MICROSCOPY RESEARCH AND TECHNIQUE 00:00–00 (2014)

Molecular Ultrastructure of the Urothelial Surface: Insights from a Combination of Various Microscopic Techniques 3 3  ZUPANCI  C,  1 ROK ROMIH,1 HORST ROBENEK,2 KRISTINA ZU  ZEK    DASA ROZMAN, ZORAN SAMARDZIJA, 4 1  ROK KOSTANJSEK, AND MATEJA ERDANI KREFT * 1

Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia University Clinical Centre of M€ unster, Albert-Schweitzer-Campus 1, M€ unster, Germany 3 “Jozef Stefan” Institute, Ljubljana, Slovenia 4 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia 2

KEY WORDS

atomic force microscopy; freeze fracturing; immunoelectron microscopy; urothelium; urinary bladder

ABSTRACT The urothelium forms the blood–urine barrier, which depends on the complex organization of transmembrane proteins, uroplakins, in the apical plasma membrane of umbrella cells. Uroplakins compose 16 nm intramembrane particles, which are assembled into urothelial plaques. Here we present an integrated survey on the molecular ultrastructure of urothelial plaques in normal umbrella cells with advanced microscopic techniques. We analyzed the ultrastructure and performed measurements of urothelial plaques in the normal mouse urothelium. We used field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), transmission electron microscopy (TEM) on immunolabeled ultrathin sections (immunoTEM), and freeze-fracture replicas (FRIL). We performed immunolabeling of uroplakins for scanning electron microscopy (immuno-FESEM). All microscopic techniques revealed a variability of urothelial plaque diameters ranging from 332 to 1179 nm. All immunolabeling techniques confirmed the presence of uroplakins in urothelial plaques. FRIL showed the association of uroplakins with 16 nm intramembrane particles and their organization into plaques. Using different microscopic techniques and applied qualitative and quantitative evaluation, new insights into the urothelial apical surface molecular ultrastructure have emerged and may hopefully provide a timely impulse for many ongoing studies. The combination of various microscopic techniques used in this study shows how these techniques complement one another. The described advantages and disadvantages of each technique should be considered for future studies of molecular and structural membrane specializations in other cells and tissues. Microsc. Res. Tech. 00:000– 000, 2014. V 2014 Wiley Periodicals, Inc. C

INTRODUCTION Functions of various cells are accomplished with molecular and structural specializations of their plasma membrane. In superficial urothelial cells, i.e., umbrella cells, of the urinary bladder in mammals, urothelial plaques that cover the apical surface (Koss, 1969; Min et al., 2003) contribute to a blood–urine barrier function (Hicks, 1975; Negrete et al., 1996). The urothelial plaques contain numerous 16-nm intramembrane particles consisting of four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa (Wu et al., 1990, 1994). Two additional uroplakins UPIIIb and UPIIIc were recently identified in mammals (Desalle et al., 2014), however their involvement in intramembrane particles has not been determined yet. Hexagonally ordered intramembrane particles slightly bend the membrane which results in the formation of concave urothelial plaques (Derganc et al., 2011; Kachar et al., 1999). Urothelial plaques are interconnected by narrow ’hinge’ regions, which give flexibility to the apical plasma membrane during distension–contraction cycles of the urinary bladder. However, neither the distance between the highest C V

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point of the hinge region and the lowest point of urothelial plaque (urothelial plaque depth), the length of the curvature of the concavity (curve length), nor the hinge-to-hinge diameter of urothelial plaques has ever been systematically measured. Scanning electron microscopy (SEM) is routinely used to show the presence of urothelial plaques encircled by hinge regions, the latter seen as microridges. These features are considered as markers of the advanced differentiation stage of umbrella cells and functional urothelium (Kreft et al., 2005; Romih et al., 2002; Veranic et al., 2004). Although field Additional Supporting Information may be found in the online version of this article. *Correspondence to: M. E. Kreft; Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia. E-mail: [email protected] Disclosure: The authors declare that there is no conflict of interests regarding the publication of this paper. Received 14 April 2014; accepted in revised form 15 July 2014 REVIEW EDITOR: Prof. Alberto Diaspro Contract grant sponsor: The Slovenian Research Agency; Contract grant number: P3-0108. DOI 10.1002/jemt.22412 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com).

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Fig. 1. FESEM image of the urothelial luminal surface coated with carbon show the urothelial plaques (asterisks) in the apical plasma membrane of umbrella cells (A). Immuno-FESEM image of urothelial surface coated with platinum demonstrates uroplakins (18 nm colloidal gold particles, arrows) within the urothelial plaques (B). Note heterogeneity of the urothelial plaque sizes. Scale bars 500 nm.

emission scanning electron microscopes (FESEM) enable analyses at resolutions down to 0.4 nm, they have not been applied for the studies of urothelial surfaces yet. Another possibility to observe urothelial plaques is atomic force microscopy (AFM). With additional mathematical software AFM would enable us to reveal precise topography of urothelial plaques. The localization of uroplakins in urothelial plaques was demonstrated by immunolabeling of ultrathin sections (Romih et al., 2002; Wu et al., 1990; Zupancˇicˇ et al., 2011) and by freeze-fracture replica immunolabeling (FRIL) (Kreft and Robenek, 2012). FRIL targets the proteins localized on an outer surface of the sample and also epitopes within cells or tissues (Robenek et al., 2009; Severs and Robenek, 2008). Another possibility is the detection of immunolabeled proteins with FESEM (immuno-FESEM). This approach would enable the visualization of uroplakins on cell surfaces, by exploiting secondary antibodies conjugated with gold particles. The aim of our study was to analyze urothelial apical surfaces by FESEM, immuno-FESEM, AFM, FRIL, TEM, and immuno-TEM and to compare the information about structural and molecular characteristics of urothelial plaques obtained by these techniques. We measured the maximal hinge-to-hinge diameter and the depth of the urothelial plaques, calculated their curves and curve lengths. Our results obtained by different techniques give a comprehensive insight into the molecular ultrastructure and the variability of the urothelial plaques at the umbrella cell apical surface. MATERIALS AND METHODS The experiments were approved by the Veterinary Administration of the Slovenian Ministry of Agriculture and Forestry (permit no. 34401-1/2010/6) in compliance with the Animal Health Protection Act and the Instructions for Granting Permits for Animal Experimentation for Scientific Purposes. Eight adult male mice; strain B6 were used. Animals were euthanized by CO2 inhalation and the urinary bladders were immediately excised, cut open, and dissected into pieces (samples).

FESEM Samples of one urinary bladder were fixed for 2 h in 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. After rinsing in cacodylate buffer and postfixation with 1% OsO4, samples were dehydrated through graded series of ethanol. They were critical point dried, placed on a polished carbon sample holder, coated with thin amorphous carbon layers (4 nm) in a Gatan PECS ion-beam coating apparatus, and examined in a FESEM JSM-7600F (JEOL) at 3.0 kV accelerating voltage. Immuno-FESEM Samples of one urinary bladder were washed in 3% BSA in PBS and incubated with rabbit polyclonal antibodies against total uroplakins (Wu et al., 1990), a kind gift from Professor Dr. T-T Sun, diluted 1:1000 for 30 min at 16 C. For negative controls, the incubation step with primary antibody was omitted. After rinsing, goat antirabbit IgG conjugated to 18 nm colloidal gold (1:40) (Jackson Immunoresearch, West Grove, NA) were applied for 30 min at 20 C. For negative controls, the incubation with the primary antibody was omitted. After rinsing in PBS, samples were fixed and further treated as described for FESEM, except that they were coated with a platinum layer (7 nm) in a Bal-Tec SCD 050 coating apparatus and examined in a FESEM JSM-7500F (JEOL) at 3.0 kV. AFM Samples of two urinary bladders were processed as described for FESEM, but without coating. Samples were then adhered to the carbon adhesive tape with the superficial urothelial side up and examined in AFM Digital instruments-dimension 3100 (Veeco) using a pyramidal Si tip with a radius of 10 nm. AFM images were recorded in tapping mode. FRIL Full description of the technique has been presented previously (Kreft and Robenek, 2012). Briefly, urothelial cells were scrapped from the two urinary bladders, Microscopy Research and Technique

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rapidly frozen, freeze-fractured, and platinum-carbon replicas were made. Replicas were treated with Trisbuffered 5% sodium dodecyl sulphate with 30 mM sucrose, pH 8.3, overnight at room temperature. After washing in PBS, the replicas were incubated in 5% BSA. Uroplakins were then localized with antiuroplakin antibodies; diluted 1:5000 in 1% BSA in PBS, for 1 h at RT. Negative controls were done by omitting the primary antibodies. After rinsing in PBS, goat antirabbit IgG conjugated to 18 nm colloidal gold (1:20) (Jackson Immunoresearch) were applied for 1 h at RT. Immunolabeled replicas were observed in a Philips 410 electron microscope at 80 kV. TEM and Immuno-TEM The urothelial samples were prepared as described previously (Hudoklin et al., 2011). The urothelium was isolated from two urinary bladders and high pressure frozen in a Balzers HPM 010 apparatus. For morphological studies, samples were freezesubstituted first with acetone containing 2% OsO4 and then with 100% acetone in a Leica AFS apparatus. Samples were embedded in Epon. For immunolabeling, frozen samples were freezesubstituted in pure acetone and embedded in Lowicryl HM20. Ultrathin sections were cut. Nonspecific labeling was blocked by the PBS buffer containing 0.1% fish gelatine, 0.8% BSA, and 5% FCS. Sections were incubated with anti-uroplakin antibodies, diluted 1:50,000. For negative controls, the incubation step with primary antibody was omitted. After washing, sections were incubated with goat antirabbit IgG conjugated to 10 nm colloidal gold (Jackson Immunoresearch), diluted 1:50. Sections were washed, counterstained, and viewed in a Philips CM100 electron microscope at 80 kV. Statistical and Mathematical Analysis We measured the hinge-to-hinge diameters of randomly sampled urothelial plaques imaged by FESEM (carbon) (n 5 20), FESEM (platinum) (n 5 20), AFM (n 5 20), and FRIL (n 5 20) in AxioVision Rel. 4.8 software (Zeiss). The average and the standard errors were calculated for each technique. An one-way ANOVA on ranks and then Tukey’s test for pairwise comparison of the means, i.e., FRIL vs. FESEM (carbon), FRIL vs. FESEM (platinum), FRIL vs. AFM, AFM vs. FESEM (carbon), AFM vs. FESEM (platinum), FESEM (carbon) vs. FESEM (platinum) were performed (SigmaPlot 11.0). For the AFM images, an additional mathematical analysis was applied. The WSxM software was used (Horcas et al., 2007) to obtain the curve representing the Z/X axis, e.g., the depth (Z) and the diameter (X) of a single urothelial plaque. Each of the 20 curves was mathematically analyzed and the length of each curve was calculated by the Mathematica software (Wolfram Research, Mathematica, Version 8.0, Champaign, IL, 2010). The average and the standard error were determined and the AFM curve lengths were compared to the results of hinge-to-hinge diameter measurements obtained from AFM images by the Student’s t test. RESULTS In FESEM, the appearance of the apical surface of umbrella cells differed between carbon (Fig. 1A) and Microscopy Research and Technique

Fig. 2. Three-dimensional (A) and two-dimensional (B) AFM images of urothelial apical surfaces. Urothelial plaques in the apical plasma membrane are denoted with asterisks. The curves represent the depth (Z) and the diameter (X) of urothelial plaques 1 and 2 (C). Scale bar 300 nm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

platinum coating of the sample (Fig. 1B). The hinge regions look thicker in platinum-coated samples (Fig. 1B) in comparison to carbon-coated samples (Fig. 1A). In immuno-FESEM, uroplakins were detected in urothelial plaques, but not on hinge regions (Fig. 1B). In both FESEM approaches, the variability of urothelial plaque sizes was seen. AFM showed that the urothelial surface is fully covered with urothelial plaques separated by topographically higher hinge regions (Figs. 2A and 2B). Calculated curves showed variations in plaque topography. Curves were more flat in central regions and toward hinges they became steeper (Fig. 2C and Supporting Information Fig. 1). With AFM topography

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analyses, we additionally confirmed that plaques have very variable sizes. The FRIL method enabled us to observe intramembrane particles in the fracture plane of the apical plasma membrane. Numerous uroplakin-positive intramembrane particles formed urothelial plaques interconnected with hinge regions (Fig. 3). In the hinge regions, a few uroplakin-negative intramembrane particles were seen (Fig. 3). High pressure frozen samples revealed the scallopshaped apical plasma membrane of umbrella cells (Fig. 4A). Strong immunogold labeling confirmed the presence of uroplakins in the apical plasma membrane (Fig. 4B). However, there was no clear distinction between urothelial plaques and hinge regions. The statistical analyses are summarized in Figure 5. Analysis of hinge-to-hinge diameter measurements obtained by FESEM (carbon), FESEM (platinum), AFM, and FRIL showed that means are statistically different (P < 0.001; one-way ANOVA on ranks, since

variances were not equal). The pairwise comparisons of means (Tukey’s test) showed no statistically significant differences between the hinge-to-hinge diameters of urothelial plaques measured on the FESEM images after carbon coating (524 6 28 nm), those measured on the FESEM images after platinum coating (579 6 25 nm) and those measured on AFM twodimensional images (579 6 29 nm) (Fig. 5). The mean hinge-to-hinge diameter of urothelial plaques calculated from all three techniques was 560 6 16 nm (n 5 60). In comparison, hinge-to-hinge diameters of urothelial plaques were significantly larger (795 6 48 nm) in FRIL images. We found significant differences in the following pairwise comparisons: FRIL vs. FESEM (carbon), FRIL vs. FESEM (platinum), and FRIL vs. AFM (Tukey’s test; P < 0.05) (Fig. 5). The mathematical analyses of AFM images enabled us to graphically represent the curves of urothelial plaques in their maximal dimension (Fig. 2C Supporting Information Fig. 1). AFM showed a mean urothelial plaque depth of 140 6 9 nm (n 5 20) and a mean curve length of 652 6 31 nm (n 5 20). Table 1 summarizes minimal, maximal and average hinge-to-hinge diameters of urothelial plaques measured by different microscopic techniques, and in addition for AFM technique the minimal, maximal and average curve length dimensions are presented. There were no statistically significant differences between the urothelial plaque curve length and the hinge-to-hinge diameter measurements in AFM images (Student t test, P > 0.05). DISCUSSION We have examined the apical surface of the normal urothelium with different microscopic techniques. Microscopic techniques used in this study complement one another and gave a new insight into the urothelial apical plasma membrane molecular ultrastructure and morphology.

Fig. 3. The FRIL image shows a fracture plane of the apical plasma membrane of an umbrella cell. The immunogold labeling of uroplakins (black dots) is seen within the urothelial plaques on the E-face of the apical plasma membrane. Urothelial plaques are variable in size and pleomorphic. Note sparse intramembrane particles in hinge regions (asterisks). Inset shows four times enlarged urothelial particles, which are tightly-packed in urothelial plaques. Scale bar 500 nm.

Urothelial Apical Plasma Membrane Ultrastructure To upgrade our knowledge on the ultrastructure of the apical surface we have used and compared FESEM, AFM, and FRIL. FESEM images of the samples coated with carbon and platinum revealed similar general appearances of the urothelial surface

Fig. 4. The ultrathin section image reveals the scallop-shaped apical plasma membrane of an umbrella cell (A). The immuno-TEM image shows strong immunogold labeling of uroplakins (10 nm colloidal gold particles, arrows) on the apical plasma membrane. In the cytoplasm, uroplakin labeling is seen on the membranes of fusiform vesicles (B). L: lumen of the urinary bladder. Scale bars 500 nm.

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regardless of coating procedures. However, hinge regions look thicker in platinum-coated samples as compared to carbon-coated samples. This difference may arise from different specimen preparation routes and thicker platinum-coating applied. Also, the details in platinum-coated specimens are more visible due to higher secondary electron yield of platinum vs. carbon, i.e., d(Pt) > d(C) (Goldstein, 1992). The AFM enables image recording in different modes. The urothelial surface was previously recorded in contact mode, which gave a blurry appearance of urothelial plaques (Kreplak et al., 2007). We used the tapping mode, since it is advantageous for the imaging of the biological specimens as it limits the alteration of

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the surface by minimizing the lateral forces and the contact time between the sample and the tip (Morandat et al., 2013), which gives sharp images. In the FESEM, AFM and conventional SEM, the samples are chemically fixed. For FRIL the cells are cryofixed before fracturing and replica preparation. This makes FRIL the technique where no alteration of chemical composition of the samples is introduced. In replicas, the urothelial plaques with tightly packed intramembrane particles are clearly distinguished from the hinge regions that contain only sparse intramembrane particles. FRIL also reveals the complexity and diversity of urothelial plaques. However, no data about membrane curvature can be acquired by FRIL, which can be complemented by AFM. AFM images recorded in contact mode gave a mean hinge-to-hinge diameter of 600 nm (Kreplak et al., 2007). Our measurements reveal that there are no significant difference of a mean hinge-to-hinge diameter between the results obtained by the FESEM (carbon coating), FESEM (platinum coating), and AFM. Moreover, the mean urothelial plaque diameter of the normal mouse urothelium measured in our study using FESEM (carbon), FESEM (platinum), and AFM analyses was 560 6 16 nm, which is in the middle of the range previously reported (Kachar et al., 1999; Kreft and Robenek, 2012; Severs and Hicks, 1979). Besides the visualization of the surfaces and the measurement of urothelial plaque diameter, AFM enables the calculation of the heights/depths and the length of each urothelial plaque curve. The mean curve length (652 6 31 nm) was in average for a 1.2-fold longer than the mean hinge-to-hinge diameter measured by AFM (579 6 29 nm), although there were no significant differences. However, the mean AFM curve length was shorter than the mean diameter measured by FRIL (794 6 48 nm). One possible reason for that is scrapping of urothelial cells from the urinary bladder wall TABLE 1. Minimal, maximal, and average hinge-to-hinge diameters of urothelial plaques measured by different microscopic techniques Microscopic technique

Fig. 5. The mean hinge-to-hinge diameters (6SE) of urothelial plaques, obtained by different techniques (n 5 20, i.e., in each technique the number of hinge-to-hinge diameters was 20). There are statistically significant differences between pairwise comparisons of the means, i.e., FRIL vs. FESEM (carbon), FRIL vs. FESEM (platinum), and FRIL vs. AFM (Tukey’s test; *P < 0.05).

TABLE 2.

FESEM (carbon) FESEM (platinum) AFM AFM curve length FRIL

Average (nm)

768 867 782 838 1179

524 579 579 652 795

For AFM also the urothelial plaque curve length dimensions are presented.

Advantages

FESEM

- Overview of large apical surfaces

Immuno-FESEM AFM

- Immunolabeling of unfixed samples - Overview of large apical surface - Depth/height measurements - Overview of large apical surface and intracellular membranes - Intense immunolabeling - Simultaneous visualization of cell surface and interior - Intense immunolabeling

TEM Immuno-TEM

Max (nm)

358 403 332 360 471

The advantages and disadvantages of various microscopic techniques in analysis of the urothelial apical plasma membrane

Technique

FRIL

Min (nm)

Disadvantages - Only apical surface of the cell - Variable results due to preparation protocols - Weak immunolabelling - Only apical surface of the cell - Minor alteration of apical surface appearance and dimensions - No overview of large surface areas

In this study, the same anti-uroplakin antibodies were used in all techniques. The optimal dilution of primary antibodies was experimentally determined for individual technique and were least diluted in the case of immuno FESEM.

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during replica preparation. These cells therefore lack the normal tension from smooth muscle layer of the bladder wall and their apical plasma membrane might therefore be distended due to processing technique. For planning the study it is therefore crucial to be aware of the advantages and disadvantages of different techniques and when analyzing the results one must be aware of technique limitations (Table 2). Uroplakin Distribution in the Apical Plasma Membrane of Umbrella Cells To explain the variability of urothelial plaque structure and sizes, the study of the distribution of integral membrane proteins, uroplakins, is crucial. ImmunoTEM, immuno-FESEM and FRIL have all confirmed the presence of uroplakins in urothelial plaques (Min et al., 2003; Wu et al., 1994). The optimal dilution of primary antibodies was experimentally determined for each individual technique. Immuno-TEM and FRIL gave intense labeling, while immuno-FESEM labeling was weaker, although anti-uroplakin antibodies were in this case the least diluted. One of the reasons could be the less accessible binding sites for these antibodies in immuno-FESEM technique. Furthermore, FESEM and AFM did not show 16-nm uroplakin particles within urothelial plaques, while they were seen by FRIL. Another significant advantage of FRIL was that it enabled defining of the borders between urothelial plaques and hinge regions. Our results, that urothelial plaques and hinge regions are both highly pleomorphic, supports our previous observations (Kreft and Robenek, 2012) and also show that gradual aggregation or segregation of urothelial plaques takes place in the apical plasma membrane of umbrella cells. Our study of urothelial apical plasma membrane molecular ultrastructure therefore contributes additional insights into the dynamics of the urothelial apical surface. This knowledge provides the framework for understanding normal urothelial function, since the maintenance of the blood–urine barrier depends on the molecular ultrastructure of the urothelial plaques (Hu et al., 2000, 2002). Moreover, it was previously shown that small urothelial plaques are characteristic for urothelial preneoplastic lesions (Zupancˇicˇ et al., 2011) and our results therefore should be considered in diagnostic validation of urothelial pathogenesis. CONCLUSIONS We present how various microscopic techniques contribute to our understanding of the apical plasma membrane structure of urothelial cells. We combine our results with the goal to stimulate debate on usefulness of unique advantages of all the techniques used in this study and to show how these techniques complement one another. The examples discussed illustrate the impact of microscopic techniques in advancing our understanding of selected aspects of urothelial cell biology. The information that the microscopic techniques discussed here have provided is unique and further exploitation of these approaches may be expected in future urological studies and research of other membranes and surfaces. ACKNOWLEDGMENTS We thank Prof. Dr. Tung-Tien Sun for the generous gift of uroplakin antibodies. We express gratitude to

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