Golgi-to-ER exchange kinetics indicate an approximately 90:10 Golgi-to-ER distribution
The Cisternal Maturation model requires that glycosyltransferases ‘resident’ in the Golgi apparatus actively recycle during each round of transport. Much of this recycling must be within the Golgi cisternal stack. However, a portion may well be recycled between the Golgi apparatus and ER as part of an ongoing need to balance membrane flows [for review, see (14)]. Hence, a quantifiable fraction of fully processed Golgi enzymes would be normally found in the ER. Fluorescence microscopy data from Zaal et al. (1) suggest that approximately 67% would be Golgi localized and the remaining 33% in the ER. Such a high ER value was surprising given that Golgi-specific glycosyltransferases and glycosidases do not normally modify ER-resident glycoproteins [for review, see (16)]. We, therefore, looked at quantifying this distribution using direct kinetic rate measurements.
Assuming the transport time between the Golgi and the ER is much shorter than the residence times within, we consider the Golgi and the ER to be two separate compartments connected by first-order transport processes (Figure 1A). The change in ER enzymes as a function of time can be described by
where E is the relative concentration of proteins in the ER and kGA and kER are the rate constants for transport between the Golgi and the ER in the retrograde and anterograde directions, respectively. Degradation and synthesis are neglected in our analysis due to experimental conditions investigated. The general solution of the differential equation can be written as
where the integration constant C is determined from the initial condition. The steady-state solution, Ess, is given by a ratio of the rate constants:
Using GalNAcT2-GFP transfected HeLa cells, we previously collected fluorescent microscopy data in the presence of cycloheximide (CHX), with and without mSar1pdn protein as an ER exit block, that can now be used to determine the steady-state distribution (5). In the presence of mSar1pdn, GalNAcT2-GFP loss from the Golgi and accumulation in the ER were recorded and quantified as previously described. For this analysis, kER was set equal to zero by definition, and the final steady state was reached when all proteins relocated at the rate kGA into the ER (Ess = 1). A value of 0.57 ± 0.04/h for kGA was reported. In the absence of mSar1pdn, FRAP experiments were done following photobleaching of approximately 35% of ER fluorescence. The intensity recovery data from the time-lapse image set was then fit to equation 2, and from three separate measurements, we found kGA + kER to be 4.00 ± 0.42/h (Figure 1B). Substituting these measured values into equation 3 yields an ER pool value for GalNAcT2-GFP of 14 ± 2% and, hence, a distribution of approximately 90:10 Golgi to ER. Given these measured rate constants, one can say that GalNAcT2-GFP would exit the Golgi apparatus at a rate of 1.0%/min and exit the ER at a rate of 5.7%/min. The reciprocals of these values yield the mean residence times of 106 ± 8 min in the GA and 17.8 ± 2.2 min in the ER for a total cycle time of 124 ± 8 min for GalNAcT2, in contrast with previously estimated total Golgi and ER residence times of 84.6 ± 11.3 min for GalT (1).
Figure 1. Two-compartment model for Golgi glycosyltransferase cycling.A)Schematic diagram of the two-compartment kinetic model. The endoplasmic reticulum (ER) and the Golgi apparatus are considered as two separate compartments connected by first-order transport processes. The rate constants kER and kGA are the ER-to-Golgi (anterograde) and Golgi-to-ER (retrograde) transport rates, respectively. Golgi-to-ER transport rate kGA = 0.57 ± 0.04/h was measured from an ER exit block experiment using mutant Sar1pdn proteins by Miles et al. (5).B)Measured fluorescence recovery after 35% photobleaching of the ER in HeLa cells expressing GalNAcT2 green fluorescent protein. Relative ER fluorescence was averaged over three separate HeLa cells. Data are redrawn from Miles et al. (5). The solid curve reflects the fit of the data points to the exponential equation shown. The fitted parameter m2 in the table inset represents the sum of rate constants kER + kGA. Both rate measurements were done in the presence of protein synthesis inhibitor cycloheximide.
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Immunogold electron microscopy indicates approximately 90:10 distribution of GalNacT2 between the Golgi apparatus and ER
Given the difference in distribution values between the kinetic model analysis of fluorescent microscopy data for GalNAc-T2 and the previously reported direct fluorescent microscopy results for GalT (1), we chose to determine the distribution between the Golgi and ER of GalNAcT2 using immunogold labeling of thawed cryosections combined with electron microscopy. In contrast to light microscopy, the resolution of the electron microscope is high relative to the underlying structures. On the basis of our kinetic modeling results, we expected the level of ER labeling to be low for any given glycosyltransferase. Therefore, for the immunogold experiments, we chose to use stably transfected GalNAcT2-VSV (VSV, P5D4 epitope tag from vesicular stomatitis virus G Protein). At fivefold overexpression, GalNAcT2-VSV has the same cellular distribution as that of an endogenous protein (19) and has been found repeatedly to have the same cycling kinetics between the Golgi apparatus and ER as other Golgi enzymes (3,5,6).
Two sets of electron micrographs were taken, one at magnification of 34 000 to quantify immunogold-labeling density and a second at lower magnification to score overall Golgi and ER areas. For immunogold labeling, cryosections were incubated sequentially with anti-VSV primary antibody/10 nm immunogold to localize GalNAcT2 and with anti-protein disulfide isomerase (PDI)/5 nm immunogold to identify ER. GalNAcT2, positive regions were photographed at random and scored morphometrically (3). For overall area stereology, cells were photographed at random at a magnification of 10 000 or 16 000 and organelles identified by their characteristic morphology. Consistent with previous results (3,19), 10-nm GalNAcT2 labeling was heavily concentrated over Golgi cisternae (arrowhead, Figure 2A) and associated tubules (arrows, Figure 2A) with occasional 10-nm GalNAcT2 labeling (arrowhead, Figure 2B) in association with the PDI-positive ER (arrow, Figure 2B). As shown in Figure 2C, Golgi cisternal stacks (arrowheads) and ER (arrow) could be readily distinguished morphologically in the lower magnification micrographs.
Figure 2. Electron micrographs of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2)-VSV HeLa cells with immunogold labeling (A, B) and without (C).A)Electron micrograph acquired at 34 000 magnification showing Golgi cisternae (arrowhead) and Golgi tubules (arrow) where 10-nm gold particles indicate VSV labeling as described in the Materials and Methods.B)Thirty-four thousand magnification-acquired image depicting endoplasmic reticulum (ER) immunolabeled for GalNAcT2 with 10-nm (arrowheads) and 5-nm gold particles labeling protein disulfide isomerase, an ER marker (arrow).C)An example of a randomly selected image acquired at 16 000 magnification and illustrating Golgi stacks (arrowheads) and ER ribbon (arrow). Note that the Golgi tubules are not readily apparent, and spacing between cisternae is difficult to reliably distinguish at this magnification.
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As expected, the 10-nm GalNAcT2 labeling density was much higher over Golgi cisternae and associated tubules than over the ER, mitochondria, or nucleus. We took the average of both mitochondrial and nuclear labeling as an indicator of non-specific reactivity. The level of non-specific labeling was approximately 30% of that observed over the ER. The mitochondrial/nuclear labeling density was subtracted from all values to give a corrected Golgi apparatus- and ER-labeling density for GalNAcT2-VSV. Labeling density over the Golgi apparatus was approximately 80–100 higher than that over the ER. In order to convert labeling density to the total distribution of GalNAcT2-VSV between Golgi apparatus and ER, we determined the relative area of ER and Golgi apparatus (cisternae plus tubules) in low- magnification, random cell sections. As summarized in Table 1, the Golgi apparatus area was approximately 12% of the ER. In total, 90% of the GalNAcT2 labeling was associated with the Golgi apparatus and 10% with the ER (Table 1).
Table 1. GalNAcT2-VSV relative protein distribution by immunogold labeling
| ||Golgi cisternae||Golgi tubules||ER|
|Particle density (number/µm2)||227||166||2.86|
|Relative area average (n = 2)||5.57 ± 0.4||7.09 ± 0.5||100|
|Relative protein average (n = 2)||90 ± 1.5||10 ± 1.5|
Best-practice widefield fluorescence microscopy yields approximately 90:10 Golgi-to-ER steady-state distribution for Golgi glycosyltransferases
The EM and kinetic analysis distribution values indicated an approximately 90:10 distribution for GalNAcT2 leading us to investigate whether a similar steady-state distribution of GalNAcT2 between the Golgi apparatus and ER would be obtained using fluorescence microscopy. HeLa cells are fairly thin, approximately 6–7-µm thick, when imaged as fixed, plastic-mounted samples. As such, widefield images focused for bright Golgi apparatus sampling should contain intensity information from the full cell volume; the Golgi apparatus is located at approximately mid-cell height. This was essentially the approach used by Zaal et al. (1) in quantifying GalT-GFP distribution in a single plane image collected with a laser-scanning fluorescence microscope operated with the pinholes wide open. For our studies, all widefield images were collected at a resolution sufficient for deconvolution analysis (1.4 numerical aperture objectives and approximately 2× oversampling to give images that met Nyquist sampling criteria).
GalNAcT2-VSV, GalNAcT2-GFP and GalT all distributed in a similar manner by widefield fluorescence microscopy (Figure 3). Antibody staining was used to reveal GalNAcT2-VSV and GalT distributions and the inherent fluorescence of the GFP moiety to reveal the distribution of GalNAcT2-GFP. In images displayed with a normal grayscale range (100–3000 grayscale levels, 12 bit camera), all had a distinct juxtanuclear Golgi-like fluorescence distribution with little detectable fluorescence observed over the cytoplasm (Figure 3A,B,C). When the same images were displayed with a compressed grayscale range that accentuated the display of low-intensity fluorescence (100–300 grayscale levels), three striking distributional features were now apparent (Figure 3A′,B′,C′). First, the bright, juxtanuclear Golgi apparatus area was now much larger. This was an expected outcome for out-of-focus plane Golgi intensity (i.e. blur) and light spread by the objective, also known as point-spread function. Second, there was cytoplasmic fluorescence apparent for all three. Third, particularly obvious for GalNAcT2-GFP (Figure 3B′) and GalT (Figure 3C′), the fluorescence pattern gave a rim-like fluorescence staining at the nuclear envelope, typical of an ER distribution. For GalNAcT2-VSV, this was less obvious. However, this is not unusual for antibody staining as even antibodies against bona fide ER proteins such as p63 often give a somewhat granular staining pattern [see e.g. Figure 1 in (6)]. In double label experiments with Sec61p, an ER marker, near-complete overlap between the cytoplasmic GalNAcT2-GFP fluorescence and Sec61p staining was observed (data not shown). We conclude from this that there is a low quantifiable level of both GalNAcT2 and GalT present in the ER. At first glance, the intensity of GalNAcT2-VSV staining over the ER was slightly more intense than that of GalNAcT2-GFP or GalT (compare Figure 3A′,B′,C′). The overall background brightness of the GalNAcT2-VSV labeling, however, was slightly higher than that for GalNAcT2-GFP or GalT, and the net distribution is the same (summarized in Table 3).
Figure 3. Golgi enzymes are found in the endoplasmic reticulum (ER) at lower concentrations by widefield light microscopy. HeLa cells stably expressing GalNAcT2-VSV (A, A′) stained for VSV, expressing fluorescent GalNAcT2-GFP (B, B′) fixed and mounted, and wild-type cells stained for endogenous GalT (C, C′) are shown as single plane widefield images. Identical images are shown at normal (A – C, linear mapping of 100–3000 grayscale levels of 0–4095) and high(A′–C′, 100–300 grayscale levels of 0–4095) brightness. Note that the nuclear envelope (arrowheads) typical of the ER distribution only becomes visible under high brightness (A′–C′). Bar, 10 µm.
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Table 3. Comparison of grayscale fluorescence intensity parameters by three fluorescence microscopy methods
|Method||Marker||Wildtype||ER||Golgi (vis)||Signal-to-noise (Golgi)||n|
|Widefield||T2-VSV||20.7 ± 7.8||48.4 ± 21||1392 ± 383||190 ± 50||30|
|T2-GFP||4.1 ± 2.0||13.7 ± 5.9||732 ± 508||110 ± 70||30|
|GalT||0||15.3 ± 6.7||1260 ± 391||200 ± 60||30|
|Laser scanning||T2-VSV||0.83 ± 0.50||1.30 ± 0.29||31.6 ± 6.6||3.0 ± 0.6||10|
|Spinning disk||T2-VSV||13.2 ± 4.3||25.8 ± 8.6||701 ± 90||30 ± 6||25|
|GalT||0||4.0 ± 1.1||409 ± 68||54 ± 9||30|
As a first approximation to determining the apportionment of GalNAcT2 and GalT between the Golgi apparatus and ER, wildtype (WT) and tagged HeLa cells were imaged in the same field at a constant illumination intensity and exposure that did not saturate the CCD camera (Figure 4A). To draw organellar and cell boundaries, we displayed images nonlinearly (technically, gamma = 0.4), and cell boundaries and the Golgi apparatus were outlined by eye (visual threshold, Figure 4B). Under these conditions, the boundaries of cytoplasmic fluorescence were apparent, and little detail was lost due to image display saturation. We then calculated the fraction of total fluorescence intensity from the Golgi apparatus compared with that of the whole cell to be approximately 63–64% for either GalNAcT2 (epitope or GFP tagged) or GalT (Table 2). Values were the same whether or not the cells had been cultured for 4 h in the presence of CHX to inhibit protein synthesis. Note that all values were corrected for non-specific background fluorescence. For tagged cells, the correction was based on the mean fluorescence level found in co-cultured and co-imaged WT cells (Figure 4A,B). For endogenous GalT, the level of intensity found between cells was subtracted. Quantitatively, these results are essentially the same as those found by Zaal et al. (1) for the visual quantification of a concatenated GalT-GFP-GFP-GFP chimera using widefield microscopy (laser-scanning microscope operated with wide-open pinholes). Wild-type cytoplasmic intensity was about 40% of the total signal found over the ER region of GalNAcT2-VSV and 30% of GalNAcT2-GFP (Table 3). As widefield microscopy is a high signal-to-noise ratio technique (>100, Table 3), the background-corrected ER values are significant.
Figure 4. Single plane widefield images were analyzed before and after deconvolution using visual and calculated thresholds.A)Shown is a fluorescence image of HeLa cells stably expressing GalNAcT2-VSV polypeptide N-acetylgalactosaminyltransferase 2-VSV stained for VSV, as well as a wild-type HeLa (WT). A nonlinear intensity display (gamma = 0.4) was used to bring out dimmer structures. Bar, 10 µm.B)Cell outlines are shown as well as the Golgi apparatus, as identified visually (visual threshold, arrowhead).C)Shown is the surface plot of the fluorescence intensity. The visual threshold (arrowhead) underestimates the Golgi intensity peak, and the calculated threshold (arrow) is needed to account for the total intensity of the peak.D)The average ER intensity + 2SD in the region (arrowhead) far from the Golgi was used for the calculated threshold (arrow). E, F) Shown are the image and its surface plot after five iterations of deconvolution. The Golgi apparatus as determined from the visual (arrowhead) and calculated (arrow) thresholds of intensity is outlined or marked. Note the overlap between visual and calculated thresholds. All fluorescence images are shown with gamma = 0.4 while surface plots are drawn with actual pixel values (gamma = 1.0).
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Table 2. Percentage of glycosyltransferases found in the Golgi apparatus measured from single plane widefield images
| || ||GalNAcT2-VSV||GalNAcT2-GFP||Endogenous GalT|
|Raw||Visual||61 ± 5||62 ± 5||64 ± 5||64 ± 5||65 ± 7||66 ± 4|
|Calculated||85 ± 5||84 ± 6||90 ± 3||88 ± 4||87 ± 4||89 ± 3|
|Deconvolved||Visual||85 ± 6||84 ± 8||89 ± 4||87 ± 5||88 ± 4||89 ± 3|
|Calculated||88 ± 7||87 ± 8||91 ± 4||91 ± 4||91 ± 3||91 ± 3|
As indicated in Figure 3A′,B′,C′, the actual spread of Golgi-specific fluorescence in widefield images was greater than that apparent to the eye. This was readily apparent in a quantitative manner when the image was reformatted as a surface plot of fluorescence intensity versus XY coordinates (Figure 4C). The Golgi signal surface plotted was a clustered set of intensity peaks much like a mountain range, and the visual threshold marked by an arrowhead considerably underestimated the perimeter of the mountain range. To include the whole Golgi signal within the boundary, we measured the average ER intensity within a region far from the Golgi (arrowhead, Figure 4D), calculated a standard deviation (SD) and used the average intensity value + 2SD as a calculated threshold. Negligible amounts of the ER in dispersed spots were above this calculated threshold as tested in HeLa cells stained for Sec61p, an ER marker (data not shown). Moreover, the Sec61p distribution indicated that the ER distribution was uniform about the cytoplasm (data not shown). Such scattered spots were excluded from a Golgi intensity calculation by setting a minimum area limit. The Golgi intensity within the area defined by the calculated threshold was measured and corrected for maximal underlying ER contribution by subtracting the average ER intensity value from each pixel. This approach yielded a Golgi fraction of almost 90% for both the transfected/overexpressing GalNAcT2-VSV and GalNAcT2-GFP and the endogenous GalT (Table 2, raw, calculated). Addition of CHX did not affect the results.
We thus reasoned that the discrepancy between visual and calculated threshold was an outcome of the objective point-spread function. If so, it should be resolved by deconvolution, a mathematical method to reassign light to its source (20). After applying iterative maximum likelihood estimation deconvolution to the raw image, we observed that the gap between visual and calculated thresholds was greatly reduced and the two almost overlapped in the XY fluorescence image (Figure 4E). The surface plot showed sharper peaks with reduced perimeter and near overlap of the two thresholds (Figure 4F). Golgi fractions obtained from the deconvolved widefield images using a visual threshold were 85–89% for GalNAcT2 whether VSV- or GFP-tagged and endogenous GalT, plus and minus CHX, and 87–91% using a calculated threshold (Table 2, deconvolved). This confirmed our hypothesis that objective light spread was responsible for the difference in two different methods for raw images and more importantly indicates that the correct ratio for distribution of Golgi glycosyltransferases between the Golgi apparatus and ER was approximately 90:10. As the same result is obtained in the presence of CHX, we infer that this is a steady-state measurement and only a negligible portion of the ER pool is from new synthesis.
Technically, these results strongly indicate that visually identifying the Golgi apparatus from raw images underestimates the Golgi fraction of the glycosyltransferases at approximately 65% [present work and Zaal et al. (1)]. The use of a calculated threshold and/or deconvolution produced a decidedly higher outcome, approximately 90%, which agreed with both the EM and kinetic-modeling quantification. On the basis of our results, the best practice for widefield microscopy would be to determine a calculated threshold on a deconvolved image, as it applies an objective method to the corrected image (Table 2, bold). Using a calculated threshold on raw images provided a good practical approximation, as it gave only a slightly lower number than the best-practice result and can be applied to undersampled images. In support of the assumption of these experiments that single plane, widefield images focused on bright Golgi apparatus captured fluorescence intensity in a representative manner from the full depth of fixed HeLa cells (approximately 6–7 mm), we found no statistically significant change in total fluorescence intensity following BFA treatment, a condition that disperses Golgi glycosyltransferases into the ER (data not shown).