Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this investigation, we employed cryoelectron microscopy to elucidate the three-dimensional structure of the uroplakin complex embedded within the porcine AUM at a resolution of 3.5 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.


Introduction
The urothelium, which lines the urinary tract, performs a dual function: it serves as a permeability barrier, effectively preventing the leakage of urine components into surrounding tissue while undergoing morphological changes to adapt to the distention and contraction that occurs during the micturition cycle Wu et al., 2009;Apodaca, 2004;Khandelwal et al., 2009). The apical surface of the urothelium is covered by numerous structurally rigid membrane plaques, known as asymmetric unit membranes (AUM), as observed by electron microscopy (Hicks, 1965;Hu et al., 2000;Jenkins and Woolf, 2007). The AUMs are composed of hexagonally arranged 16-nm uroplakin complexes containing four major proteins, UPIa, UPIb, UPII, and UPIIIa (Lee, 2011). UPIa and UPIb have four transmembrane domains and belong to the tetraspanin family (Yu et al., 1994). Meanwhile, UPII and UPIIIa each possess a single transmembrane domain and form Ia/II and Ib/IIIa heterodimers within the endoplasmic reticulum (ER) in conjunction with UPIa and UPIb, respectively (Hu et al., 2005;Tu et al., 2002). In the post-Golgi compartment, UP heterodimers are assembled into paracrystalline arrays (Liang et al., 2001). The AUM's quasi-crystalline symmetry has been leveraged in the structural investigation of the uroplakin complex, as documented by several studies (Min et al., 2006;Min et al., 2003;Min et al., 2002;Walz et al., 1995;Zhou et al., 2001). While cryo-electron microscopy of tannic-acid-stained AUM has enabled the identi cation of the orientation of transmembrane helices in tetraspanin pairs (Min et al., 2006;Wang et al., 2009), the resolution of the resulting electron density map remains insu cient for a comprehensive study of the secondary structures of the extracellular domains or for the localization of individual UP subunits.
The uroplakin complex also plays a crucial role in the development of urinary tract infections, as it facilitates Escherichia coli's attachment to the urothelium (Hultgren et al., 1993;Hooton and Stamm, 1997). This attachment is facilitated by the interaction between the N-glycosylated UPIa's mannose residues and the FimH lectin located at the tip of the bacterium's pili . The binding of FimH to UPIa triggers phosphorylation of UPIIIa and subsequent apoptosis of the urothelial cells (Thumbikat et al., 2009). Furthermore, it has been postulated that this FimH-UPIa interaction induces conformational alterations in the transmembrane helix bundles (Wang et al., 2009). However, it is unclear whether FimH's binding to a exible carbohydrate chain can change the conformation of a rigid uroplakin complex. In addition, the positioning of the heterodimer has been inferred by comparing the uroplakin structure with and without bound FimH [Min G, 2002]. Nevertheless, the low resolution of the electron density map hinders the validity of these conclusions.
In this study, we reconstructed the 3D structure of the uroplakin complex by observing tilted AUMs using cryo-electron microscopy. By combining random conical tilt and single particle analysis (Sorzano et al., 2015;Frank et al., 1978;Righetto et al., 2019;Radermacher, 1988), we were able to build an accurate model of the uroplakin hexameric complex.

Cryo-Electron Microscopy of AUM
To determine the high-resolution structure of the uroplakin complex, we isolated the AUM from porcine urinary bladders, leveraging its resistance to sarkosyl (Liang et al., 1999), and employed cryo-electron microscopy techniques. Initially, we used cryo-electron tomography to observe the AUM; however, the obtained structure was of low resolution as a result of the irradiation-induced distortion of the specimen To our surprise, we found that the hexagonally arranged liquid-crystalline lipids were not only within the central pore but also in the inter-complex regions, spanning the entire outer lea et of the AUM (Fig. 1D, 2A, Movie S1). The crystalline lipids were tightly packed, with a distance of ~ 4.8 Å between them (Zhao et al., 2021;Ghysels et al., 2019). Further local re nement focusing on the extracellular domain revealed the additional outermost part of the lipid densities, suggesting a structural heterogeneity in the association between the lipids and the uroplakin complex ( Figure S1A). Although the head groups of most lipids were unresolved due to the insu cient vertical resolution and the random orientations, densities of some lipids were continuous with those of the side chains, suggesting possible interactions (Fig. 2C). Some lipids appeared to be stabilized by vertically-aligned bulky side chains (

Subunit arrangement of the uroplakin complex
The cryoEM structure of the uroplakin complex reveals that the extracellular domains of both UPII and UPIIIa exhibit distinctive beta-sheet structures ( Fig. 2A, B, D), which could have functional implications in protein binding or adhesion of pathogens. With these extracellular domains of UPII and UPIIIa, uroplakin heterotetramer overall adopts a Y-shaped conformation, which creates a substantial groove that may also provide a binding interface for other proteins ( Figure S2A, white arrow). Adjacent heterotetramers are connected through arches formed by the UPII and UPIIIa extracellular domains. Interestingly, beneath these arches, a channel is present, which is continuous with the central cavity space ( Figure S2A, red arrows). The biochemical robustness of the uroplakin hexameric complex, despite its abundance of inner cavities, can likely be attributed to the reinforcement provided by the lipid domains.
The prevailing hypothesis concerning the subunit arrangement of the uroplakin complex suggests that Ia/II and Ib/IIIa heterodimers form the inner and outer subdomains, as inferred from electron microscopy studies of FimH-bound AUM Min et al., 2002). However, our high-resolution reconstruction revealed that Ia/II and Ib/IIIa heterodimers constitute the outer and inner subdomains, respectively ( Fig. 2A). Additionally, it has been reported that the cleavage of UPII at Arg84 by furin results in the removal of the N-terminal pro-sequence (amino acids 25-84) from the uroplakin complex Hu et al., 2005). However, our ndings suggest that the prosequence forms the external surface of the complex through the formation of several beta sheets ( Figure S2B). Moreover, our reconstruction uncovered the presence of carbohydrate chains on six N-glycosylated residues, namely UPIa Asn169, UPIb Asn131, UPII Asn28, Asn57, UPIIIa Asn139, and Asn170 in accordance with previous studies (

Inter-subunit interactions within the uroplakin complex
Our map and model suggest that the hexameric complex is assembled through three inter-subunit interfaces: the Ia-Ib, II-IIIa, and IIIa-IIIa interfaces. The Ia-Ib interface appears similar to that reported in the PRPH2-ROM1 tetraspanin complex ( Fig. 5A) (El and Gros, 2022). The interface is stabilized by various interactions, including a hydrophobic interaction between Phe196 of UPIa and Leu155 of UPIb (Fig. 5B), and a hydrogen bond between Asn195 of UPIa and Gln130 of UPIb (Figs. 5C and 5D). The resolution of the II-IIIa interface, particularly near the exible loop 90-101 of UPII, is of lower quality. However, it appears to be stabilized by a hydrophobic cluster of a disul de bond between Cys51-80 and Pro79 of UPII, a disul de bond between Cys47-Cys110, and Leu113 of UPIIIa (Fig. 6A).
Finally, the UPIIIa extends its loop 137-161 into the groove of an adjacent IIIa, forming an interface stabilized by a hydrophobic cluster composed of Pro146 of IIIa, Leu66, and Trp182 of the adjacent IIIa (Figs. 6B). Additionally, a hydrophobic cluster between the transmembrane helices of UPIa and Ib appears to stabilize the heterotetramer (Fig. 6C). These multiple interactions are thought to contribute to the highly rigid nature of the uroplakin complex and the AUM.

Lipidomic analysis of the AUM
A lipidomic analysis was conducted to discern the lipid species comprising the characteristic paracrystalline array of the AUM. The lipid composition of the sarkosyl-insoluble fraction, primarily consisting of the AUM, was compared to that of the total urothelial membrane homogenates ( Fig. 7 and Supplemental spread sheet 1). The results, as depicted by the LC-MS pro le, revealed the enrichment of hexosylceramides in the AUM, in accordance with the previous studies (Hicks et al., 1974;Stubbs et al., 1979;Watanabe et al., 2022). We also observed enrichment of ceramides, which is likely to be fragmentation products of ionization. These results suggest that the hexagonally-arranged lipid domain is composed of hexosylceramides.

Discussion
The structures of the AUM and the uroplakin have been extensively analyzed utilizing electron microscopy (Min et al., 2006;Min et al., 2003;Min et al., 2002;Walz et al., 1995;Zhou et al., 2001). One of the studies achieved a resolution of approximately 6 Å in the membrane plane, yet the vertical resolution was limited to 12.5 Å, thus failing to uncover the residue-level information (Min et al., 2006). This limited structural information led to incorrect subunit assignment of the uroplakin (Min et al., 2002). The Ia/II heterodimer has long been believed to form the inner subdomain of the uroplakin hexameric complex based on the observation of additional densities in the difference map between the uroplakin structures with and without bound FimH, which interacts with glycosylated UPIa (Zhou et al., 2001). However, our reconstruction revealed that these "additional densities" are likely artifacts of the low-resolution EM maps, as the Ia/II heterodimer actually constitutes the outer subdomain of the uroplakin ( Fig. 2A and  S2B). Reports indicate that FimH binding to UPIa induces conformational changes in the uroplakin's transmembrane domains (Wang et al., 2009). We also solved the structure of FimH-bound uroplakin, but no structural changes were observed ( Figure S3A). The high exibility of the carbohydrate chain and the fast on/off rate of FimH-mannose binding make it challenging to visualize the attached FimH and the . This observation holds true for the UPII prosequence. The question arises as to why only a single UPII band was observed in the electrophoresis of the AUM. One potential explanation is that the estimated molecular weight of the glycosylated UPII prosequence is roughly 14 kDa, and thus it may align with the 15 kDa band. Another possibility is that the heterogeneous nature of the glycosylation chains, which make up over half of the prosequence's mass, may not produce a distinguishable band in the electrophoresis.
The epithelial lining of the bladder demonstrates a distinct impermeability to water, protons, and urea, effectively preventing the in ltration of urinary substances into the surrounding tissue (Negrete et al., 1996;Lavelle et al., 1998). Depletion of uroplakin complexes from the urothelial apical membrane by UPIII knockout leads to a two-fold increase in water permeability. Yet, the urothelial barrier maintains a substantial degree of resistance to water and urea permeation (Hu et al., 2002). These observations imply that the lipids and the uroplakin exert complementary in uences on the barrier properties of the urothelial apical membrane. Our lipidomic analysis revealed an enrichment of hexosylceramide in the AUM ( . It is noteworthy that the uroplakin-free hinge regions that connect the neighboring AUM plaques also exhibit resistance to sarkosyl and alkali treatments, indicating highly specialized structures (Liang et al., 1999). These hinge regions may serve to prevent the penetration of small molecules at the boundary between the plaques.
While our team was successful in constructing a model of the uroplakin complex, we encountered a limitation in the resolution of the vertical dimension, which hindered our ability to con dently determine the precise positioning of the side chains. To overcome this challenge, we propose the utilization of highresolution subtomogram averaging, as it has the capability to bypass the overlap of molecules in the high-tilt views. However, to effectively implement this technique, it is imperative to devise an advanced algorithm that can accurately rectify the signi cant deformation of the AUM caused by irradiation.

Isolation of porcine AUM
Fresh porcine bladders were obtained from a local slaughterhouse. The AUM was isolated according to the previous study with modi cations (Liang et al., 1999). The urothelium was scraped from the luminal surface with a medicine scoop and suspended in ice-cold PBS. After centrifugation at 1,500 ×g at 4 ℃ for 5 minutes, the pellets were homogenized with a Dounce glass homogenizer in buffer A (10 mM Hepes, pH 7.4, 1 mM EDTA, and protease inhibitor cocktail). After centrifugation at 2,500 × g at 4 ℃ for 10 minutes, the pellets were resuspended in buffer A, loaded onto a 1.6 M sucrose cushion, and centrifuged at 46,000 ×g at 4 ℃ for 30 minutes. The membrane fraction concentrated at the interface was collected, loaded onto a 1.6 M sucrose cushion, and centrifuged once again. The collected membrane fraction was resuspended in buffer A plus 2% sarkosyl and incubated at room temperature for 10 minutes. Sarkosylinsoluble membranes were collected after centrifugation at 18,000 ×g at 4°C for 30 minutes. The pellets were further washed by resuspension in 25 mM NaOH, and centrifuged at 18,000 ×g at 4 ℃ for 10 minutes. The pellets of the AUM were washed twice with buffer A and proceeded to electron microscopy.

Puri cation of FimH-FimC complex
The cDNA of mH and its chaperone mC genes were cloned from E. coli DH5a into pACYCDuet-1 bicistronic plasmid (Sigma-Aldrich, Burlington, MA), and the resulting co-expression plasmid was introduced to E coli. BL21 (DE3) (Pellecchia et al., 1999). The bacteria were grown at 37℃ in LB medium containing chloramphenicol (34 µg/ml). At an OD600 of 0.7, IPTG was added to a nal concentration of 0.5 mM. The cells were further grown for 18 hours at 15℃, harvested by centrifugation, washed by PBS, and disrupted by sonication. After removing cell debris by centrifugation, the supernatant was applied to Ni-NTA resin (Qiagen, Germantown, MD) and eluted with 20 mM Tris-HCl pH 8.0, 0.3 M imidazole. Fractions containing FimH-FimC were pooled and loaded onto a gel ltration column (ProteinArk, Rotherham, UK) equilibrated with 20 mM Hepes-NaOH pH 7.4, 150 mM NaCl. Fractions containing FimH-FimC were dialyzed against buffer A and were concentrated using Vivaspin 2 (Sartorius, Göttingen, Germany). For cryo-EM, two times molar-excess FimH-FimC was mixed with the AUM (0.05 mg/ml) and incubated for 1 hour at 4℃ before plunge-freezing.
Cryo-electron microscopy of the AUM The AUM was suspended in buffer A at a concentration of 0.05 mg/ml. 3 µl of the sample was applied to freshly glow-discharged holey carbon grids, Quantifoil R1.2/1.3 Cu/Rh 200 mesh (Quantifoil Micro Tools GmbH, Großlöbichau, Germany), blotted from both sides for 3 seconds at 4 ℃ under 99% humidity and plunge frozen in liquid ethane using Vitrobot Mark IV (Thermo Fisher Scienti c, Waltham, MA). Images were recorded using a Titan Krios G4 microscope at the University of Tokyo (Thermo Fisher Scienti c) at 300 keV, a Gatan Quantum-LS Energy Filter (Gatan, Pleasanton, CA) with a slit width of 20 eV, and a Gatan K3 BioQuantum direct electron detector in the electron counting mode. The nominal magni cation was set to 64,000 × with a physical pixel size of 1.35 Å/pixel. Movies were acquired using the SerialEM software (Mastronarde, 2005), and the target defocus was set to 2.5-4.5 µm for tomography and 1-3 µm for single particle analysis (SPA). For tomography, each movie was recorded for 0.18 seconds with a total dose of 1.26 electrons/ Å 2 and subdivided into 10 frames. The angular range of the tilt series was from − 60° to 60° with 3.0° increments using the dose-symmetric scheme (Hagen et al., 2017) or the continuous scheme. The total dose for one tilt series acquisition is thus 50 electrons Å 2 . For SPA, each movie was recorded for 6.7 seconds with a total dose of 50 electrons/ Å 2 and subdivided into 50 frames. Specimens were tilted at 0, 30, 45, and 55°. The ratio of tilted images was 1:1:2:2 for 0, 30, 45, and 55°, respectively.

Data processing for tomography
Movies were subjected to beam-induced motion correction, image alignment, CTF correction, and reconstruction by back-projection using the IMOD software package (Kremer et al., 1996). Tomograms were 8 × binned and the centers of each uroplakin particle were manually selected using 3dmod tool.
Volumes with 30-pixel 3 dimensions were extracted from 8 × binned tomograms and were averaged using the PEET software (Nicastro et al., 2006). A randomly selected subtomogram was used for the initial reference. Alignments were repeated twice for 8×binned and once for 4×binned tomograms with 60-pixel 3 dimensions. The averaged subtomogram was used for the reference in the subsequent SPA.
Data processing for SPA Image processing was conducted using CryoSPARC v4.2.1 (Punjani et al., 2017) ( Figure S1A). 4 × binned 128 pixel 2 images were picked and extracted from motion-corrected and CTF-corrected micrographs using the projections of the averaged subtomogram as references. The particle stack was cleaned by ve rounds of 2D classi cation and three rounds of heterogeneous re nement. Following local motion correction and CTF re nement, homogenous re nement of unbinned 512 pixel 2 particles yielded a global resolution of 5 Å. At this point, the crystalline lipids were not observed. To increase the resolution, six uroplakin particles surrounding the central particle were subtracted, and the subsequent local re nement yielded a global resolution of 3.5 Å, visualizing the crystalline lipids. The edges of the AUM were often folded, which lled the distribution of orientations. However, the overlapping molecules compromised the precision of the side view alignment. The resolutions in the transverse and the vertical direction were calculated using the 3DFSC function of CryoSPARC.

Model building
The initial model was generated using AlphaFold-multimer (
No manual curation was performed for individual identi cations, as the results were only used to estimate lipid classes abundant in AUM preparation. Peak intensities were normalized using built-in function of MS-DIAL, based on total signal for identi ed lipids (mTIC). R version 4.2 software was used for differential analysis using normalized peak intensity data exported from MS-DIAL. Fold enrichment values were calculated as a ratio for AUM versus control using triplicate, independently prepared samples.

Declarations Data availability
The map and the model are available on the EMDB under the following accession numbers: EMD-36340 , and PDB 8JJ5.

Ethics & Inclusions statement
This research strictly followed the ethical guidelines and standards established by our institutions, University of Yamanashi and the University of Tokyo, located in Japan. The entire research process, from study design and implementation to data ownership and the authorship of publications, was carried out exclusively by the authors listed, all of whom are researchers at the aforementioned institutions. As this research was conducted in Japan, by Japanese researchers, it is both locally relevant and compliant with local standards and regulations. The roles and responsibilities of each researcher were established and agreed upon at the outset of the research process. We did not undertake capacity-building plans for external researchers, as our team had all the necessary capabilities to conduct this research. The research has not been restricted or prohibited within our local context. Therefore, there was no need for any special exceptions or permissions to be sought from local stakeholders. Our research does not involve any elements that could lead to stigmatization, incrimination, discrimination, or personal risk to participants.
It also does not pose any health, safety, security, or other risk to the researchers involved. The research did not involve the transfer of any biological materials, cultural artefacts, or associated traditional knowledge out of Japan. Hence, no bene t sharing measures were discussed or required. In the formulation of our research and the analysis of our results, we have considered and cited local and regional research relevant to our study.   Interactions between the UPIa/Ib transmembrane helices and the lipids.Vertically-aligned bulky side chains appear to be incorporated into the crystalline lipids. Top (left) and side (right) views are shown.   Lipidomic analysis of the AUM. The chart displays the fold-enrichment of each lipid species detected in the sarkosyl-insoluble AUM fraction relative to the whole bladder scrape. Lipids were extracted from both sample types and analyzed using liquid chromatography-mass spectrometry (LC-MS). The peak heights were normalized by dividing them by the total signal for all identi ed lipids for each experiment. The foldenrichment values indicate the difference in relative abundance of each lipid species in the sarkosylinsoluble AUM fraction compared to the whole bladder scrape, with higher values suggesting an enrichment in the AUM fraction. The result for positive ion mode is shown. Data represent the mean foldenrichment and standard deviation from three independent experiments (n = 3).