First published online September 12, 2003
doi: 10.1242/10.1242/jcs.00811
Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle
Guangwei Min1,
Ge Zhou1,
Matthieu Schapira1,
Tung-Tien Sun2 and
Xiang-Peng Kong1,*
1 Structural Biology Program, Skirball Institute of Biomolecular Medicine, Departments of 1Biochemistry, New York University School of Medicine, New York, NY 10016, USA
2 Dermatology, 2Pharmacology and 2Urology, 2Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016, USA
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Fig. 1. Mouse urothelial plaques. (A) Concave urothelial plaques make up almost the entire mouse urothelial apical surface (arrows) as well as large cytoplasmic fusiform vesicles (arrowheads). Scale bar: 100 nm. (B) A negatively stained, isolated mouse urothelial plaque showing a crystalline hexagonal array of 16 nm uroplakin particles. Scale bar: 100 nm. (C) The calculated diffraction pattern of a frozen-hydrated mouse urothelial plaque. Each spot with a signal/noise ratio of >1 is shown as a square; the size of the square is proportional to its signal/noise ratio. The number in each square is the `IQ' number of the diffraction spot [IQ of 1 to 7 correspond to signal-to-noise ratio of 7 to 1, respectively (Henderson et al., 1990 )]. Circles are drawn at contrast transfer function (CTF) correction zeros; one spot at 6.9 Å (IQ=3) resolution is marked with an arrow.
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Fig. 2. Projection maps of the mouse urothelial plaque. (A) A map calculated from six untilted crystals, including all data of up to 7 Å resolution. (B) Another projection map calculated using data of up to 12 Å resolution. For comparison, the outlines of the 16 nm particles according to the 3D reconstruction (shown in Fig. 4A) are marked by unbroken white lines; the symmetry elements of twofold (ovals), threefold (triangles) and sixfold (central hexagon) axes are indicated.
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Fig. 3. The phase and amplitude variations along some of the high resolution lattice lines. The data points included all the spots with IQ<6 after merging all the data from the tilt series. The fitting curves were computed using the LATLINE program from the MRC software suite (Agard, 1983).
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Fig. 4. The 3D structure of the 16 nm mouse uroplakin particle at 10 Å resolution. (A) The top view of the 3D density map of a mouse 16 nm uroplakin particle that is contoured at the 1.5 level. The boundary of a `subunit' consisting of an inner and an outer subdomain is outlined in blue. (B) The particles as seen in a hexagonal crystalline array with one unit cell illustrated. (C) The side view of the 16 nm particle showing, from top to bottom, the joint (J), trunk region (TK), transmembrane domain (TM) and cytoplasmic domain (C). (D) One of the six inverted U-shaped subunits of the 16 nm particle, consisting of an inner subdomain (left) connected to an outer (right) subdomain via a (top) horizontal joint (j; outlined in blue). This inverted U-shaped subunit presumably represents a fundamental building block of the 16 nm particle (Staehelin et al., 1972; Hicks et al., 1974). (E) The inner subdomains of two neighboring subunits are connected via a minimal contact between them (double arrowhead). In A, the stellate-shaped particle has a maximal diameter of 17.5 nm and has a large lipid-filled, central hole ( 6 nm in diameter); in C the particle is about 12 nm tall with a 6.5 nm extracellular and a 0.5 nm cytoplasmic region [from atomic force microscopy data (Min et al., 2002)]. All panels except B are to the same scale; bar (in A) 2 nm. The unit cell in B is 16.5 nm in length.
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Fig. 5. Cross-sections of the 16 nm mouse uroplakin particle and placement of the transmembrane helices. (A) A 16 nm particle with the positions of the six sections (20 Å) shown in C-H (top to bottom) indicated by the horizontal lines. (B) A vertical section of one outer subdomain. A tunnel is present that appears to traverse through the entire outer domain (arrowheads in B,E-H). In F, the minimal link between the inner subdomains of neighboring subunits (arrow). (I) Five transmembrane helices, each represented by a 1 nm circle, can fit in the middle of the transmembrane zone (as shown in H). Although F-H seem to indicate the presence of a tunnel also in the inner subdomain, this tunnel does not appear to traverse through the entire height of the subdomain. (J) Hypothetical assignment of uroplakin pairs Ia/II and Ib/III to the inner and outer subdomains, respectively, of the 16 nm urothelial particle. The outlines of the 16 nm particle are drawn in thin blue lines in C-J.
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Fig. 6. A schematic model of the 16 nm uroplakin particle. (A) A model in which the inner subdomains, outer subdomains and the joints of the 16 nm urothelial particle are represented by blue cylinders, green cylinders and yellow flexible hinges, respectively. The transmembrane zone is indicated with darker colors. (B) A tilted view of the model. (C) A hypothetical scheme in which the bending of the joint region (yellow) caused by bacterial binding leads to a twist between the inner and outer subdomains; conformational changes such as this may play a role in the transmembrane signal transduction causing urothelial umbrella cells to undergo morphological and physiological changes.
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© The Company of Biologists Ltd 2003
