) streptavidin-KDEL (ER hook), (2) SBP-GFP-CD8a-furin-WT, (3) SBP-GFP-CD8a-furin-YA, (4) SBP-GFP-CD8a-furin-AC, (5) SBP-GFP-CD8a-furin-Y+AC, (6) SBP-GFP-CD59, (7) TfR-SBP-GFP, (8) SBP-GFP-E-cadherin, (9) SBP-GFP-collagenX, (10) SBP-GFP-Tac, (11) SBP-GFP-Tac-TC, (12) SBP-GFP, (13) Ii-streptavidin (ER hook), (14) TNFα-SBP-GFP. ss, signal sequence. SBP, streptavidin binding peptide. (1) is the ER hook for RUSH reporters (2-12), while (13) is the ER Hook for the RUSH reporter (14). Key cytosolic amino acid motifs are indicated 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). Y + AC indicates mutations in both sites (Tie et al., 2016).

The intra-Golgi transport kinetics for RUSH Reporters in Golgi ministacks.

HeLa or 293T cells transiently co-expressing individual RUSH reporter and GalT-mCherry were incubated with nocodazole for 3 h. This was followed by a chase with nocodazole, cycloheximide, and 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. t 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. t plot represents the first-order derivative of its corresponding LQ vs. t 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. The LQ vs. t plot in panel G was acquired in 293T cells and analyzed by the Auto-GLIM tool, while the rest are from our previous reports (see Table 1). Panels are arranged according to their RUSH reporters’ tintra means (see Table 1). See Supplementary File 1 for the raw data.

Intra-Golgi transport kinetics of secretory RUSH reporters in Golgi ministacks.

See the legend of Figure 2 for details. #1-4 indicate independent replicates. All data were acquired from HeLa cells except for those labeled with “293T”, which were acquired using 293T cells. 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), respectively. 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 = 0.40) was calculated by Equation 3, and converted to nm/min by multiplying 274 nm per LQ unit. SD, standard deviation.

The intra-Golgi transport of SBP-GFP-collagenX quantified by GLIM and visualized by side averaging.

(A-C) GLIM. HeLa cells transiently co-expressing the RUSH reporter, SBP-GFP-collagenX, and GalT-mCherry were imaged during biotin chase (t) and subjected to GLIM. See Figure 2 legend for details. (D) Side averaging. HeLa cells transiently co-expressing the RUSH reporter, SBP-GFP-collagenX, and GalT-mCherry were incubated with nocodazole for 3 h. This was followed by a chase with nocodazole, cycloheximide, and biotin for variable time durations (t) before fixation and giantin immunostaining. Images were acquired using Airyscan microscopy and subjected to side averaging guided by giantin double puncta. Blue, red, and green horizontal lines represent the center of mass positions of giantin, GalT-mCherry, and SBP-GFP-collagenX, respectively. Scale bar, 200 nm. (E) The LQside vs. biotin chase time (t) plot shows the transition of SBP-GFP-collagenX from the cis to the trans-region of the Golgi ministack. LQside is an approximate metric corresponding to LQ (see Materials and Methods). n, the number of ministacks quantified. Error bar, SEM.

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) GFP-CD8a-TC localizes to the Golgi cisternal interior at the steady state. HeLa cells transiently expressing GFP-CD8a-TC were treated with nocodazole to induce the formation of ministacks before fixation and immunostaining for the endogenous giantin. En face averaging images of giantin and GFP-CD8a-TC are shown on the left. Scale bar, 500 nm. The radial mean intensity profile of en face averaged GFP-CD8a-TC is shown on the right. The x-axis represents the distance from the center of fluorescence mass (normalized to the giantin radius), and the y-axis represents the radial mean intensity (normalized). n, the number of averaged ministacks. (C) 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 on the right. n, the number of cells analyzed. Scale bar, 10 µm.

The organization of Golgi ministacks under the BFA treatment.

(A-E) HeLa cells transiently co-expressing GalT-mCherry and indicated GFP or CD8a-tagged Golgi proteins were incubated with nocodazole for 3 h. This was followed by additional treatment with nocodazole and 5 µM BFA for variable durations before fixation. Endogenous TGN46 was immunostained. Images were acquired by wide-field microscopy. Representative images at 0 and 30 min are shown. Scale bar, 10 µm. (F) LQs of different Golgi proteins were determined using GLIM. The dotted lines represent LQs for GM130 (LQ = 0.00) and GalT-mCherry (LQ = 1.00). n, the number of analyzed Golgi ministacks. (G-H) HeLa cells transiently expressing GalT-mCherry alone (G) or together with CD8a-furin (H) were incubated with nocodazole for 3 h. This was followed by additional treatment with nocodazole and 5 µM BFA for variable durations before immunostaining endogenous giantin or GM130. After Airyscan imaging, images were subjected to side averaging. Horizontal color lines indicate centers of mass of corresponding side-averaged Golgi proteins. d(GM130-GalT-mCherry) and d(GalT-mCherry-CD8a-furin) in nm are plotted against the BFA treatment time in the right panels. n, the number of ministacks quantified. Error bar, SEM. Scale bar, 200 nm.

GLIM and the Auto-GLIM tool.

(A) A diagram that illustrates the principle behind GLIM. In the Golgi ministack, three proteins, GM130 (endogenous, cis-Golgi marker), GalT-mCherry (transfected, trans-Golgi marker), and protein X (transfected or endogenous, Golgi protein of interest) are fluorescently labeled. The centers of fluorescence mass (centers) of the three proteins can be calculated. The Golgi axis is defined as the vector from the center of GM130 to that of GalT-mCherry. The distance from the center of GM130 to that of GalT-mCherry is denoted as d1, while the distance from the center of GM130 to that of protein X is dx. The LQ is then calculated as dx/d1. (B-E) An example that illustrates the study of intra-Golgi transport kinetics using the RUSH reporter (SBP-GFP-Tac) and GLIM. HeLa cells co-expressing SBP-GFP-Tac and GalT-mCherry were treated with nocodazole for 3 h, followed by a biotin chase for various durations. Cells were then immunostained for endogenous GM130 and imaged using wide-field microscopy (B). Scale bar, 10 µm. The image acquired after 20 min biotin chase was selected to demonstrate the image processing for GLIM. The background-subtracted image is displayed in binary intensity to enhance the visualization of ministacks with varying intensities (C). Analyzable ministacks (outlined by yellow contours) were manually selected after intensity thresholding (D). A gallery of 31 analyzable ministacks from (D) and the corresponding LQ histogram (n = 1 cell) are shown in (E) and (F), respectively. The LQ histogram of n = 8 cells is shown in (G). (H-M) Assessing the Auto-GLIM tool. HeLa cells co-expressing GalT-mCherry and SBP-GFP-Tac-TC were treated with nocodzole for 3 h, fixed, and immunostained for endogenous GM130. The image in (H) was acquired by wide-field microscopy. It is subjected to the Auto-GLIM tool with a frame of 72 × 72 pixels to detect the cell contour (marked by the yellow line) (I), conduct background-subtraction (J), and select analyzable Golgi ministacks (marked by yellow lines) (K). Scale bar, 10 µm. The resulting histogram of LQs is shown in (L). We also acquired LQs using the conventional manual method (M), demonstrating strong agreement between the two approaches.

Additional intra-Golgi transport kinetics of RUSH reporters.

LQ vs. t plots in (A-D, G, J, and M-O) are generated in this study while the rest are from our previous reports (see Table 1). See the legend of Figure 2 for details.

Example images used for side averaging in Figure 3D.

See the legend of Figure 3D for details. Scale bar, 10 µm.

Acquiring Golgi residence times of SBP-GFP-CD59 and GFP-CD8a-TC.

(A) HeLa cells were transiently transfected to co-express RUSH reporter, SBP-GFP-CD59, and GalT-mCherry. Cells were incubated with biotin and 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 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. t1/2 is expressed as mean ± SEM. n, the number of quantified cells. Scale bar, 10 µm.

The effect of BFA treatment on Golgi markers.

(A) Arf1 quickly dissociates from the Golgi under the treatment of nocodazole and BFA. HeLa cells transiently expressing Arf1-GFP and ST6GAL1-DMyc were treated with nocodazole for 3 h. This was followed by additional treatment with nocodazole and 5 µM BFA for 5 min before fixation and immunostaining of Myc-tag and endogenous giantin. Images were acquired by Airyscan microscopy. (B) HeLa cells transiently expressing GalT-mCherry were treated with nocodazole for 3 h. This was followed by additional treatment with nocodazole and 5 µM BFA for variable durations before fixation and immunostaining endogenous GS15 and GM130. Images were acquired by spinning disk confocal microscopy. Representative images at 0 and 60 min are shown. Scale bar, 10 µm.