J. Mol. Bid. (1969) 46, 593-596

A Hexagonal Arrangement of Subunits in Membrane of Mouse Urinary Bladder

Surface membranes of mouse urinary bladder have associated with them a hexagonal structure with a unit cell side of 160 A containing subunits about 50 A in diameter packed on a p6 lattice. The structure has been confirmed and retied by analysis of electron micrographs of negatively-stained specimens using an optical diffractometer.

The free surfaces of the epithelial cells of mouse ureter and bldler are characterized by an asymmetry in the thickness of the two leaflets of the unit membrane (Hicks, 1965,1966). This phenomenon appears in the surface of the lumen and also in the disc-shaped vesicles which are found below the surface. The thickness of the inner leaflet has the usual vdue of about 30 8, while the outer one is about 50 A thick. Porter, Kenyon & Baclenhausen (1967) found a periodic structure in the thick leaflet consisting of a set of striations about 140 A apart. We have now found that this periodicity is due to a hexagonal structure in the outer surface of the membrane and have resolved subunits about 50 i% in diameter, arranged in a surface lattice with the symmetry of the plane group p6 (International Tables for X-ray Crystallography (1952). Vol. 1. Birmingham: Kynoch Press).

Plate I is an electron micrograph of a thin section of mouse bladder showing mem- branes of the lumen (L) and of the disc-shaped vesicles. Where the membranes are sectioned obliquely, a hexagonal pattern (H) can be seen consisting of three sets of striations at 120” to each other. The striations are about 140 A apart; and thus, if seen end-on, could account for the rows of clots (D) 140 d apart, seen occasionally in cross-sections of the thick leaflet (Porter et al., 1967). A similar hexagonal structure has been observed by using the freeze-fracture method (Plate II). Here also the distance between the rows forming the pattern is about 140 A.

The structure is seen most clearly in negatively-stained material. For these prepa- rations, membrane fragments were obtained either by scraping the surface of the epithelium with a needle or by mild sonic&ion, rtnd negatively stained with neutral- ized phosphotungstate.

An electron micrograph of a negatively-stained fragment is shown in Plate III. The structure consists of rings packed in a hexagonal array with center-to-center spacing of about 160 8, so that the distance between the rows is about 140 8. Occa- sionally the rings are seen to be made up of six subunits forming a, hexagon, as in the circled area. The center-to-center distance between subunits is about 50 A, and the hexagon on which they lie is skewed with respect to the main rows of hexagons. As a result of this skewing, the subunits lie on rows forming a set of fine striations about 50 d apart, which make an angle of 19” to the main rows. These striations can best be seen by viewing the micrograph obliquely along the direction of the long arrow. (Two other (fainter) sets of these striations are visible in directions at 120”

593

594 J. VERGARA, W. LONGLEY AND J. D. ROBERTSON

to the arrow.) An arrangement of subunits which accounts for these appearances is depicted by the model shown in Plate IV(d) and is described in detail later. (The illustrations in this paper are printed so that the direction of skewing of the subunits is the same in each. The absolute sense of this rotation is unknown, since it depends on which side up the membranes lie on the supporting substrate and we have no way so far of determining this.)

A clearer picture of the structure can be obtained by the use of optical diffraction (Klug & Berger, 1964) and by optical filtering (Klug & De Rosier, 1966). Plate IV(b) shows the optical diffraction pattern obtained from the electron micrograph repro- duced in Plate IV(a). The diffraction pattern consists of a hexagonal array of spots having sixfold symmetry. The intense spot marked with an arrow, and its five symmetry-related fellows, are produced by the 50 A striations seen on the micro- graphs. Plate IV(c) shows the optically reconstructed image obtained by using an opaque mask with holes which permitted only those rays forming the hexagonal array (including the central ray) to pass through, thus removing background “noise”. The structure is now clearly seen to consist of rings of six subunits lying on the ver- tices of hexagons which are rotated about 19” with respect to the rows of hexagons.

Drawings were made of the structure and reduced photographically for optical diffraction. The subunits were represented as white circles of a diameter such that t,he intensities on the diffraction pattern fall to zero at a radius corresponding to 35 A. This is the spacing at which the diffraction pattern of the micrograph fades out. The circles were placed at the vertices of hexagons rotated away from the lines join- ing the centers of adjacent hexagons (i.e. the edge of the hexagonal cell). Various models were drawn with hexagons of different radii and different angles of skew and their diffraction patterns were photographed. The diffraction pattern which most closely matched that of Plate IV(b) is shown in Plate IV(e), and the model from which it was obtained is reproduced in Plate IV(d). The correspondence between the two diffraction patterns is reasonably close; thus the model can be taken as a good repre- sentation of the structure visualized by electron microscopy. In this model the centers of the subunits were placed at a radius corresponding to about 50 A, and were rotated 19” away from the cell edge. This position is near to that for closest packing of circles in this lattice though the radius is less than that required for perfect closepacking, so that the subunits cluster into separate rings of six. In fact, the hexagons in the membrane are probably touching each other, though penetration of the stain be- tween them makes them appear to be separate. The structure is best explained as a result of packing identical subunits on a p6 plane lattice as shown in Figure 1 (taken from Casper & Klug, 1962). This drawing shows how asymmetrical subunits may be arranged with approximate close packing on a p6 lattice and also indicates how the striations at 19” to the cell edge are produced. The letters indicate the different sorts of contacts between the subunits.

Hexagonal patterns have been observed before in sections or negatively-stained preparations of cell membranes (Benedetti & Emmelot, 196Su) and in bacterial cell walls (e.g. Watson & Remsen, 1969). The unit cell sides in these cases are of various magnitudes between 90 and 160 A. No substructure has previously been reported in these patterns. However, in some of the published photographs fine striations of the type found in bladder can be observed, suggesting that these structures will also turn out to be constructed of subunits as in the lattice described here. For example, in the paper by Benedetti t Emmelot (19683), Figure 3 shows such striations. In this

hATE 1. A thm section of mouse bladder fixed in glutaraldehyde and osmium t&-oxide nn(L stained uith lead citrate and uranyl acetate, showing the lumen (L) and the disc-shaped vesicle.+. The outer leaflet of the membrane is thicker than the inner and is sometimes seen to consist of POU ;i of dots (D) spaced at about 140 A. In oblique section, the membrane shows a hexagonal pattern (H) formed of 3 setv of striations at 120” t,o each other. These st,riations are about 140 A apart and thu-: if seen entl-on would account for the rows of (lots. x 100,OOO.

PLATE III. A fragment of mouse bladder membrane negat,ively stained with phosphotungstic acid. The stain appears black in this reproduction.

The structure consists of rings lying on three sets of rows about 140 A apart, forming a hexagonal array. The center-to-center distance between the rings (i.e. the side of the unit hexagonal cell) is about 160 A. Occasionally, each ring is seen to be composed of 6 subunits t.he centers of which form a hexagon with an edge length of about 50 A (circled). The subunits are lined up on striations that run across the patt’ern at an angle of 19” to the main rows of t)he pattern. These striat,ions are best seen by viewing t,ho plat,e obliquely in the direction of thp arrow. x 200,000.

PLATE I\-. (a) A srdl (magnified) portion of the rlcctron micrograph of Ire~;tti\t,l~-staillc,l( bladdrr membranr~ which was used to form thix pattern in (b). x .iIKJ,tKNl.

(b) The optical diffraction pattern from (a). The spot marked by the arr~nv is produr~l by thv 30 A% striations. The white cross is due to diffraction from the edges of the r&angular ma-k.

(c) Reconstructed Image obtained by mwking the patt,ern of(b) so that ~rrlly thts rays forming t hv hexagonal p&tern were admitted.

(d) A model of the structure incorporating widence from thr &ctron micrographs and from the, vx<)nstruct,ed image. This model gave the diffraction pattern which most rlorely rrwmblvll that of the txloct~ron micrographs.

(v) Diffraction pattern of (d) rrproduccd to the same scale as (h).

LETTERS TO THE EDITOR 595

FIG. 1. A drawing of asymmetric units pecked on ap6 lattice. The units are placed approximately at the positions of closest packing and 8s s result, lie in rows making an angle of 19’ to the cell edge. The letters indicate the different classes of contacts between units. (Taken from Caspar & Klug, 1962.)

case the diameter of the subunits would be about 30 A. A suggestion of subunits can also be seen in a recently published micrograph of a negatively-stained preparation of a bacterial cell wall (Watson & Remsen, 1969) where fine striations can again be seen by viewing the print obliquely.

The nature of the material in the hexagonal lattice of bladder membranes is still unknown. It has been suggested (Hicks, 1965) that the thick leaflet contains keratin, but this could hardly account for the appearance of globular particles as seen here. However, if the subunits are protein, they could be expected to have a molecular weight of around 50,000. It is also unknown whether the structure is an integral part of the unit membrane, replacing the non-lipid components of the usual outer leaflet, or whether it is an extra layer assembled upon the membrane surface.

This work was supported by National Institute of Health grant no. 5ROl NB 07107 and National Science Foundation grant no. GB 6964 to one author (J.D.R.) and by funds from the Damon Runyan Memorial Fund for Cancer Research under grant DRG 884 AT to another (J.V.).

Department of Anatomy Duke University Medical Center Durham, North Carolina, U.S.A.

JUAN VERUARA %kLIAMLONULEY J. DAVIDROBERTSON

Received 9 June 1969, and in revised form 2 September 1969

REFERENCES

Benedetti, E. L. & Emmelot, P. (1968a). In The Me&runes, ed. by Albert J. Dalton, ~01.4, p. 33. New York: Academic Press.

Benedetti, E. L. & Emmelot, P. (19683). J. Cell Biol. 38, 16. C~~XW, D. L. D. & Klug, A. (1962). Cold Spr. Harb. Syrnp. Qwnt. Biol. 27,l. Hicks, R. M. (1965). J. CeU Biol. 26, 26,

596 J. VERGARA, W. LONGLEY AND J. II. ROBERTSO?;

Hicks, R. M. (1966). J. Cell Biol. 28, 21. Klug, A. & Berger, J. E. (1964). J. Mol. BioE. 10, 565. Klug, A. & De Rosier, D. J. (1966). Nature, 212, 29. Porter, K. R., Kenyon, K. & Badenhausen, S. (1967). Protophma, 63, 262. Watson, S. W. & Remsen, C. C. (1969). S&ewe, 163, 686.