Quantitative Analysis of VSVG–GFP Trafficking
The kinetic properties of VSVG–GFP transport through the secretory pathway were studied using confocal time-lapse imaging techniques in single living cells. COS cells expressing VSVG–GFP at 40°C were shifted to 32°C in the presence of the protein synthesis inhibitor cycloheximide to synchronously release VSVG–GFP from the ER into the secretory pathway. Images were captured every 0.5–2 min for 3–10 h under conditions that minimized both the photobleaching of VSVG–GFP and the differences in its detection efficiency during transport (Materials and Methods).
The changes in subcellular distribution of VSVG–GFP upon shift from 40° to 32°C are shown in Fig. A
(obtained at 0, 40, and 180 min after shift to 32°C; see Quicktime movies for full sequence available at http://dir.nichd.nih.gov/cbmb/pb7labob.html
). VSVG–GFP molecules which were localized to widely dispersed ER membranes at 40°C redistributed into the juxtanuclear Golgi complex within 40 min of shift to 32°C. By 180 min, nearly all the molecules had been exported out of the Golgi complex and delivered to the plasma membrane. Our imaging conditions had the entire cell depth residing within the center of the focal plane (Materials and Methods), so that all fluorescent VSVG–GFP molecules could be detected throughout the time course of the experiment. This population corresponded to ~2 × 107
VSVG–GFP molecules for the cell shown in Fig. A
, calculated by comparison of cellular VSVG–GFP fluorescence with known concentrations of GFP (Materials and Methods).
To study quantitatively the changes in distribution of VSVG–GFP during its transport to the cell surface, fluorescent intensities within ROI containing the juxtanuclear Golgi compartment and the entire cell (Fig. B, areas enclosed by yellow and blue lines, respectively) were measured and plotted for each digital image acquired during the experiment. As shown for a single cell in the graph in Fig. C, Golgi-associated fluorescence rose sharply within the first 5–30 min after the temperature shift. It peaked at ~40 min with ~2.5 × 106 VSVG–GFP molecules in the Golgi complex, and then steadily declined over the next 100 min. The time course of distribution of Golgi fluorescent intensities among nine other cells expressing 1.4– 2.0 × 107 molecules of VSVG–GFP (Fig. D) indicated that different cells exhibit similar VSVG–GFP transport kinetics.
Loss of total cellular fluorescence in these experiments was not the result of photobleaching from repetitive scanning. The total fluorescence over time remained constant when using identical imaging conditions with fixed cells or with cells treated with brefeldin A to retain VSVG–GFP in the ER (data not shown). A potential explanation for the fluorescence loss therefore is cellular degradation of VSVG–GFP after delivery to the plasma membrane.
Modeling of VSVG–GFP Kinetics
We examined whether the kinetic data obtained above could be fit to a simple model for VSVG–GFP transport (Fig. A), consisting of three compartments, ER, Golgi, and plasma membrane, arranged in series. In this model VSVG–GFP is transported out of each compartment into the succeeding one at a rate equal to a rate constant (i.e., KER, KG, and KPM) multiplied by the amount of VSVG– GFP in the originating compartment. Simple linear first order kinetics through a series of homogeneous compartments with no backflow is assumed. Total VSVG–GFP fluorescence is conserved at each transfer step, until VSVG–GFP is internalized from the plasma membrane and is degraded.
To see whether the model could account for the data, and if so, to extract rate parameters, we used generalized least squares optimization to simultaneously fit to the model Golgi and total fluorescence intensities with time for each of 32 cells (Materials and Methods). Fig. B shows an optimal fit for a typical cell imaged for 10 h after a shift to permissive temperature. Solid and empty circles are the experimental data points for Golgi and total fluorescence, respectively. The two lines represent the corresponding model solution. In fitting the model to the data we assumed that all VSVG–GFP was initially in the ER at the time of temperature shift from 40° to 32°C. The Golgi fluorescent intensity values are nonzero initially because a small fraction of the ER overlaps the Golgi ROI. Similarly, at late times the Golgi fluorescent intensity values are nonzero, because a fraction of the plasma membrane falls within the Golgi ROI. This overlap was accounted for by fitting the measured Golgi fluorescent intensity values against the Golgi compartment plus small contributions from ER and plasma membrane.
The levels of VSVG–GFP in each of the three model compartments is shown in Fig. C as calculated by the fit in Fig. B. Note that arrival of VSVG–GFP molecules on the plasma membrane occurred before all of the VSVG– GFP had left the ER. Indeed, more than 100 min were required to completely empty the ER, whereas the Golgi complex was not completely emptied until 200 min after a shift to the permissive temperature. The final slope of the total and plasma membrane curves represents the loss of VSVG–GFP after its delivery to the plasma membrane.
On a cell by cell basis, the model fits of the data were very good. The average coefficient of variation for the effective ER rate constant, KER, was only 2.13% (over all cells). For the Golgi rate constant, KG, it was 2.46%, whereas for the plasma membrane rate constant, KPM, it was 5.01%. Coefficients of variation for the fluorescence sampling efficiencies were similarly low; all were less than 5%. This means that for each individual cell, the three rate constants and three sampling efficiencies were readily and accurately determined.
Table summarizes the mean rate constants (± standard error) from the model solutions obtained from analysis of the 32 cells (in five separate experiments). The mean rate constant for VSVG–GFP export out of the ER (KER) was 2.84 ± 0.20% per minute, whereas export out of the Golgi complex (KG) was 3.03 ± 0.26% per minute. Degradation (KPM) of VSVG–GFP occurred at a rate of 0.25 ± 0.03% per minute from plasma membrane-derived membranes.
To illustrate the sensitivity of the fit to changes in rate constants, curves of simulated data are shown in Fig. D where we increased either KER (red curve) or KG (blue curve) by a factor of 1.8 and allowed the remaining parameters to readjust. The discrepancies in these fits to the experimental data are substantial. For comparison, the black lines show the optimized fit. The standard error in Table and Fig. D, therefore, reflects cell-to-cell variability, rather than the error in any one fit.
The average time spent by a single VSVG–GFP molecule in the ER, Golgi, or plasma membrane (i.e., mean residence time) as it moved through the secretory pathway was obtained by inverting the optimized rate constants cell by cell and then averaging (Table ). We found that a VSVG–GFP molecule spends on average about the same time in the Golgi complex as in the ER compartment; mean residence time was ~40 min for each. In contrast, mean residence time in the plasma membrane and its associated membranes before degradation was ~700 min.
The data fit also allowed us to estimate the fluxes between compartments. As shown in Fig E, for a cell containing ~2 × 107 VSVG–GFP molecules, the peak ER-to-Golgi flux was 7,000 molecules/s (15–20 min after shifting to permissive temperature), whereas the peak Golgi-to-plasma membrane flux was 4,000 molecules/s at 40–45 min. The peak flux of degradation from the plasma membrane and its associated membranes was 700 molecule/s at a time of 125–150 min.
The accuracy of the linear kinetic model in fitting the data is a preliminary indication that in our experiments, no rate-limiting transport step is saturated by the expression levels of VSVG–GFP. To ask more quantitatively whether we were saturating any rate-limiting transport step with high expression levels of VSVG–GFP, we enlarged the model to include standard Michaelis–Menten kinetics (Materials and Methods). The Michaelis constants, Km, obtained from the fits for both the ER and Golgi steps, were 10–100 times the maximum levels of VSVG–GFP expression in our cells. This meant that the processes underlying VSVG–GFP trafficking in our experiments were operating on the linear portion of their Michaelis–Menten curves. Thus, no effects of saturation occurred even though each step of the secretory pathway was confronted with a wide range of VSVG–GFP levels (i.e., from 2 × 107 at the start of imaging to nearly zero by the end of the experiment). We also examined a different population of cells incubated for shorter periods of time at 40°C (4–5 compared with 20 h), resulting in lower levels of VSVG–GFP expression (i.e., 2–5 × 106 VSVG–GFP molecules/cell). The linear model was again sufficient to fit the data. The rate constants increased somewhat (i.e., 1.4-fold) (see Discussion), but the quality of the fits to linear kinetics were comparable to those shown above. Thus, using the currently available data no greater complexity was required than the simple linear model of Fig. A and its first order rate constants, KER, KG, and KPM to characterize VSVG–GFP trafficking in our cells.
Perturbants of VSVG–GFP Transport
The above methodology was used to analyze the effect on VSVG–GFP transport of different pharmalocogical reagents that have been reported to interfere with secretory protein trafficking. These reagents included: cytochalasin B (cyto B), which disrupts the actin cytoskeleton and thereby blocks membrane transport along actin filaments; aluminum fluoride, which causes persistent activation of heterotrimeric G proteins (Gilman, 1987
), and induces binding of peripheral coat proteins to Golgi membranes (Melancon et al., 1987
; Barr et al., 1991
; Donaldson et al., 1991
; Robinson and Kreis, 1991; Bomsel and Mostov, 1992
; Ktistakis et al., 1992
); and nocodazole, which interferes with microtubule polymerization, blocking microtubule-dependent translocation of membrane transport intermediates (Rogalski et al., 1984; Presley et al., 1997
In cells treated with cyto B to depolymerize actin, changes in Golgi fluorescence intensities over time after shift to permissive temperature could be effectively fit to the model of Fig. A (see Fig. A). However, the fits required different values of KER, KG, and KPM compared with untreated cells; KER was 1.2-fold lower, KG was 1.6-fold lower, and KPM was 2.4-fold higher (Table ). The calculated mean residence time for VSVG–GFP was 46 min in the ER, 57 in the Golgi complex, and 179 on the plasma membrane. For comparison, the times in untreated cells were 39, 42, and 709 min, respectively (Table ).
Changes in VSVG–GFP levels in ER, Golgi, and plasma membranes as a function of time in cyto B and untreated cells calculated from the model fits are shown in Fig. B (dashed curves, cyto B-treated cells; solid curves, untreated cells). Cyto B treatment only slightly slowed export of VSVG–GFP out of the ER compartment system. The effect on Golgi egress, however, was significant with VSVG–GFP requiring 300 instead of 200 min to completely empty out of the Golgi complex. VSVG–GFP also spent a much shorter time in plasma membrane-derived membranes before being degraded. This data suggests that cyto B affects the processes whereby VSVG–GFP is exported out of the Golgi complex, as well as trafficking events at the plasma membrane.
The effects of AlF and nocodazole treatment on Golgi egress of VSVG–GFP were also examined (Fig. C). In these experiments, AlF and nocodazole were added 40 min after a shift to permissive temperature to allow VSVG–GFP to accumulate in the Golgi complex before addition of the drug. VSVG–GFP was not exported out of the Golgi system and remained concentrated in centralized Golgi membranes in AlF-treated cells. By contrast, nocodazole treatment had very little effect on Golgi to plasma membrane trafficking of VSVG–GFP. These results are shown quantitatively in Fig. D, which plots changes in Golgi-associated fluorescence due to export to the plasma membrane in AlF- and nocodazole-treated cells, as well as in untreated cells, as a function of time after addition of the drugs.
Morphological Analysis of Post-Golgi Transport: Budding from the Golgi Complex
To visualize post-Golgi trafficking of VSVG–GFP, confocal images of cells were collected at short time intervals and at high magnification when VSVG–GFP flux out of the Golgi complex was greatest (i.e., after 50 min of shift from 40° to 32°C as shown in Fig. E). The time-lapse sequences in Fig. A show VSVG-GFP–containing membranes pulling off from the Golgi complex as tubular processes that extended several microns in length. VSVG–GFP was accumulated at the tips of these tubules, appearing as a ball-like mass. After a variable time the enlarged tip regions detached and moved outward as separate post-Golgi elements, whereas the remaining membrane stalk retracted back to the Golgi body (Fig A, arrows). Tubule growth and detachment occurred repeatedly and appeared to be an important mechanism for the Golgi export of VSVG–GFP. The Golgi-derived tubules were seen in cells expressing either high or low levels of VSVG–GFP and were also observed in HeLa, NRK, CHO, primary rat glial astrocytes, and MDCK cells. In all cases, VSVG-GFP– containing tubule membranes pulled off as entire domains from the Golgi complex, detached, and then subsequently moved to the cell periphery.
Figure 4 Budding of post-Golgi carriers from the Golgi complex. (A) Cells expressing VSVG–GFP at 40°C were shifted to 32°C. The sequence shows the Golgi region after 50 min at 32°C. The images were taken at 2.6-s intervals with (more ...)
To further characterize the membrane tubules involved in Golgi export of VSVG–GFP, double-labeling experiments were performed with antibodies to resident proteins of the Golgi and TGN (Fig. B
). These included: the coat proteins β-COP (COP-I) (Pepperkok et al., 1993
) and AP1 (Stamnes and Rothman, 1993
), which cycle on and off Golgi membranes and potentially regulate budding and sorting events (Kreis, 1992); GM130, a Golgi matrix protein (Nakamura et al., 1995
); and furin, a proteolytic enzyme that cycles between the cell surface, endosomes and TGN (Bosshart et al., 1994). Although significant overlap in the distribution of these proteins and VSVG–GFP occurred in the Golgi body (Fig. B, yellow
from overlap in the merged images of red
antibody labeling and green VSVG–GFP), no colocalization was detected in VSVG-GFP–containing tubule elements extending out or detached from the Golgi complex (arrows
). These findings suggest that VSVG–GFP is actively sorted into discrete domains of the Golgi complex during export, consistent with electron microscopic studies showing a nonuniform distribution of proteins at the TGN (Ladinsky et al., 1994
; Geuze et al., 1987
; Narula and Stow, 1995
; Ikonen et al., 1996
). Such domains elongate as tubules before detaching from the Golgi body as diagrammed schematically in Fig. C
and do not contain the known coat proteins AP1 or β-COP.
Size and Dynamics of Post-Golgi Carriers
After detaching from the Golgi complex, tubule elements containing VSVG–GFP typically underwent dramatic shape changes as they translocated to the cell periphery. In confocal sections captured at high speed they could be seen to bifurcate (Fig. A
, short arrow
, a bifurcating element still attached to the Golgi, see also Quicktime movie available at http://dir.nichd.nih.gov/cbmb/pb7labob.html
), and they showed elastic properties, including extension and retraction during movement (Fig. A
, long arrows
). Tubules sometimes broke in half when one region was pulled through the cytoplasm whereas another region was held behind (Fig. A
). They also often rounded up into spherical shapes. These dynamic properties yielded a population of post-Golgi carriers (PGCs) with diverse morphologies (Fig. B
) that could be observed at both high and low VSVG–GFP expression levels. When tubules extended off the spherical body of a PGC, they usually pointed in the direction of movement as if they were being pulled. Under these conditions, the spherical domain (i.e., varicosity) often changed position along the PGC length (see Quicktime movie sequence at http://dir.nichd.nih.gov/cbmb/pb7labob.html
for Fig. C
). Similar looking spherical domains have been found associated with fluorescently labeled tubular endosomes imaged in living cells (Hopkins et al., 1990
), suggesting they are a common feature of tubule transport intermediates.
Figure 5 Structure and dynamics of post-Golgi carriers. (A) Inverted images of a VSVG-GFP–expressing cell 50 min after shift from 40° to 32°C. Images were captured every 2 s at high magnification using a confocal microscope. Short (more ...)
PGCs were large and carried significant amounts of VSVG–GFP cargo. An average-sized PGC occupied an area of 1.3 μm2 corresponding to 32 pixels (with each pixel 0.2 × 0.2 μm), as shown in Fig. D. For comparison, a 100-nm fluorescent bead shown at the same magnification and imaging conditions occupied a single bright pixel (Fig. D, inset). Based on the measured conversion factor between fluorescence and number of VSVG–GFP molecules (Materials and Methods), ~10,000 VSVG–GFP molecules were contained within an average-sized PGC at peak VSVG–GFP flux out of the Golgi in a cell expressing in the order of 2 × 107 VSVG–GFP molecules. A 100-nm vesicle at an equal surface density as found in the PGCs, by comparison, would carry no more than 100 VSVG– GFP molecules.
Microtubule-dependent Translocation of PGCs
The path and velocity of PGCs were analyzed from time-lapse sequences captured with a video camera system between 60 and 90 min after temperature shift to 32°C in VSVG-GFP–expressing cells. At this time numerous, fluorescently labeled PGCs were seen moving out from the Golgi region. The movement of two such structures taken from images captured with a high speed SIT camera is plotted in Fig. A
, revealing their saltatory motion and velocities of up to 2.7 μm/s. PGCs usually moved along straight or curvilinear tracks toward the cell periphery, although sometimes they reversed directions (Fig. C
, red arrows
, paths of three PGCs in an untreated cell). The rapid outward movement of PGCs is illustrated in Fig. B
where eight images, each 10 s apart, are overlaid. The position of one representative PGC is highlighted over time by boxes. Over 70 s, this PGC moved more than 60 μm from the central Golgi region to the tip of a plasma membrane extension (see Fig. B
, see also Quicktime movie at http://dir.nichd.nih.gov/cbmb/pb7labob.html
Figure 6 Translocation of PGCs. (A) Velocity of two PGCs (red and black). Images were collected with a SIT camera at a rate of 8 images/s and the velocity measured as the distance between the pixel coordinates in consecutive images. The graph shows a trend (more ...)
Microtubules were responsible for the directed transport of PGCs. This was demonstrated in experiments where VSVG-GFP–expressing cells were treated with nocodazole after PGCs had formed and moved out of the Golgi region (i.e., 60 min after a temperature shift to 32°C). Under these conditions PGCs stopped their movement and remained stationary in the cell periphery (Fig. C, nocodazole-treated cell). This contrasted with the effects of cyto B– and AlF treatment. Neither drug blocked translocation of PGCs through the cytoplasm (Fig. C, cyto B– and AlF-treated cells), even though both drugs affected the formation of PGCs from Golgi membranes (Fig. ). In nocodazole-treated cells, VSVG–GFP was delivered to the plasma membrane over time despite the lack of directed movement of PGCs (Fig. ). This may occur by random diffusion to and fusion of PGCs to nearby top or bottom cell surfaces, since COS cells viewed in reconstructed cross-section (Fig. D) revealed these surfaces were less than 1 μ apart in many areas. A likely role for microtubule dependent transport of PGCs, therefore, is in delivery of PGCs to specific domains of the plasma membrane.
Quantitation of VSVG–GFP Delivery to the Cell Surface by PGCs
Although a single PGC could carry up to 10,000 VSVG– GFP molecules, we wanted to quantify the overall contribution to post-Golgi trafficking made by these large structures. In particular, we wanted to know what fraction of the total Golgi to plasma membrane transport of VSVG–GFP is by PGCs, rather than by some alternate pathway (for example, by small 100-nm-diam vesicles). We approached this question by applying kinetic analysis of the precursor–product relationship of PGC fluorescence and plasma membrane fluorescence. First we quantified the time course of accumulation of VSVG–GFP fluorescence on the plasma membrane, and then compared it with the rate of appearance of PGCs containing VSVG– GFP and their fusion with the plasma membrane. This made it possible to determine the fraction of the product (plasma membrane fluorescence) that is contributed by the measured precursor (PGC fluorescence).
Using the imaging tools described in Materials and Methods, we designed a protocol in which VSVG–GFP was first allowed to transit through the secretory pathway for 40 min, accumulating substantial fluorescence in the Golgi complex. The entire cell excluding a strip containing the Golgi region was then subjected to repetitive photobleaching to remove fluorescence in all areas outside the Golgi complex (Fig. A
, see Quicktime movie at http: //dir.nichd.nih.gov/cbmb/pb7labob.html
). High-resolution digital images of the entire cell were then collected every 17 s for ~30 min to characterize VSVG–GFP transport out of the Golgi complex and into the photobleached area. The aim of this procedure was to measure the time course for appearance in the photobleached region of both total VSVG–GFP and VSVG–GFP contained within PGCs. These two measurements could then be used for precursor–product analysis of VSVG–GFP delivery to the plasma membrane. For data analysis, we used an ROI made up of the most peripheral half of one of the bleached areas (Fig. A
, ROI). PGCs were defined operationally as fluorescent structures having a projection area larger then 0.2 μm2
(corresponding to a 0.45 × 0.45-μm square box).
A kinetic model of Golgi-to-plasma membrane trafficking was constructed which included two independent pathways of delivery of VSVG–GFP into the bleached ROI (Fig. C). One pathway used PGCs that translocated into the ROI and then fused with the plasma membrane. The other pathway encompassed all other routes, including small vesicles that might translocate into the ROI and fuse, as well as lateral diffusion within the plasma membrane of VSVG–GFP that was delivered to the plasma membrane outside the ROI. Because the experiment provided time-course data on the PGC fluorescence (the precursor), we could determine the minimum contribution made by PGCs to Golgi to plasma membrane trafficking by examining the fraction of total plasma membrane fluorescence that could be accounted for using the measured PGCs as precursor.
The data to be fitted included total VSVG–GFP fluorescence and VSVG–GFP fluorescence associated with PGCs in the ROI as a function of time after the photobleach (Fig. D
and solid circles
, respectively). Total fluorescence continually increased over time, whereas fluorescence associated with PGCs reached a pseudo steady state due to new PGCs arriving in the ROI at approximately the same rate as others fused with the plasma membrane (Fig. B
, see Quicktime movie at http://dir.nichd.nih.gov/cbmb/pb7labob.html
). The slight decline in PGC fluorescence seen reflected the slow rate of Golgi emptying. Fitting simultaneously total and PGC fluorescence as a function of time using least squares optimization yielded an optimal fit (Fig. D
, smooth lines
) when 61.5% of the VSVG–GFP was transported via PGCs and 38.5% was transported via the other pathway. The coefficients of variation on these estimates were 5.2% respectively, yielding 95% confidence limits of 57.2–65.8% for the PGC contribution. Thus, the majority of VSVG–GFP trafficking to the plasma membrane occurred via PGCs. Interestingly, the fit also required very rapid transport in the other pathway (i.e., 17 times faster than the rate for PGC translocation and fusion). A possible source of this rapid transport is lateral diffusion of VSVG–GFP into the ROI from plasma membrane outside the ROI where other PGCs might have fused. The fact that VSVG–GFP is highly mobile in the plasma membrane (see Fig. ) is consistent with this idea. Thus, our results of 61% for the PGC pathway should be viewed as a minimum estimate.
Figure 8 Fusion and dispersion of VSVG–GFP at the plasma membrane. (A) Inverted images of VSVG-GFP–expressing cell collected with a SIT video camera at a rate of 8 images/s ~50 min after shift from 40° to 32°C. The sequence (more ...)
Lifetime and Fusion of PGCs with the Plasma Membrane
The average lifetime of a PGC before fusing with the plasma membrane was calculated to be 3.8 min based on the PGC kinetic modeling data above. During this period PGCs showed no overlap with endosomal structures labeled with Texas red-conjugated transferrin, or with structures labeled with antibodies to β-COP, or with the adaptor complexes, AP1, AP2, or AP3 (data not shown). This indicated that the PGCs did not intersect with any other compartments before delivering their cargo to the cell surface.
Fusion of PGCs with the plasma membrane was studied using a SIT camera system to continuously collect images. Fig A shows a video sequence taken at 8 frames/s of a single PGC (black arrows) moving toward the edge of the cell, remaining stationary for 14 s, and then rapidly dispersing its fluorescence in the plasma membrane. The fact that fluorescence could be seen spreading across the plasma membrane as it was lost from the PGC indicated VSVG–GFP was highly mobile in the plasma membrane and that fusion was occurring, rather than movement out of the focal plane. A different PGC (Fig. A, white arrow) that did not fuse with the plasma membrane remained visible over this period. The rate of dispersion of VSVG– GFP fluorescence upon fusion with the plasma membrane is shown quantitatively for this experiment in Fig. B. Dispersal of PGC fluorescence occurred in ~2 s (Fig. A, solid circles). For comparison, the PGC identified by the white arrow in Fig. A, showed no change in fluorescent intensity (open circles). An example of an irregularly shaped PGC fusing with the plasma membrane is shown in Fig. C. Note that the entire structure rapidly delivered its cargo to the plasma membrane, indicating that a single, continuous structure was involved.