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Negative staining of coated vesicles
Micron, 1977, Vol. S: 233-235. Pergamon Press. Printed in Great Britain.
Negative s t - ; - ; - g of coated vesicles C. D. OCKLEFORD*I", A. WHYTE* and D. E. BOWYER*
*Department of Pathology, Universityof Cambridge, TennisCourtRoad, CambridgeCB2 1QP and ~Department of Anatomy, University of Leicester, University Road, Leicester,LE1 7RH, England It has been suggested that the coated vesicles of rat vas deferens fall into two distinct size classes and that the members of these two classes perform separate functions. It has also been proposed that the smaller ones, less than 75nm diameter, perform Golgi-related or secretory functions whereas the larger ones, over 100nm diameter, undertake selective uptake of protein by micropinocytosis (Friend and Farquhat, 1967). The h u m a n trophoblast exhibits m a n y ultrastructural features typical of cells with both absorptive and secretory functions and, therefore, might be thought to be comparable with the vas deferens. The trophoblast has a surface area at term of between 10 and 13m 2. Within its cytoplasm there are of the order of 9 x 1012 coated vesicles. Secantially-sectioned placental coated vesicles appear to be covered by a polygonal latticework of clathrin, a structure which appears in median sections as a series of projections (presumably the origin of the misnomer 'bristle coat'). This polygonal lattice is a prominent feature of negatively-stained coated vesicles isolated from tissue using the method devised by Pearse (1975). We have analyzed the morphology of 500 vesicles from one isolate. Vesicles vary in size, shape and in the presence or absence of a central electron-lucent structure which has been interpreted as a phospholipid bilayer. The range of volumes forms a skewed distribution with a relatively sharp m i n i m u m cut off at about 0.65 x 105nm 3, which is also approximately the m i n i m u m volume of a spaceenclosing pattern of polygons of the size which compose the lattice. The distribution of vesicles of highest volume tapers more gradually (maximum volume 15.8 x 105nmS). The corresponding range of the longest axes across the vesicles is from 45 to 130nm, the longest measured from this sample being about 130% of the m a x i m u m linear dimension of coated vesicles quoted in several recent reviews. These and other measures of coated vesicle
size all give rise to unimodal distributions. In addition, the modal value of both the longest and shortest cross-sections of coated vesicles from h u m a n placenta is about 80nm, a size where it has been claimed there are no coated vesicles in the vas deferens. T h e correlation of specific distinctly separate sizes of coated vesicles with separate functions does not, therefore, appear to be a general rule for all cell types with secretory and micropinocytic functions because the distribution of coated vesicle sizes is entirely different in different tissues. Some isolated clathrin lattices contain electron-lucent structures which are presumably the collapsed phospholipid bilayer membranes of the coated vesicles. The ratio of their occurrence to absence is about 2.7: 1.0. There is a connection between bilayer possession and vesicle size. The mean volume of bilayercontaining lattices is 3.7 × 105nm 3, whereas that of empty lattices is 2.4 x 105nm s (difference significant P < 0.001). I f the absence of bilayer in some vesicles is not an artefact of the preparation method, this observation m a y be relevant to coated vesicle function. Apparently vesicles lose their coats in vivo prior to fiuion with other membraneous compartments. The fact that the average empty lattice is smaller than the average bilayer-containing lattice m a y indicate that they originated as parts of larger vesicle lattices which had left the bilayer and were in progress of breaking down by stages, being recycled to the cell surface or being transported to some other part of the syncytium. Vesicles approximate either to spherical or to prolate spheroidal form (see Fig. 1). The ratio or occurrence is about 2.5:1.0 respectively. The prolate vesicles are on average (3.99 x 105rimS), significantly (P < 0.001) larger than the spherical vesicles (3.12 x 105nmS). Apparently, increase in volume of the vesicles is achieved at least in part by a change in the ratio of the polygons in the lattice wall. T h a t this ratio varies in vesicles from the same sample must be considered in the development of mode'.s of
233
234
(l. l). Ockleford, A. Whyte and I). E. Bowww
8
d
f Fig. 1. Examples of negatively stained coated vesicles isolated from human placenta. The variation in size and shape is apparent. The vesicle (a) is one of the type classed as prolate spheroidal in the text. Of the two coated vesicle lattices shown in micrograph (d) one contains a phospholipid bilayer whereas the other does not. "x 140,000.
vesicle f o r m a t i o n (Ockletbrd a n d W h y t e , 1977). I t is r a t h e r simply e x p l a i n e d b y models w h i c h invoke l a t e r a l m o v e m e n t o f m e m b r a n e c o m p o nents because the r a n d o m a r r i v a l of different polygons or f o r m a t i o n of different polygons from subunits at the site of i n v a g i n a t i o n is expected to give rise to a range of vesicle sizes a n d shapes. W h a t d e t e r m i n e s the choice of f o r m a t i o n of p o l y g o n type from clathrin subunits is p u r e
speculation, but could be a function of the type or c o n c e n t r a t i o n of m a t e r i a l s to be taken up or of some other m a t e r i a l s w h i c h p r o m o t e uptake. H o w the v a r i a b i l i t y in ratio arises is certainly not a p r o p e r t y w h i c h is easily e x p l a i n e d in the m a n n e r in which the assembly of m o r e pred i c t a b l e structures such as c e r t a i n viruses can be explained. Self assembly m a y be involved but a p p a r e n t l y with far less control over the o r d e r
Negative Staining of Coated Vesicles and precision of assembly than is c o m m o n in m a n y other recently described systems.
REFERENCES Friend, D. S. and Farquhar, M. G., 1967. Functions of coated vesicles during protein absorption in the rat vas deferens. 07. Cell Biol., 35: 357-376.
235
Ockleford, C. D. and Whyte, A., 1977. Differentiated regions of human placental cell surface associated with exchange of materials between maternal and foetal blood: coated vesicles. J. Cell So/., 25: 293312. Pearse, B. M. F., 1975. Coated vesicles from pig brain: purification and biochemical characterisation. 07. Mol. Biol., 97: 93-98.
Negative s t - ; - ; - g of coated vesicles C. D. OCKLEFORD*I", A. WHYTE* and D. E. BOWYER*
*Department of Pathology, Universityof Cambridge, TennisCourtRoad, CambridgeCB2 1QP and ~Department of Anatomy, University of Leicester, University Road, Leicester,LE1 7RH, England It has been suggested that the coated vesicles of rat vas deferens fall into two distinct size classes and that the members of these two classes perform separate functions. It has also been proposed that the smaller ones, less than 75nm diameter, perform Golgi-related or secretory functions whereas the larger ones, over 100nm diameter, undertake selective uptake of protein by micropinocytosis (Friend and Farquhat, 1967). The h u m a n trophoblast exhibits m a n y ultrastructural features typical of cells with both absorptive and secretory functions and, therefore, might be thought to be comparable with the vas deferens. The trophoblast has a surface area at term of between 10 and 13m 2. Within its cytoplasm there are of the order of 9 x 1012 coated vesicles. Secantially-sectioned placental coated vesicles appear to be covered by a polygonal latticework of clathrin, a structure which appears in median sections as a series of projections (presumably the origin of the misnomer 'bristle coat'). This polygonal lattice is a prominent feature of negatively-stained coated vesicles isolated from tissue using the method devised by Pearse (1975). We have analyzed the morphology of 500 vesicles from one isolate. Vesicles vary in size, shape and in the presence or absence of a central electron-lucent structure which has been interpreted as a phospholipid bilayer. The range of volumes forms a skewed distribution with a relatively sharp m i n i m u m cut off at about 0.65 x 105nm 3, which is also approximately the m i n i m u m volume of a spaceenclosing pattern of polygons of the size which compose the lattice. The distribution of vesicles of highest volume tapers more gradually (maximum volume 15.8 x 105nmS). The corresponding range of the longest axes across the vesicles is from 45 to 130nm, the longest measured from this sample being about 130% of the m a x i m u m linear dimension of coated vesicles quoted in several recent reviews. These and other measures of coated vesicle
size all give rise to unimodal distributions. In addition, the modal value of both the longest and shortest cross-sections of coated vesicles from h u m a n placenta is about 80nm, a size where it has been claimed there are no coated vesicles in the vas deferens. T h e correlation of specific distinctly separate sizes of coated vesicles with separate functions does not, therefore, appear to be a general rule for all cell types with secretory and micropinocytic functions because the distribution of coated vesicle sizes is entirely different in different tissues. Some isolated clathrin lattices contain electron-lucent structures which are presumably the collapsed phospholipid bilayer membranes of the coated vesicles. The ratio of their occurrence to absence is about 2.7: 1.0. There is a connection between bilayer possession and vesicle size. The mean volume of bilayercontaining lattices is 3.7 × 105nm 3, whereas that of empty lattices is 2.4 x 105nm s (difference significant P < 0.001). I f the absence of bilayer in some vesicles is not an artefact of the preparation method, this observation m a y be relevant to coated vesicle function. Apparently vesicles lose their coats in vivo prior to fiuion with other membraneous compartments. The fact that the average empty lattice is smaller than the average bilayer-containing lattice m a y indicate that they originated as parts of larger vesicle lattices which had left the bilayer and were in progress of breaking down by stages, being recycled to the cell surface or being transported to some other part of the syncytium. Vesicles approximate either to spherical or to prolate spheroidal form (see Fig. 1). The ratio or occurrence is about 2.5:1.0 respectively. The prolate vesicles are on average (3.99 x 105rimS), significantly (P < 0.001) larger than the spherical vesicles (3.12 x 105nmS). Apparently, increase in volume of the vesicles is achieved at least in part by a change in the ratio of the polygons in the lattice wall. T h a t this ratio varies in vesicles from the same sample must be considered in the development of mode'.s of
233
234
(l. l). Ockleford, A. Whyte and I). E. Bowww
8
d
f Fig. 1. Examples of negatively stained coated vesicles isolated from human placenta. The variation in size and shape is apparent. The vesicle (a) is one of the type classed as prolate spheroidal in the text. Of the two coated vesicle lattices shown in micrograph (d) one contains a phospholipid bilayer whereas the other does not. "x 140,000.
vesicle f o r m a t i o n (Ockletbrd a n d W h y t e , 1977). I t is r a t h e r simply e x p l a i n e d b y models w h i c h invoke l a t e r a l m o v e m e n t o f m e m b r a n e c o m p o nents because the r a n d o m a r r i v a l of different polygons or f o r m a t i o n of different polygons from subunits at the site of i n v a g i n a t i o n is expected to give rise to a range of vesicle sizes a n d shapes. W h a t d e t e r m i n e s the choice of f o r m a t i o n of p o l y g o n type from clathrin subunits is p u r e
speculation, but could be a function of the type or c o n c e n t r a t i o n of m a t e r i a l s to be taken up or of some other m a t e r i a l s w h i c h p r o m o t e uptake. H o w the v a r i a b i l i t y in ratio arises is certainly not a p r o p e r t y w h i c h is easily e x p l a i n e d in the m a n n e r in which the assembly of m o r e pred i c t a b l e structures such as c e r t a i n viruses can be explained. Self assembly m a y be involved but a p p a r e n t l y with far less control over the o r d e r
Negative Staining of Coated Vesicles and precision of assembly than is c o m m o n in m a n y other recently described systems.
REFERENCES Friend, D. S. and Farquhar, M. G., 1967. Functions of coated vesicles during protein absorption in the rat vas deferens. 07. Cell Biol., 35: 357-376.
235
Ockleford, C. D. and Whyte, A., 1977. Differentiated regions of human placental cell surface associated with exchange of materials between maternal and foetal blood: coated vesicles. J. Cell So/., 25: 293312. Pearse, B. M. F., 1975. Coated vesicles from pig brain: purification and biochemical characterisation. 07. Mol. Biol., 97: 93-98.