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(The FASEB Journal. 1999;13:S251-S256.)
© 1999 FASEB

Secretory pathway kinetics and in vivo analysis of protein traffic from the Golgi complex to the cell surface

KORET HIRSCHBERG and JENNIFER LIPPINCOTT-SCHWARTZ

Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

1Correspondence: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 18T, Room 101, 18 Library Dr., Bethesda, MD 20892, USA. E-mail: jlippin{at}helix.nih.gov


   INTRODUCTION
TOP
 INTRODUCTION
GOLGI-TO-PLASMA MEMBRANE...
 QUANTITATIVE ANALYSIS AND...
 REFERENCES
 
EUKARYOTICCELLS, FROM yeast to human, have the ability to synthesize and secrete highly processed and complex molecules. This enables them to exquisitely modify their cell surface and extracellular space, and thereby to respond to stimuli in their environment and to communicate with other cells. This property depends on the correct functioning of the secretory pathway, which is comprised of distinct membrane-bound compartments (including the endoplasmic reticulum [ER], Golgi complex and plasma membrane) and vesicle/tubule transport intermediates. Newly synthesized protein and lipid enter this pathway in the ER and are transported to the Golgi complex for further processing and maturation by glycolipid and glycoprotein trimming enzymes. On reaching the trans-Golgi network, they are sorted and packaged into post-Golgi transport intermediates that deliver them to the plasma membrane.

Cell biologists have made significant progress in elucidating the biochemical requirements for secretory transport and the role of transport intermediates (1 , 2) . Less understood are the morphological and kinetic properties of secretory traffic, including how transport intermediates are formed and their path and fate during transport. In addition, it is not clear how long cargo resides in a particular compartment and the rate of cargo influx and efflux out of a given compartment. This lack of understanding is largely because of the inherent limitations of traditional approaches. For example, biochemical assays using either in vitro or permeabilized systems (3 , 4) often have difficulty identifying and/or characterizing intermediate steps because the amount of cargo carried by transport intermediates within the secretory pathway is usually quite small. Immunofluorescence and electron microscopy approaches, which can be used to identify carriers, provide only static snapshots of cells and therefore are unable to reveal the origin/fate of transport intermediates and their dynamic properties.

A recent technique that overcomes many of the limitations inherent in studies of in vitro or fixed specimens exploits the intrinsic fluorescence of the green fluorescent protein (GFP) as a tag for the production of fusion proteins (5) . GFP fusion proteins are easily expressed and visualized within living cells. They are remarkably photostable to repetitive imaging using low illumination levels and, in many cases, do not perturb normal trafficking or function of the parent protein (6 , 7) . They also can be quantitated by correlating their fluorescence to a known standard solution of GFP molecules (8 , 9) . It is not surprising, therefore, that GFP fusion proteins have been used as fluorescent reporters in a wide variety of applications in living cells, including time-lapse imaging studies to follow changes in protein distribution (10 , 11) , quantitative imaging experiments to measure the amount of protein localized at a particular site (12 , 13) , and biophysical assays such as photobleaching to measure protein mobility (14) .

To study protein trafficking through the secretory pathway, we have used a fusion protein where GFP was attached to the temperature sensitive protein known as ts045 VSVG from vesicular stomatitis virus (11) . The ts045 VSVG protein is a type I transmembrane protein that has been widely used for studying secretory membrane traffic because it contains a mutation that leads to its reversible misfolding and retention in the ER at 40°C (15 16 17 18 19) . On temperature reduction (e.g., to 32°C) the protein folds correctly and is exported out of the ER and through the secretory pathway as a synchronous pool. This property makes ts045 VSVG an ideal tool for following protein traffic within cells. Addition of the GFP tag to the cytoplasmic tail of ts045 VSVG did not alter either the folding or transport properties of this protein (11 , 19) . Recently, GFP-tagged ts045 VSVG (VSVG-GFP) was used to visualize ER-to-Golgi traffic in living cells (11 , 19) . Release from the 40°C temperature block by shifting to 32°C resulted in the ER-localized chimera first concentrating in large, pleiomorphic pre-Golgi structures (known as the intermediate compartment) scattered throughout the cytoplasm. These structures, rather than small vesicles, then translocated as units along microtubules to the Golgi complex. Because these studies revealed that pre-Golgi structures are large, complex structures that deliver cargo directly to the Golgi complex, they have provided valuable insight into the functioning of the early secretory pathway.

Here, we describe results using VSVG-GFP to characterize the transport intermediates involved in membrane traffic from the Golgi complex to the plasma membrane (13) . We address how such structures bud off from the Golgi complex, how they translocate through the cytoplasm, and how they fuse with the plasma membrane. We also present findings from kinetic modeling of VSVG-GFP transport data that reveal how long VSVG-GFP cargo resides within the Golgi complex and its rate of influx and efflux out of this compartment (13) .


   GOLGI-TO-PLASMA MEMBRANE TRANSPORT OF VSVG-GFP
TOP
 INTRODUCTION
 GOLGI-TO-PLASMA MEMBRANE...
QUANTITATIVE ANALYSIS AND...
 REFERENCES
 
Little is known regarding the properties of Golgi-to-plasma membrane transport intermediates because of the difficulty in identifying these structures within cells. However, it has generally been assumed that they are small 60–100 nm vesicles. To study Golgi-to-plasma membrane transport intermediates in living cells, confocal images of cells expressing VSVG-GFP were collected at short time intervals and at high magnification when VSVG-GFP transport out of the Golgi complex was greatest (i.e., after 50 min of shift from 40 to 32°C) (13) . The time-lapse sequences showed VSVG-GFP-containing membranes pulling off from the Golgi complex as tubular processes that extended several microns in length (Fig. 1A ). VSVG-GFP was often concentrated in a globular domain at the tips of these tubules. After a variable time, the enlarged globular region detached and moved outward as a separate post-Golgi element, while the remaining membrane stalk retracted back to the Golgi body.



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  		Figure 1. VSVG-GFP trafficking through the secretory pathway. 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 with a narrow pinhole on a confocal microscope. Inverted images are displayed at the indicated time intervals. Arrows point to a tubule budding off from the Golgi body and its tip region detaching. Bar = 5 µM. B) VSVG-GFP expressing cells were incubated for 20 h at 40°C, shifted to 32°C, and imaged every 30 s for 3 h using a confocal microscope. Shown are inverted images at 0, 40, and 180 min after temperature shift. Overlaid are the regions of interest (ROIs) used for quantitative kinetic analysis of transport. Outer line represents total cell fluorescence; inner line represents Golgi fluorescence. C) The three compartment model for trafficking of VSVG-GFP found to be sufficient for accurate simulation of the experimental data and used for extracting kinetic parameters. D) VSVG-GFP expressing cells incubated for 20 h at 40°C were shifted to 32°C and imaged every 120 s for 10 h. The fluorescent intensities with time within the Golgi ROI (filled circles) and total cell ROI (empty circles) are plotted. Lines are the simulated data generated using the model in panel C. E) Profile of passage of VSVG-GFP moving through each compartment. VSVG-GFP levels in ER, Golgi, and plasma membrane were calculated by the fit shown in panel C.

The Golgi-derived tubules were not an artifact of VSVG-GFP expression, because they were observed at both high and low expression levels of VSVG-GFP and in diverse cell types. Indeed, previous time-lapse imaging studies of living cells labeled with NBD-ceramide have revealed numerous tubule processes extending out of Golgi elements (20) , while electron microscopy studies have commonly found tubules emerging out of the trans face of the Golgi complex (21 22 23) . These observations, together with our own, suggest that tubule growth and detachment are an important mechanism for membrane export from the Golgi complex.

To further characterize the membrane tubules involved in Golgi export of VSVG-GFP, double-labeling experiments were performed with antibodies to peripheral membrane proteins of the Golgi and trans-Golgi network (TGN). These included the coat protein, ß-COP (24) , and the adaptor complexes, AP1 and AP3 (25) . No colocalization of these proteins with VSVG-GFP-containing tubule elements was observed even though significant overlap in the distribution of these proteins and VSVG-GFP occurred in the Golgi body. This suggested that these proteins are not directly involved in the formation of VSVG-GFP-containing tubules. Whether or not other peripheral protein complexes will be identified that regulate the formation and/or function of these tubules remains to be elucidated. However, alternative mechanisms for the budding of these structures to those based simply on coat recruitment and envelopment are possible. Recent ideas involving lipid based sorting at the TGN (26) provide a potential explanation for how sorting of VSVG-GFP into specific membrane domains might be coupled to tubule budding.

Factors underlying the formation and detachment of Golgi tubules containing VSVG-GFP were investigated in cells treated with nocodazole, cytochalasin-B (cyto-B), or aluminum fluoride (AlF) (13) . Depolymerization of microtubules with nocodazole did not significantly affect export of VSVG-GFP from the Golgi complex. This implied that the tubules involved in Golgi export do not require microtubules to form or detach from the Golgi complex (although microtubules are important for the peripheral extension and translocation of these structures, as discussed below). Treatment of cells with cyto-B, which depolymerizes the actin cytoskeleton, in contrast, led to a delay of VSVG-GFP egress from the Golgi complex. This implied that actin-based cytoskeletal elements facilitate protein export from the Golgi complex as suggested previously (27 , 28) . The fact that Golgi-derived tubules that formed in cyto-B-treated cells were significantly longer than in untreated cells further suggested that actin-based elements could be involved in the detachment of these transport intermediates. Treatment of cells with AlF, which persistently activates heterotrimeric G proteins, abolished all export of VSVG-GFP from the Golgi complex. This suggested that AlF-sensitive components underlie Golgi protein export.

After detaching from the Golgi complex, tubule elements containing VSVG-GFP typically moved out to the cell periphery and underwent dramatic shape changes during transport. In confocal sections captured at high speed, we observed these structures bifurcating, extending, and retracting during movement. Their motion was saltatory and occurred at maximal speeds of 2.7 µM/s. Treatment of cells with nocodazole inhibited this movement, suggesting that the post-Golgi carriers (PGCs) moved along microtubule tracks. These findings are similar to those obtained from other studies examining transport of secretory cargo to the cell surface (10 , 29) . In those studies, as well as our own, the net movement of post-Golgi structures was outward from the Golgi region to the cell periphery. This suggested the involvement of a plus end-directed microtubule motor protein (e.g., kinesin-like).

We found that PGCs enriched in VSVG-GFP could deliver their cargo to the cell surface in the absence of microtubules. A likely explanation for this type of delivery is via random diffusion of PGCs and their fusion with nearby plasma membrane sites. This is because the cells used in our experiments (i.e., COS) were flat with a distance of only a few micrometers between Golgi elements/PGCs and the plasma membrane.

PGCs enriched with VSVG-GFP were large and carried significant amounts of cargo. We found that an average-sized PGC occupied an area of 1.3 µM2 corresponding to 32 pixels (with each pixel 0.2 x 0.2 µM). In contrast, a 100 nm fluorescent bead observed at the same magnification and imaging conditions occupied a single bright pixel. In a cell expressing ~20 million VSVG-GFP molecules, we calculated that ~10,000 VSVG-GFP molecules were carried by a single PGC at peak VSVG-GFP flux out of the Golgi complex (based on conversion of VSVG-GFP fluorescence with fluorescence from a known standard solution of GFP). For comparison, a 100 nm vesicle could carry no more than 200 membrane proteins when maximally packed in a bilayer (30) . PGCs remained as large structures throughout their short lifetime, which we calculated to be 3.8 min on average. During transport, we found that PGCs never intersected with other intracellular transport pathways.

Fusion of PGCs with the plasma membrane in COS cells expressing VSVG-GFP was studied using an intensified video camera system to continuously collect images. After docking at the plasma membrane for >=10 s, PGCs fused with this surface. After fusion, dispersal of VSVG-GFP fluorescence across the plasma membrane was rapid (i.e., 2 s). This indicated VSVG-GFP was highly mobile on the plasma membrane. The fact that the entire VSVG-GFP content of a PGC dispersed on fusion with the plasma membrane indicated that PGCs were a single, continuous structure rather than a chain or cluster of vesicles.

These findings suggest that Golgi-to-plasma membrane traffic is mediated by large tubulovesicular structures (i.e., PGCs) rather than by small vesicles. These structures bud as entire domains from the Golgi complex and move along microtubules without intersecting other pathways before fusing with the plasma membrane. They thus represent a unique intracellular transport organelle that conveys protein cargo from the Golgi complex directly to the plasma membrane. Future work needs to address how proteins sort into these structures, what regulates their formation, and how they move along microtubule tracks.


   QUANTITATIVE ANALYSIS AND KINETIC MODELING OF SECRETORY TRANSPORT
TOP
 INTRODUCTION
 GOLGI-TO-PLASMA MEMBRANE...
 QUANTITATIVE ANALYSIS AND...
REFERENCES
 
The kinetic properties of VSVG-GFP transport into and out of the Golgi complex were studied using confocal time-lapse imaging techniques in single living cells (13) . Our goal was to quantitate the changes in cellular distribution of VSVG-GFP during transport in order to measure the residency time and flux (number of molecules per second) of VSVG-GFP through various compartments of the secretory pathway. Toward this end, we acquired digital time-lapse imaging sequences from VSVG-GFP-expressing cells on temperature shift from 40 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 where all fluorescent VSVG-GFP molecules could be detected at any time and under conditions of minimal photobleaching. Temporal changes in the amount of VSVG-GFP fluorescence within regions of interest (ROIs) encompassing the juxtanuclear Golgi region and entire cell were measured and plotted (Fig. 1B ). Computer software that used generalized least squares optimization algorithms was then used to simultaneously fit Golgi and total fluorescence intensities with time to a simple model for VSVG-GFP trafficking (Fig. 1C ).

Fitting the time course data to our trafficking model did not require a changing rate constant as the concentration of VSVG-GFP in various compartments went from high (early in the experiment) to low (late in the experiment). Rather, we found that a series of first order rate laws connecting the ER, Golgi complex, and plasma membrane was sufficient to accurately account for the data, indicating that secretory transport machinery was never saturated during VSVG-GFP transport. This allowed us to calculate VSVG-GFP flux (i.e., molecules per second) into and out of the Golgi complex and delivery to the plasma membrane in individual cells. For ER-to-Golgi transport of VSVG-GFP, the mean rate constant was 2.8%/min; for Golgi-to-plasma membrane transport it was 3.0%/min; and for transport from the plasma membrane to a degradative site it was 0.25%/min (see Fig. 1D ). For a cell containing ~2 x 107 VSVG-GFP molecules, the rate constants predicted a peak ER-to-Golgi flux of 7,000 molecules per second (15–20 min after shifting to permissive temperature), a peak Golgi-to-plasma membrane flux of 4,000 molecules per second (at 40–45 min), and a peak plasma membrane to lysosome flux of 700 molecules per second (at 125–150 min). A VSVG-GFP molecule was found to spend 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. These transport rates are the first to be reported for a specific protein population within single living cells.

Previous kinetic measurements of the secretory pathway have used radiolabeled amino acid pulse-chase kinetics combined with cell fractionation and immunoprecipitation (31 32 33) . These measurements are significantly less precise than the above estimated values using GFP imaging methods for several reasons. First, traditional biochemical analyses typically use only 6–10 data points (because cell fractionation is so labor intensive), and each point represents the average value from thousands of different cells. In contrast, our sampling involves hundreds of data points collected from the same cell. Second, our Golgi "fraction" (i.e., a spatial site within a single cell) is exceptionally well-defined in contrast to biochemical isolation of Golgi fractions from gradients where contamination with other organelle fractions is unavoidable.

We believe that kinetic analysis of secretory trafficking using GFP imaging techniques can be used to study much more complex transient situations, such as physiological or pharmacological perturbations or even cell-cycle-induced changes in protein trafficking pathways, that simply cannot be approached using traditional steady-state tracer kinetic methods. We have found, for example, that in cells treated with cyto-B, which disrupts the actin cytoskeleton, there is a significant decrease in the Golgi exit rate constant and an increase in the rate of internalization of VSVG-GFP from the cell surface. These changes in rate constant for VSVG-GFP trafficking suggest that there are regulatory changes in Golgi export mechanisms and plasma membrane trafficking on disruption of the actin cytoskeleton.

The fact that linear rate laws could summarize the diverse trafficking events occurring between the ER and Golgi, and between the Golgi and plasma membrane, suggests that within each of these sets of events, the transport processes were effectively characterized by a single rate-limiting step and that neither the identity nor quantitative features of this rate-limiting step was perturbed by VSVG-GFP trafficking. With additional imaging approaches, we anticipate that it will be possible to refine our model of VSVG-GFP trafficking to characterize specific pathways in more detail. For example, we have used photobleaching combined with high-resolution imaging to study the role of transport intermediates involved in Golgi-to-plasma membrane trafficking (13) . By kinetically modeling the data, we were able to calculate the fraction of total Golgi-to-cell surface transport of VSVG-GFP that occurs by large post-Golgi structures rather than small vesicles, and to estimate the lifetime of these structures within cells.

In conclusion, these studies demonstrate that GFP imaging methods combined with computational analysis provide a new approach for dissecting the control and regulation of the secretory pathway in single, intact, living cells. Numerous questions relevant to the regulation and functioning of the secretory pathway can thereby be addressed, providing insight into how molecular machinery functions on a system-wide basis within secretory membranes of living cells.


   ACKNOWLEDGMENTS
 
K. H. is funded by the Human Frontiers Science Program.


   REFERENCES
TOP
 INTRODUCTION
 GOLGI-TO-PLASMA MEMBRANE...
 QUANTITATIVE ANALYSIS AND...
 REFERENCES
 

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