Immunofluorescence Microchamber Technique for Characterizing Isolated Organelles

Analytical Biochemistry 305, 55– 67 (2002) doi:10.1006/abio.2002.5655, available online at http://www.idealibrary.com on

Immunofluorescence Microchamber Technique for Characterizing Isolated Organelles John W. Murray, Eustratios Bananis, and Allan W. Wolkoff Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461

Received October 8, 2001; published online April 25, 2002

We describe a rapid technique for the localization and quantitation of specific proteins on organelles bound to microscope chambers. Disposable chambers are constructed from glass slides and provide a platform for the binding of organelles and subsequent immunofluorescence and biochemical assays. Several studies are presented to demonstrate the utility of this technique. Kinesin was visualized in postnuclear supernatants. Golgi and endoplasmic reticulum bound quantitatively to chambers. Endocytic vesicles prepared from rat liver that had been injected in situ with Texas red-labeled asialoorsomucoid allowed for simultaneous detection of asialoorosomucoid, asialoglycoprotein receptor, caveolin 1, and microtubules. Asialoglycoprotein receptor colocalized with asialoorosomucoid-containing vesicles, whereas many of the caveolin 1 structures had no asialoorosomucoid or asialoglycoprotein receptor. The microchambers were also used to measure the binding to endocytic vesicles of exogenously added Rab5 and to monitor the ATP-dependent acidification of endocytic vesicles using the fluorescent dye acridine orange. © 2002 Elsevier Science (USA)

Key Words: immunofluorescence; endocytosis; digital imaging; microscopy.

During our ongoing investigations of microtubulebased endocytic processing we have used glass motility chambers that allow for observation and experimental manipulation of samples on a microscope stage (1, 2). Various designs of such chambers have appeared over the years (3–5) and these have facilitated the understanding of cytoskeletal motor protein function as well as cell and organelle movement. Recent advances in the types of fluorescence probes and digital imaging and microscope technologies have increased the use of microscopes for quantitative assessment of biological samples. 0003-2697/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

The optical microchamber described has several important features: (i) it is inexpensive and easy to construct, (ii) it has a small chamber volume of 3–5 ␮l, (iii) it is disposable, and (iv) it allows for rapid and complete exchange of internal contents. We have laid groundwork for the use of such chambers for the rapid quantitative characterization of preparations of cellular membranes and associated proteins. Several issues must be considered when quantifying biological samples by fluorescence microscopy. High magnification images correspond to small amounts of material and are subject to large signal variation and noise. Small shifts in the focal plane can dramatically affect signal intensity. Excitation light sources can be unstable. Microscope optics have a complicated distribution of signal intensity over distance at high resolution (point spread function), especially along the Zaxis. Fluorescence bleaching, quenching, and other molecular effects are potential limitations. One means around these problems is to employ fluorescence standards for all samples and report data as fluorescence ratios (6, 7). However, under appropriate conditions, fluorescence intensity from a microscope can be shown to be proportional to fluorophore concentration without the necessity of ratioing. Many groups have used direct measurements of fluorescence intensity to quantify levels of proteins (8, 9). The microchamber employed in our studies is amenable to such quantitation and several examples of quantitation are demonstrated. METHODS

Reagents. Microtubules and membrane fractions such as postnuclear supernatants (PNS) 1 were diluted into Assay Buffer (35 mM Pipes, 5 mM MgCl 2, 1 mM 1 Abbreviations used: PNS, postnuclear supernatant; ASOR, asialoorosomucoid; HA, Helix pomatia agglutinin; IPTG, isopropyl ␤-Dthiogalactopyranoside; ASGPR, asialoglycoprotein receptor; AO, acridine orange; Baf, bafilomycin.

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EGTA, 0.5 mM EDTA, 4 mM dithiothreitol, 20 ␮M Taxol, 2 mg/ml bovine serum albumin, pH 7.4). Antibodies were diluted into Blocking Buffer (Assay Buffer plus 5 mg/ml casein). Fluorescence images were recorded after 3 ⫻ 15 ␮l washes of Anti-bleach Buffer (Assay Buffer, containing 10 mM glucose, 52 units/ml catalase, 26 units/ml glucose oxidase). Immunostaining of nuclei and PNS was performed in Blocking Buffer plus 0.01% Triton X-100 to attain limited membrane permeabilization. Rhodamine–tubulin and unlabeled tubulin were purchased from Cytoskeleton (Denver, CO) and polymerized as in (2). To allow microtubule binding, coverslips were incubated with 20 ␮g/ml DEAE-dextran and then washed in distilled water, prior to the construction of chambers. Bafilomycin A 1 and acridine orange were from Sigma. Endocytic vesicles, PNS, nuclei. Endocytic vesicles were isolated as in (1). Procedures were approved by theUniversityAnimalUseCommittee.Briefly,Sprague– Dawley rats (Taconic Farms, Germantown, NY) were injected via the portal vein with Texas red-labeled asialoorosomucoid (ASOR). After 5 min, the liver was removed, homogenized, and centrifuged free of nuclei for 10 min at 3000 rpm in an Eppendorf 5810R centrifuge. The resulting PNS was applied to a Sephacryl S200 gel filtration column and the excluded volume was subjected to a 1.4/1.2/0.25 M sucrose float up step gradient. Endocytic vesicles were pooled from the 1.2/ 0.25 M sucrose interface (1). Nuclei were obtained by washing and recentrifuging the 3000-rpm pellet twice in Assay Buffer. Antibodies and probes. These included antibodies to kinesin heavy chain (monoclonal, Chemicon MAB1613); caveolin-1 (monoclonal, Transduction Laboratories No. 2297); asialoglycoprotein receptor (polyclonal, affinity purified (10)), calnexin (Polyclonal, Stressgen No. SPA-860-D), and appropriate Cy2-, Cy3-, and Cy5-labeled secondary antibodies (Jackson Laboratories). FITC-labeled Helix pomatia agglutinin (HA) lectin was from Sigma (No. L1034). All probes recognized cytosolic exposed domains (i.e., solution accessible) except HA-lectin and calnexin, where 0.01% Triton X-100 was used to attain limited membrane permeabilization. Control experiments included immunostaining in the absence of primary antibody and in the absence of antigen. Primary and secondary antibodies were used at concentrations of 10 –100 ␮g/ml. These high concentrations allowed for rapid incubations and a consistent fluorescence signal across the chamber surface. Microscopy. All microscopy was performed at the Analytical Imaging Facility of the Albert Einstein College of Medicine (http://www.aecom.yu.edu/aif/). A 60X, 1.4-NA planapo objective was used on an Olympus 1X70 inverted microscope with mercury lamp illumi-

nation containing automatic excitation and emission filter wheels connected to a Photometrics CCD camera run by IPLab Spectrum software (Scanalytics) running on a Power Macintosh. Fluorescence excitation/emission/beam splitter filters were from Chroma Technology Corp. and allowed the examination of fluorescence from DAPI/FITC/TRITC/Cy5. Some images were recorded directly to videotape and digitized later with a Scion Image frame grabber. Digital images were transferred to a PC computer and analyzed with Scion Image software. Specific macros for image quantitation can be obtained by contacting J. W. Murray. Acridine orange imaging was performed on a Bio-Rad Radiance 2000 confocal mounted on a Nikon Eclipse epifluorescent microscope heated to 35°C. Rab5. GST-Rab5 was expressed and isolated from Escherichia coli. In brief, Rab5 cDNA was obtained from Dr. Ira Mellman, digested, and inserted in frame into a pGEX-6P-1 vector (Amersham Pharmacia). Bacterial expression of GST-Rab5 was induced with IPTG. Bacteria were lysed by sonication and Rab5 was bound to and eluted from glutathione–agarose beads according to the manufacturer’s protocol. Purity was determined by SDS–PAGE and protein concentration by the Coomassie plus protein assay (Pierce). GST-Rab5 was diluted into Assay Buffer plus 4 mM GDP and, after 10 min, incubated for 5 min in chambers that had been preincubated with the endocytic vesicle preparation. After washes and primary and secondary antibody incubations, the chambers were sealed with glycerol. Images were collected within 1 h of the experiment. Data were fit to a Langmuir binding isotherm. Appropriate interpretation required a low concentration of Rab5 binding sites relative to total Rab5 such that the total Rab5 approximately equals the free concentration of Rab5; otherwise the apparent affinity represents an upper limit. RESULTS

Overview of the Immunofluorescence Microchamber Technique A schematic diagram of the chamber along with an overview of the immunofluorescence technique used to characterize organelles is shown in Fig. 1. The design shown is for an inverted microscope. The microchamber consists of a glass sandwich where the internal volume is bounded by a large coverslip (22 ⫻ 40 mm, Corning No. 2940-244) on the bottom, a thin piece of glass (⬃22 ⫻ 8 mm) on the top (created by scoring and breaking standard glass slides (e.g., Fisher No. 12-55010)), and double-stick tape on the sides (Scotch 3M No. 665, 0.009 cm thick). This creates an internal volume of ⬃4 ␮l (0.5 ⫻ 0.8 ⫻ 0.009 cm (length ⫻ width ⫻ height) ⫽ 3.6 ␮l). The internal volume is accessible by the 0.009 ⫻ 0.5 cm gaps at either end. The chamber is

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ganelles bind to both top and bottom of the chamber, but only the bottom surface is imaged. Fluorescence from top-bound organelles and light scatter from upper or lower surfaces of the chamber may contribute to the diffuse, background intensity of the image. A typical immunofluorescence microchamber protocol is as follows: four microliters of a concentrated (e.g., ⬎2 mg/ml total protein) suspension of organelles is

FIG. 1. Overview of “optical microchamber” construction and immunofluorescence protocol. Two pieces of half-width double-stick tape are applied to a large coverslip. A narrow piece of glass cut from a microscope slide is placed across the tape to create a chamber holding approximately 4 ␮l. Suspensions of organelles are perfused into the chamber, incubated, washed, and stained with selected antibodies or probes. Anti-bleach buffer is added, and the lower surface of the chamber is viewed in high magnification with a widefield fluorescent microscope and low-light CCD camera.

held together by double-stick tape and additionally secured with nail hardener at the tape/cut glass joints. Such a chamber functions as a preassembled microscope slide. Aqueous solution applied to either side will be drawn inside through capillary action. This fluid can be replaced by simultaneously pipetting solution at one side and wicking away solution from the other. Evaporation at the two gaps is rapid, depending on the ambient humidity. To avoid dehydration, the chamber is placed in a humidified box, e.g., a container with standing water. Or, to seal the chamber, a drop of glycerol is added at each gap. Biological samples are added to the chamber and incubated, and unbound material is washed away. The remaining bound material forms a thin layer attached to the glass that minimizes optical noise from out of focus material. We have found, as will be demonstrated, that cellular membranes bind directly to untreated glass coverslips. We refer to these membranes as “organelles.” The exact mechanism for glass binding is unclear but it has been shown that purified phospholipids and protein–phospholipid mixtures bind to untreated glass or glass that is hydrocarbon coated (11, 12). We have not encountered organelles that will not bind to untreated glass. On the contrary, we have had difficulty preventing excessive binding to glass when, for example, the binding of organelles to microtubules in preference to glass is sought. However, some macromolecules will not bind to glass, and glass pretreatment protocols have been developed for this purpose, e.g. (13, 14). For instance, pretreatment of glass with 20 ␮M DEAEdextran prior to assembly of the microchamber allows the binding of microtubules as shown below. The or-

FIG. 2. Immunofluorescence of postnuclear supernatant (PNS) within chambers. PNS was perfused into chambers and stained for kinesin. The immunofluorescence procedure outlined in Fig. 1 with 5-min antibody incubations allowed the detection of kinesin on distinct subsets of the phase-dense material (A, B). Arrows indicate two phase-dense lobes of similar appearance, one of which labeled for kinesin.

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perfused into the chamber, incubated for 10 min, and then washed 3 ⫻ 15 ␮l with blocking buffer. The sample is then incubated with primary antibody for 5 min, washed, incubated with fluorescent secondary antibody for 5 min, and then washed and perfused with anti-bleach buffer. Fixatives are avoided to allow subsequent demonstration of biological activity. A potential drawback is that material bound loosely to the organelles may be removed by the washing. Small volumes of concentrated antibody solutions allow for rapid incubation times, and lamellar flow allows for the thorough exchange of chamber content and disruption of solution boundary layers (15). Immunofluorescence Studies of PNS Figure 2 demonstrates the localization of the motor protein kinesin within rat liver postnuclear supernatant using the microchamber. Rat liver PNS was perfused into chambers and stained with primary antibody followed by Cy-2-labeled secondary antibody. By phase contrast it can be seen that membrane structures of varied sizes and optical densities bind to the glass; however, only specific subsets of these label for kinesin. For example, by phase contrast a large structure in the center of the field is visible and has two lobes of similar size (A, arrows), yet only one of these lobes is apparent in the anti-kinesin stain (B, arrows) and within this lobe smaller foci of staining are seen. Control experiments without primary antibody showed no staining under identical camera settings. Similar experiments were also performed on isolated rat liver nuclei, demonstrating that nuclei can also attach and remain bound during immunofluorescence procedures (not shown). Multiple Channel Fluorescence Figure 3 shows an example in which four probes have been simultaneously colocalized in the same sample in three fluorescence channels. Fluorescent microtubules were prepared by polymerizing a mixture of rhodamine-labeled and unlabeled tubulin. Microtubules were bound to the chamber, which was then washed. Isolated early endocytic vesicles containing Texas red ASOR were added to the chamber, incubated for 10 min, and washed, and primary and secondary antibodies to ASGPR (asialoglycoprotein receptor) and

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caveolin-1 were added successively. The fluorescent microtubules could be easily distinguished from the brightly fluorescent Texas red ASOR vesicles within the TRITC fluorescence channel. The merged view of Fig. 3 reveals fluorescent vesicles in blue, green, and red, indicating nonoverlapping stains, or yellow, aqua, and white, indicating partial or complete overlapping stains. It is seen that nearly all ASOR-containing vesicles, most of which bound microtubules, also contain the receptor ASGPR. Few vesicles stain green only, indicating that receptor is localized to shared compartments that also contain ligand and/or caveolin-1. However, many blue-only vesicles are visible, indicating that caveolin-1 frequently occurs in compartments that are separate from either ASOR or ASGPR. These data indicate that the endocytic vesicles are in a presegregated stage in endocytic processing of receptor and ligand and that a separate organelle compartment may be present within the preparation that labels with caveolin 1. Quantitation of Fluorescence Localized to Golgi and ER To determine whether specific proteins on cellular organelles could be assessed quantitatively using the microchamber technique, we first determined that fluorescence intensity was proportional to fluorophore concentration within the intensity range of typical experiments. Figure 4 shows a dilution series of fluorescent-labeled secondary antibody in anti-bleach buffer perfused into a chamber. Digital photos were collected at each concentration and the modal (most common) pixel value was plotted versus the concentration of antibody. This yielded a straight line, demonstrating that fluorescence signal is proportional to fluorophore concentration. To quantify fluorescence emanating from organelles, varying concentrations of PNS were incubated in separate chambers and stained for organelle-associated protein markers. Following incubation for 6 min, chambers were washed and stained with antibodies to calnexin, an ER resident protein (16), and HA lectin (Helix pomatia agglutinin), which recognizes Golgi (17). A series of 12-bit (i.e., pixel intensity between 0 and 2 12), 568 ⫻ 517 pixel images was captured for each dilution of PNS with exposure time and gain selected to

FIG. 3. Four fluorescent probes localized within a single field of endocytic vesicles. Consecutively added to chambers were rhodaminelabeled microtubules (red), endocytic vesicles containing Texas red asialoorosomucoid (ASOR) (bright red), primary and fluorescent-labeled secondary antibodies to asialoglycoprotein receptor (green), and caveolin-1 (blue). Two representative sets of images (A, B) are displayed in pseudo-color, and the probes are indicated on the left for clarity. The merged view (overlay of the pseudocolor images) shows fluorescent vesicles in blue, green, and red, indicating nonoverlapping stains, or yellow, aqua, and white, indicating partial or complete fluorescence overlap. ASOR vesicles bound to microtubules, and nearly all of the ASOR vesicles also contain receptor (ASGPR) (merged view, yellow or white), while many caveolin vesicles contain neither ASGPR nor ASOR (merged view, blue).

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avoid signal saturation. Representative images, as shown in Fig. 5, demonstrate that the amount of material bound to the coverslip decreases with greater dilution of PNS, as seen in both fluorescence (Fig. 5A) and phase contrast (Fig. 5B) (milligrams per milliliter of total protein indicated at the left). For quantitation, pixels above an intensity threshold were set to white while the rest were set to black, and the number of white pixels for each image was summed. This “binary image, pixel sum” was used to represent the amount of protein present. The threshold value for binarization was selected to avoid a diffuse appearance of white pixels and so that the “no primary antibody” control was completely black. The number of calnexin- (Fig. 5C) or HA-staining (Fig. 5D) pixels increased with increasing concentration of PNS. The amount of fluorescence staining as well as the amount of material seen by phase contrast appeared to level off at high concentrations. We fit the calnexin and HA fluorescence to hyperbolic curves to demonstrate this trend. Although some experiments showed considerable scatter, differences between low and high concentrations are significant (P ⬍ 0.005, Fig. 5C; P ⬍ 0.05, Fig. 5D). In separate experiments, we also found that increasing the concentration of KCl from 0.07 to 2 M did not decrease the amount of cellular material bound to glass or the associated calnexin staining (data not shown). We also quantified fluorescence signal intensity using an “intensity sum” (or “volume”) technique where the intensities from all pixels are summed. This type of measurement gave high variation compared to the binary sum technique as the data were skewed by the presence of very brightly fluorescing, large structures that appeared to contain a significant amount of light scattering as well as 3-dimensionality. Fluorescence must emanate from material of constant path length to be compared quantitatively. The 3-dimensionality of organelle material defies this constant path length requirement. However, the binary sum method appears to minimize this effect. Spatial Localization of Proteins A major advantage of the technique is the ability to examine the spatial localization of specific proteins along organelles. This information can indicate functional interaction and can be used to identify and classify isolated organelles. A semi-automated method to quantify protein colocalization using a Scion Image macro program is shown in Fig. 6. Panels from Fig. 3B showing ligand (ASOR) and receptor (ASGPR) fluorescence are displayed in modified form as Figs. 6A and 6B. The background cutoff was raised in the ASOR panel to eliminate microtubules, which fluoresce at a lower intensity. Both ASOR (A) and ASGPR (B) were

converted to binary images (i.e., pixels to black or white) using an automatically chosen threshold. Each ASOR vesicle was then manually selected with the computer mouse and outlined by Scion image. The outlines were copied to the ASGPR panel (B) and the fraction of white pixels gave the “colocalization” of ASGPR to ASOR for each vesicle. For reference, the outlines were copied to a third image (Fig. 6C) and numbered, and the percentage of colocalization is displayed in parentheses for each vesicle. The colocalization of several probes is presented in Table 1. Two categories were used to describe protein colocalization. “% Pixel Colocalization” is the average percentage of colocalization between two fluorophores (e.g., the percentage of ASGPR-positive pixels per selected ASOR pixel), while “% Vesicle Colocalization” is the percentage of the vesicles (selected outlines) that contain a minimum amount of fluorescence from the other fluorophore (e.g., the percentage of ASOR vesicles that contain at least 0.5% ASGPR staining). The percentage of vesicle colocalization will be greater than the percentage of pixel localization for a given set of images, and it is useful when fluorescence is known to arise from discrete organelles. For example, this analysis would determine how many ASOR vesicles contain at least some ASGPR (its receptor). The percentage of pixel colocalization, on the other hand, gives a direct answer as to how much the two probes overlap. Note that the colocalization of two fluorescent panels is not symmetric. That is, the colocalization of ASGPR to ASOR will be different from the colocalization of ASOR to ASGPR. The vesicle colocalization of ASGPR to ASOR was high (100 and 91%) (Table 1) and this indicates that nearly all the ASOR vesicles contain at least some ASGPR fluorescence. The pixel colocalization was also high (78%, 62%) suggesting an abundance of ASGPR at the identical location as ASOR, within the resolution limit of the microscope system. The strong colocalization data indicate that ASOR and ASGPR have yet to segregate during endocytic processing, and the isolated organelles are considered “early endocytic vesicles” (2). Control colocalization performed for different image panels (i.e., ASGPR A to ASOR B) shows that the fraction of colocalization by chance is small (14% vesicle colocalization). The colocalization of caveolin-1 to ASOR was also assessed (Table 1). Caveolin-1 is a major component of cell surface caveolae, which are membrane domains with distinct lipid and protein compositions that form an alternate endocytic pathway to the clathrin-coated pits used by ASOR (10, 18). Caveolin showed an intermediate level of colocalization to ligand (35%), and this result suggests that there is some overlap in the caveolar- and clathrin-mediated endocytic pathway once endocytosis has proceeded.

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FIG. 4. Fluorescence intensity from digital images as a function of fluorophore concentration. Fluorescent antibody (Cy2-labeled) was diluted into anti-bleach buffer and perfused into microchambers. Circles represent the modal (most common) pixel intensity from a 517 ⫻ 658 digital image. The linear relationship generated indicates that the fluorescence intensity is proportional to the antibody concentration within the range of intensities observed in these experiments.

Golgi and endoplasmic reticulum were assessed for colocalization using the images from experiments of Fig. 5. HA (Golgi marker) colocalization to calnexin (ER marker) showed moderate vesicle colocalization (⬃35%). These data indicate that about a third of the ER “vesicles” have a detectable amount of Golgi marker; however, the Golgi marker has limited spatial overlap with ER (pixel colocalization ⬃6%). It is also seen that the colocalization of HA to calnexin does not change significantly when PNS is diluted, suggesting that the probes are truly colocalized to the same structures and not merely overlapping. The control using panels from nonidentical microscope fields of view gave low (0.5%) colocalization. Binding of Exogenously Added Protein to Organelles within the Microchamber The microchamber technology may be used to quantify the binding of exogenously added proteins to organelles. One example is shown in Fig. 7. Rab5 is a small GTP binding protein that has been localized to early endosomes and can stimulate fusion of organelles from homogenized cells (19). Rabs are modified by hydrophobic prenyl groups at their C-termini, facilitating interaction with intracellular membranes (20). However, bacterially expressed, nonprenylated Rabs also appear competent for important biological interactions

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(21, 22) and, in addition, specific protein–protein contacts for membrane-bound Rab5 appears to lie outside the prenyl groups (23). As an initial characterization of Rab5– organelle interaction, we assessed whether bacterially expressed, nonprenylated GST-Rab5 could interact with isolated endocytic vesicles bound within microchambers. Endocytic vesicles were bound to chambers, washed, incubated with various concentrations of purified GSTRab5, and stained with anti-Rab5 antibodies using the immunofluorescence protocol outlined in Fig. 1. As seen in Fig. 7, the number of Rab5-staining pixels increased with increasing concentrations of added GST-Rab5 and these data were fit to a hyperbolic binding curve yielding an apparent K d of 0.5 ␮M for the Rab5– endocytic vesicle interaction. Figure 7B shows the appearance of endocytic vesicles stained for antiRab5 antibody with zero or 8.7 ␮M added GST-Rab5. Note that the zero micromolar data point is not blank, as the vesicles contain endogenous Rab5. No staining was seen under these conditions in the absence of primary or secondary antibody, and the specificity of the antibody was confirmed by Western blot. These studies demonstrate the potential for this technique in analyzing intracellular protein–protein interactions. Further investigation is required to determine the “receptor” for Rab5 on these membranes, whether the binding is specific to subpopulations of endosomes, and the functional impact of this binding. Microbiochemistry The protocols presented herein avoid the use of fixatives and therefore allow for the assessment of biological activity of organelles bound within the chambers. We and others have demonstrated the use of microchambers for evaluating the activity of motor proteins, e.g., (1, 24, 25). Here we demonstrate that the ATPdependent acidification of endosomes can be monitored in a microchamber in real time using the fluorescent dye acridine orange (26). This dye concentrates in acidic organelles and forms dimers and higher order aggregates that fluoresce with a peak at 565 nm (27). Figure 8 shows endocytic vesicles bound within microchambers in the presence of acridine orange before and 12 min after the addition of 2 mM ATP (Fig. 8A). The ATP-dependent increase in fluorescence was inhibited by the specific vacuolar pump inhibitor, bafilomycin A 1 (Fig. 8B). In Fig. 8C, the change in acridine-orangestaining pixels after ATP addition was plotted over time in the presence (closed circles) and absence (open circles) of bafilomycin A 1. In the absence of inhibitor, there was an initial rapid increase in fluorescence followed by a stage of slower fluorescence increase. The increase in fluorescence likely represents the aggregation of acridine orange within the vesicles and is there-

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FIG. 5. Quantitation of marker proteins bound within microchambers. PNS at various concentrations was bound to chambers and immunostained for both HA (a Golgi marker) and calnexin (ER marker). (A) Examples of merged color images showing Golgi (red) and ER (green) are displayed along with the corresponding phase contrast images (B). (C, D) A series of such images was quantified and shows an increase in fluorescence signal as the amount of PNS is increased for both calnexin and HA antigens. These were fit to binding curves to demonstrate the trend of the data. Each data point represents a single 658 ⫻ 517, 12-bit image.

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ATP-dependent acidification. The increase in acridine orange fluorescence also demonstrates that membrane integrity is maintained within the endosomes bound to glass in these experiments. DISCUSSION

In the present article, we present a microscopy technique to characterize cellular organelles that makes use of a disposable glass chamber. The “microchamber” described is inexpensive, easy to construct, and of small volume (3– 4 ␮l) and can be used to determine quantitative, spatial information about biological material. Cellular organelles bind to untreated glass and remain bound without fixation during fluid exchange, allowing for immunofluorescence, protein binding, and optical biochemistry assays. The glass also can be pretreated and/or blocked to allow binding of specific material, such as microtubules. We demonstrate using probes to Golgi- and ER-specific epitopes that organelles from homogenized cells bind quantitatively to glass. Concentration-dependent binding of organelles was confirmed by observing the levels of cellular material bound to chambers using phase contrast microscopy. Algorithms were developed to quantify the spatial colocalization of different proteins along organelles. Such techniques require assumptions about the sample material, and quantitative modeling (e.g., deconvolution methods) may be necessary for assigning accurate fluorescence intensity values to a particular organelle surface, especially for structures/organelles that are smaller than the resolution of the microscope (i.e., diameters ⬍0.2 ␮M) (28, 29). However, we have found under high magnification and resolution (60X objective, 1.4 NA) that the immunofluorescence signal occurs in discrete clusters, or vesicles, on biological material that is bound to glass. In contrast, smooth gradients of fluorescence at this

FIG. 6. Colocalization algorithm used to quantify the spatial overlap of two proteins. Panels from Fig. 3B were modified and redisplayed as binary (black and white) images of the asialo-ligand (ASOR) (A) and its receptor (ASGPR) (B). ASOR vesicles were manually selected with a computer mouse and auto-outlined, and the identical region was assessed for white-staining pixels in the ASGPR channel. Outlines were copied to a third image (C) for reference and the amount of pixel colocalization in each vesicle is displayed in parentheses. The application of this algorithm toward several different fluorescent probes is presented in Table 1.

fore related to the activity of the H ⫹-ATPase and the aggregation/polymerization parameters of the dye. No increase in fluorescence was seen in the presence of bafilomycin A 1. These experiments demonstrate that the endosomes, which are active in microtubule motility and receptor-ligand sorting assays (2), also undergo

TABLE 1

Results of Colocalization for ASOR, ASGPR, and Caveolin as well as for HA and Calnexin in Dilutions of PNS Images for colocalization Panels A, B from Fig. 3 ASGPR A to ASOR A ASGPR B to ASOR B Caveolin A to ASOR A ASGPR A to ASOR B (Control) PNS dilutions from Fig. 5 HA to calnexin 50 mg/ml HA to calnexin 10 mg/ml HA to calnexin 2.5 mg/ml HA to calnexin 0.5 mg/ml Control 50 mg/ml

% Pixel colocalization

78 62 15

% Vesicle colocalization

100 91 35

0.9

14

6 4 8 6 0.5

42 39 31 29 6

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FIG. 7. Binding of Rab5 to endocytic vesicles using the microchamber technique. Endocytic vesicles were bound within a series of chambers, incubated with varied concentrations of bacterially expressed GST-Rab5, and subjected to immunostaining for Rab5. (A) Immunofluorescence was quantified and the data were fit to a binding curve yielding a dissociation constant of 0.5 ␮M for the binding of Rab5 to endocytic vesicles. Circles represent single images subtracted from the average value detected for zero added GST-Rab5. (B) Two representative images show the appearance of immunofluorescence staining in this experiment. The number and intensity of fluorescent punctae increased significantly with the addition of 8.7 ␮M GST-Rab5.

magnification indicate either out of focus material or fluorescence from solution (i.e., diffusing fluorophores). Therefore, it appears to be appropriate and meaningful to analyze the colocalization of particular proteins to fluorescent clusters, which presumably represent organelles or organelle domains.

Using this technique we have determined that preparations of early endocytic vesicles that contain exogenously administered ASOR also contain the asialoglycoprotein receptor, and we have shown previously that these same vesicles contain kinesins but not dynein (2). Caveolin-1 has been shown to be present in subcellular

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FIG. 8. Endocytic vesicle acidification demonstrated by increased acridine orange fluorescence. Endocytic vesicles were bound within chambers and washed in assay buffer containing (A) 2 ␮M acridine orange (AO) or (B) 2 ␮M acridine orange and 1 ␮M bafilomycin A 1 (Baf), a specific vacuolar proton pump inhibitor. Images were collected by confocal microscopy (excitation 488 (⬃FITC), emission 660 (⬃Cy5)) prior to and after the addition of 2 mM ATP. The fluorescence increase in A (bottom) indicates proton pump activity within the endocytic vesicles, and this is inhibited by bafilomycin (C). Images were quantified as in Fig. 5 and showed an increase in AO fluorescence over time (closed circles) that was not observed in the presence of bafilomycin (open circles).

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endocytic fractions of rat hepatocytes, and whole cell and tissue section immunofluorescence have revealed partial overlap of ASGPR (the ASOR receptor) with caveolin-1 (30). Using the microchamber we show that there is only partial colocalization of caveolin-1 with ASOR within isolated endocytic vesicles, potentially demonstrating a divergence of the “caveolar” endocytic pathway from the receptor-mediated pathway used by ASOR. The microchamber has been further developed into an analytical technique for the measurement of protein binding and organelle acidification. We measured the binding of bacterially expressed GST-Rab5 to glassbound endocytic vesicles by detecting an increase in Rab5 immunofluorescence in microscope images (Fig. 7). We were also able to detect the enzymatically induced acidification of endocytic vesicles by the increase in intensity of an indicator dye, acridine orange (Fig. 8). It should be possible to measure any enzymatic activity within the chamber, provided that an optical probe is available. The ability to observe signal intensity and spatial localization at high frequency could provide important insights into protein function. ACKNOWLEDGMENTS The authors thank Dr. Ira Mellman for kindly providing Rab5 cDNA in pBlueScriptKS and Ms. Pijun Wang for help in preparation of the Rab-GST fusion proteins. The work was supported by National Institutes of Health Grants DK41918, DK23026, and DK41296. Mr. Bananis is presently supported by NCI training grant CA09475. He was the recipient of a student research award and Dr. Murray was the recipient of a postdoctoral fellow research award, both from the American Association for Study of Liver Diseases.

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