THE FORMATION AND ORIENTATION OF BRUSH BORDER VESICLES FROM RAT DUODENAL MUCOSA

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1 J. Cell Set. 47, (1981) 227 Printed in Great Britain Company of Biologist! Limited 1981 THE FORMATION AND ORIENTATION OF BRUSH BORDER VESICLES FROM RAT DUODENAL MUCOSA P. R. HEARN, R. G. G. RUSSELL Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 zrx, U.K., and AND J. FARMER Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, U.K. SUMMARY An electron-microscopic study of various fractions taken during a newly developed method for preparation of brush border vesicles from rat duodenum, has shown that vesiculation of microvilli can occur in situ on the brush border membrane. This mode of formation ensures that the vesicle membranes are oriented as in vivo and that they are 'right-side-out'. The disruption of core protein-fibre appears to be a prerequisite for vesicle formation. INTRODUCTION The brush border membrane of intestinal mucosa cells is the initial barrier across which most nutrients pass into the body. There is, therefore, considerable interest in the mechanisms responsible for the transport of molecules across this specialized membrane. One way in which this is being studied is by examination of the properties of closed membrane vesicles obtained from the intestinal brush border (Eastham, Bell & Douglas, 1977; Freedman, Weiser & Isselbacher, 1977; Hearn & Russell, 1977; Hopfer, Sigrist-Nelson & Groseclose, 1976; Murer, Hopfer & Kinne, 1976; Sigrist-Nelson & Hopfer, 1974). Methods are now available which yield relatively pure brush border vesicles (BBV) (Eastham et al. 1977; Freedman et al. 1977; Kessler et al. 1978; Louvard et al. 1973) and there has been interest recently in methods of determining the orientation of the vesicles (Haase, Schafer, Murer & Kinne, 1978). Clearly it is important to be certain whether or not membrane vesicles are 'right-side-out' before interpreting transport studies in terms of their physiological significance. We have developed a simple technique for preparing brush border vesicles and have used electron microscopy to study the appearance of the brush border membrane at various stages of purification. Address for correspondence: P. R. Hearn, Department of Human Metabolism and Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield Sio 2RX, U.K.

2 228 P. R. Hearn, R. G. G. Russell and J. Fanner METHODS Male Wistar rats weighing g were killed by cervical dislocation and the first 10 cm of duodenum removed. This was opened and mucosal scrapings made using a cold glass microscope slide. For each preparation the scrapings from 4 rats were pooled and homogenized in a motor-driven Teflon-glass homogenizer (50-ml size Jencons H104/10. Clearance o-i mm) employing 5 slow up and down passages of the pestle at 300 rev/min, in 30 ml of a buffer containing 250 min sucrose, iomm MgCl^ o-i tw CaCl a, and 5 mm Tris/HCl at ph 7-4. The homogenate was then subjected to the preparative technique outlined in Fig. 1. RAT DUODENAL MUCOSA SCRAPINGS homogenize in 10 ml A per nut X1000g12min r Su Pi homogenize in 5 ml A per qut X 1000g 12 min rehomogenize X 14000g 15 min R 3 Sup 3 X g 45 min Sup 4 R 4 homogenize in 25 ml A per nut X 14000g30 min BBV Sup s Fig. 1. The scheme for preparing rat duodenal brush border membrane vesicles (BBV). 'A' refers to the sucrose buffer - see text for details. Centrifugation was at 0-4 C in an M.S.E. High speed 25 using an 8 x 50 ml fixed angle rotor. Alkaline phosphatase was used as a marker-enzyme for the brush border and was assayed by the method of Forstner, Sabesin & Isselbacher (1968). Cytochrome C-oxidase was used as a marker-enzyme for mitochondria (Scotocasa, Kuylenstierna, Erstner & Bergstrand, 1967) and cytochrome C-NADPH-reductase was used as a marker-enzyme for endoplasmic reticulum (Scotocasa et al. 1967). Both enzymes were assayed as described in these papers. Protein was determined by the method of Lowry, Rosebrough, Farr & Randall, Samples for electron microscopy were taken as follows: (1) sections of the opened duodenum, (2) sample of mucosal scrapings, (3) sample of pellet R 1( (4) sample of pellet R,, and (5) samples of pellet 'BBV. All tissue samples were taken into a cold fixative containing 3 % (wt/vol.)

3 Brush border vesicle orientation 229 glutaraldehyde in o-i M Na-cacodylate buffer. After overnight fixation the samples were washed with 3 changes of the Na cacodylate buffer before postfixation in 2 % (wt/vol.) osmium tetroxide for 2 h. Samples were dehydrated in a graded series of ethanol solutions before addition of 1 % (wt/vol.) phosphotungstic acid for 1 h followed by propylene oxide for 45 min with 2 changes of solution. After overnight soaking in propylene oxide and Araldite (1:1) the samples were finally embedded in pure Araldite. Samples were sectioned on a Reichart microtome at between 60 and 90 fim and observed under a Jeol Jem T8 microscope. RESULTS AND DISCUSSION The considerable rise in specific activity of alkaline phosphatase over the course of the isolation of BBV (see Table 1) indicates that significant purification of the brush border membrane was achieved by this method of preparation. The centrifugation of S x + S 2 (Fig. 1) was run at g for 15 min as this was found to sediment virtually all mitochondrial marker-enzyme activity. The sedimentation of brush border vesicles at high speed, to give R 4, appears to result in aggregation of the vesicles, possibly due to their coating of glycocalyx derived from the brush border (Ito, 1969). This aggregation probably explains why a purified BBV pellet can be sedimented during the slower centrifugation at g, while microsomes which have not aggregated fail to sediment at this speed. Table 1. Cumulative data on the preparation of rat brush border vesicles (BBV) BBV Initial homogenate Sp. act. % initial act. Sp. act. Alkaline phosphatase (15) (18) Cytochrome C-oxidase Cytochrome C-NADPH (6) ' (6) reductaae NADH oxidase (6) (6) (2) DNA mg/mg protein RNA /tg ribose/mg protein (6) (3) o (6) (3) Sp. act. expressed as /Jmol/min/mg protein Values in parentheses are numbers of specimens analysed. Note: Activity was only detected in 1 BBV preparation out of 6. The recovery of 2 of the major potential membranous contaminants, mitochondria and endoplasmic reticulum, was negligible in the BBV fraction (see Table 1). These findings, together with the substantial recovery of alkaline phosphatase, suggest that this preparation could be useful for solute transport studies. However, in any study which examines the transmembrane fluxes of solutes in membrane vesicles it is essential to show that the vesicles prepared have a uniform membrane orientation and also to determine that orientation. The plates obtained by electron microscopy show that it is possible to follow the process of vesicle formation in rat duodenal mucosa.

4 43* P. R. Hearn, R. G. G. Russell and J. Farmer Fig. 2. Normal rat duodenal brush border. Note regular dimensions of microvilli. Bar, i /im. Fig. 3. Normal brush border sectioned obliquely, giving an apparently vesiculated appearance. Bar, 1 fim

5 Brush border vesicle orientation 231 Fig. 2 shows the brush border of a normal rat duodeum; the microvilli are seen in longitudinal section. Within the membrane sheath of the microvillus the core proteins are seen to run in parallel down the length of the structure. When observed under the electron microscope, brush border microvilli often have the appearance of being vesiculated, as in Fig. 3, but this is caused by oblique sectioning through superimposed layers of microvilli. This can be seen to be the case in Fig. 3 where, toward the right, the sectioning becomes less oblique and longer segments of microvilli are observed which are clearly not vesicles. The waveform pattern of the segments is also suggestive of superimposed layers of microvilli which have been sectioned obliquely. In contrast, Fig. 4 shows structures which do appear to be closed membrane vesicles. The microvilli appear to have budded off into vesicles along their entire length, producing particles of remarkably uniform size, on many of which the complete enclosing membrane can be observed. In most cases in Fig. 4, though to a lesser extent in Fig. 5, the vesicles retain the linearity and spacing of the original microvilli, a feature which helps to distinguish them clearly from the 'apparent' vesicles on Fig. 3. In Fig. 5 microvilli are observed which appear to be in the process of vesiculation, pinching off and rounding as they do so. This sequence of pictures probably demonstrates how brush border vesicles are produced and how a 'right-side-out' orientation of the membrane can be preserved. Millington & Finean (1962) observed similar vesicle formation in situ, during a study of the effect of prolonged incubation of mucosal sections in normal or hypertonic saline. Both conditions led to vesiculation of microvilli after 24 h. The present results show that vesicles form rapidly during homogenization in isotonic sucrose and that they are released into the supernatant phase from which they may be harvested by high-speed centrifugation.the areas illustrated in Figs. 4 and 5, taken from pellet 1, have retained their vesicles in situ, yet it is known that vesicles are released into supernatant 1 (see Fig. 1) which are later sedimented as BBV (see Table 1). This release can be seen to have occurred in Fig. 6, which is typical of extensive regions of the mucosal surface observed in this pellet. Only a few vesicles remain along the surface, stubs of the original microvilli, while other areas (not illustrated) show only partial loss of vesicles. Fig. 7 shows a representative area of the BBV pellet in which a relatively uniform population of vesicles can be seen which are occasionaly interspersed with irregularly shaped membrane structures. Several interesting questions are raised, firstly why do microvilli vesiculate without first being broken up? Secondly, what happens to the core proteins during and after vesiculation? Thirdly, why do some vesicle populations remain in place at the mucosal surface? The first point may well reflect the fragility of the microvilli, such that when brush borders have been homogenized in isotonic sucrose buffer, the integrity of the membrane sheath is lost and it pinches off into vesicles, while the sheer close-packing of microvilli over the mucosal surface may prevent their being actually broken off at the root. It is difficult to envisage how microvilli could vesiculate whilst their core protein fibres remained intact and this point may be clarified by careful examination

6 232 P. R. Hearn, R. G. G. Russell and J. Farmer Fig 4. The brush border surface after homogenizarion in a sucrose buffer (see text). Microvilli have budded off into vesicles in situ. Bar, 1 fim.

7 Brush border vesicle orientation 233 Fig. 5. A further region showing vesiculated microviui. Bar, 1 fim. Fig. 6. A region of the vesiculated brush border which has been denuded of its vesicles. Bar, 1 fim.

8 234 P. R. Hearn, R. G. G. Russell andj. Farmer Fig. 7. A section of the brush border vesicle (BBV) pellet (see Fig. 1) showing the overall homogeneity of the vesicles formed. Bar, 1 /«n. Fig. 8. The brush border membrane after exposure to the cold sucrose buffer for 5 min only, microvilli are seen to be blistering. Bar, 1 /tm.

9 Brush border vesicle orientation 235 of Figs. 4 and 5. In Fig. 5 there is a microvillus which has not fully vesiculated though it shows signs of 'budding'. Along the length of this structure intact coreproteins can be seen which might be hindering the process of vesiculation. In Fig. 4, the very tip of a microvillus appears to be about to bud into 2 vesicles and here there is a clear separation of dark-staining contents (presumed to be core proteins) into the 2 halves of the unit. Other examples of this are seen in Fig. 4 and occasionally in Fig. 5. It appears, therefore, that the integrity of core protein fibres is lost before or during vesiculation and that this might be a prerequisite for vesicle formation. Further information can be obtained from Fig. 8 where mucosal surfaces are seen which have been exposed to the same buffer for 5 min only. Here microvilli are seen to be blistering, the membrane pulling away from the core proteins, an effect which is thought to represent swelling. The core proteins are intact. Homogenization of these membranes which, from the evidence of Fig. 8 would be already blistering, may disrupt the core protein fibres and allow vesiculation to proceed. The microfilaments within the intact microvillus have been shown to have crosslinks with the microvillus membrane (Mooseker & Tilney, 1975) and if some of these are maintained during vesiculation, then the mass of core protein material within the brush border vesicles may serve to stabilize the orientation of the membrane. The presence of these cross-linked microfilaments may also serve to stabilize any microvilli which have been actually truncated during homogenization, for when such microvilli are sedimented (as in R 2 and R 3 see Fig. 1) they are seen to contain core fibres. These observations suggest that inside-out membrane vesicles are much less likely to arise from brush border microvilli than they would be from an homogenate of cell plasma membranes. The retention of vesicles at the mucosal surface may be a property of the glycocalyx (glycoprotein matrix, Ito, 1969) which coats the brush border surface. Variability in retention of vesicle populations from one cell to another may reflect differences in composition or extent of the glycocalyx or simply different degrees of exposure to the shearing forces of homogenization. CONCLUSIONS The available information shows that closed membrane vesicles are formed from microvilli in situ in the brush border. The core-proteins in these vesicles are disarranged. In structures which appear to be forming vesicles, core-proteins are already dispersed, while wherever these fibres can be clearly seen intact, the microvillus in question has not fully vesiculated. It would appear that damage to core-protein fibres precedes vesicle formation. Vesicles are formed by budding off of the microvilli and assuming that they retain their original orientation after release from the cell surface, are, therefore, right-sideout membrane vesicles. This work was carried out at the Nuffield Department of Orthopaedic Surgery. P.R.H. was in receipt of an E.P.A. Cephalosporin scholarship at Linacre College, Oxford.

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