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Do coated pinocytic vesicles exist?
400
T I B S - N o v e m b e r 1983
(1982) European Bioenergetic* {bnterence Report 2, 163-164 16 Anderson, S., de Bruijn, M. H. L., Coulson, A. R., Eperon, 1. C., Sanger, F. and Young, I. G. (1982)J. Mol. Biol. 156, 683-717 17 Merle, P. and Kadenbach, B. (1982) Eur. J. Biochem. 125,239-244 18 Jarausch, J. and Kadenbach, B. (1982) HoppeSeyler's Z. Physiol. Chem. 363. 1133-1140 19 Piittner, I,, Solioz, M., Carafoli, E. and Ludwig, B. (1983) Eur. J. Biochem. 134, 33-37
20 Freedman, J. A. and Chan, S. H. P. (19831 J. Biol. Chem. 258, 5885-5892 13. KADENBACH, M. UNGIBAUER, J. JARAUSCH. U, BUGE a n d L. KUHN-NENTWIG
Philipps- Universittit Marburg, Fachbereich Chemie, Hans- Meerweinstrasse, D-3550 Marburg, FRG.
Do coated pinocytic vesicles exist? SIR: TWO reviews in T1BS, July 1983, present differing viewpoints on the subject of receptor-mediated endocytosis. The first, by Helenius et al.1, favoured the idea that coated pits at the cell surface give rise to free coated vesicles transporting internalized molecules to an acidic prelysosomal compartment, the endosome. T h e second, by Pastan and Willingham 2, advocated an alternative view based on the assumption that coated pits are permanently connected to the surface membrane. According to their theory the iigands are carried into the cell by a new and independent organelle, the r e c e p t o s o m e , which buds off from the neck of the coated pit. Thus, a key question in this debate is whether truly free, coated pinocytic vesicles exist. This question can only be answered by electron microscopy of very thin consecutive sections. Serial sectioning was applied to cultured mouse L-929 fibroblasts and human skin fibroblasts in a recent study at our laboratory 3. Three-dimensional reconstructions based on the consecutive sections strongly suggested that free coated pinocytic vesicles exist in these cells. Accordingly, Helenius et al. 1 write that 'recent serial section analysis by van Deurs has unequivocally demonstrated the existence of endocytic coated vesicles as independent organelles'. In an additional serial section study on the protozoon, Tetrahymena, we have similarly shown the existence of free, coated pinocytic vesicles( However, in relation to our work on fibroblasts~ and the work by Fan et al?, Pastan and WiUingham" state that 'it is still not clear that the contrast of tangentially sectioned membranes in such small necks (those connecting coated pits with the cell surface) would be sufficient to allow a clear image of these structures in every case'. We therefore feel that comments on the problems of membrane contrast and section thickness in serial section studies are timely. In serial section electron microscopy the accuracy of the three-dimensional
reconstruction depends, first of all, on the section thickness 6. The thinner the sections the more detailed the reconstructions which can be made. Consequently, the thickness of consecutive
sections must be adjusted to the dimensions of the structure under investigation. Unfortunately, there is no equation which can give optimal section thickness for a given problem; however, as a general rule, the thickness of the individual sections has to be less than the dimensions of the smallest structural detail of interest. In the differentiations between coated pits and free coated pinocytic vesicles this is the neck region of the coated pit - if some of these structures are lost, the analysis will over-estimate the number of free vesicles. The outer diameter of the smallest necks is about 25 nm; we therefore based our analysis 3 on consecutive sections of thicknesses between 15 and 20 nm. Contrast also plays an important role in the analysis of serial sections. In our
ELECTRONBEAM
PROJECTEDIMAGES
I I 1
LO sectionthickness: 8Ohm PROJECTEDIMAGES
ELECTRONBEAM
/
©
\1©
Fig. 1. Schematic drawing o f serial section electron microscopy applied to a coated pit and an adjacent free coated vesicle. (,4) The structures are partially contained in two consecutive 80 nm sections. It is not possible to reconstruct the structures from the projected images. Due to overlapping material the ' neck region will be reduced to some fuzz near the surface membrane in section 1 and at the edge of the coated pit in section 2. The coated pit appears as a free vesicle and its relation to the free coated vesicle cannot be determined. Thus, the example shows that the number of free (or apparently free) coated vesicles tends to be overestimated in a series o f such thkk sections. (B) The critical parts of the structures are contained in five consecutive 15 nm sections. The projected images clearly illustrate that these much thinner sections yield a much better topographical resolution. The free coated vesicle is easily differentiated from the coated pit and the neck region o f the coated pit can be reconstructed unequivocally.
TIBS - November
401
1983
analysis the contrast had to be sufficient to allow the membranes to be well defined, but at the same time we had to keep the general contrast level in the sections as low as possible, because we used cationized ferritin molecules as an exogenous marker of the coated pits and vesicles (necessary to document that a given profile is endocytic3). In the very thin sections, ferritin molecules can only be identified with certainty in uncontaminated sections of low contrast. This 'contrast problem' was solved by treating the cells with an intense staining en b l o c and by avoiding post-staining of the sections. After this treatment the general contrast level in sections of conventional thickness (40-50 nm) was normal 7 while in the very thin sections (15-20 nm) the general contrast w a s l o w 3. However, perpendicularly sectioned membranes were well defined. Also, membranes sectioned at angles which deviate moderately from perpendicular could be identified. This turned out to be sufficient to identify the narrowest necks. Typically, the individual coated pits were contained in six to eight sections while necks with an outer diameter of about 25
nm could be identified in only one or two sections. Thus, it was not a problem to identify the necks. The suggestion by Pastan and Willingham" that all the coated vesicles which were registered as free in our analysis~* are, in reality, provided with an unidentified neck, is therefore not justified. Criticism could be directed instead towards studies which are based on much thicker consecutive sections of high contrast. With increasing thickness the topographical resolution decreases and when the section thickness exceeds 70 nm it is questionable whether meaningful reconstructions of the coated structures can be made. This is illustrated in Fig. 1 by a schematic example in which the topographical resolution of 80 nm thick and 15 nm thick consecutive sections are compared. In conclusion, our demonstration that coated pits are not permanently surfaceassociated structures makes the 'receptosome concept' unlikely and favours the 'endosome-concept'. * 10 out of 97 profiles in L-cells, and 29 out of 81 in skin fibroblasts.
References
1 Helenius, A., Mellman, I., Wall, D. and Hubbard, A. (1983) Trends Biochem. Sci. 8, 245 2 Pastan, I. and Willingham, M. C. (1983) Trends Biochem. Sci. 8, 250 3 Petersen, O. W. and van Deurs, B. (1983) J. Cell Biol. 96, 277
4 Nilsson, J. R. and van Deurs, B. J. Cell Sci. (in press) 5 Fan, J. Y., Carpentier, J.-L., Gordon, P., Obberghen, E. V., Blackett, N. M., Grunfeld, C. and Orci, L. (1982) Proc. Nat/ Acad. Sci. USA 79, 7788 6 Bundgaard, M. (1983) Fed. Proc. 42, 2425 7 van Deurs, B. and Nilausen, K. (1982)J. Cell Biol. 94, 279 n o V A N D E U R S , O L E W. P E T E R S E N AND MAGNUS BUNDGAARD
Departments of Anatomy and Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
T I B S - N o v e m b e r 1983
(1982) European Bioenergetic* {bnterence Report 2, 163-164 16 Anderson, S., de Bruijn, M. H. L., Coulson, A. R., Eperon, 1. C., Sanger, F. and Young, I. G. (1982)J. Mol. Biol. 156, 683-717 17 Merle, P. and Kadenbach, B. (1982) Eur. J. Biochem. 125,239-244 18 Jarausch, J. and Kadenbach, B. (1982) HoppeSeyler's Z. Physiol. Chem. 363. 1133-1140 19 Piittner, I,, Solioz, M., Carafoli, E. and Ludwig, B. (1983) Eur. J. Biochem. 134, 33-37
20 Freedman, J. A. and Chan, S. H. P. (19831 J. Biol. Chem. 258, 5885-5892 13. KADENBACH, M. UNGIBAUER, J. JARAUSCH. U, BUGE a n d L. KUHN-NENTWIG
Philipps- Universittit Marburg, Fachbereich Chemie, Hans- Meerweinstrasse, D-3550 Marburg, FRG.
Do coated pinocytic vesicles exist? SIR: TWO reviews in T1BS, July 1983, present differing viewpoints on the subject of receptor-mediated endocytosis. The first, by Helenius et al.1, favoured the idea that coated pits at the cell surface give rise to free coated vesicles transporting internalized molecules to an acidic prelysosomal compartment, the endosome. T h e second, by Pastan and Willingham 2, advocated an alternative view based on the assumption that coated pits are permanently connected to the surface membrane. According to their theory the iigands are carried into the cell by a new and independent organelle, the r e c e p t o s o m e , which buds off from the neck of the coated pit. Thus, a key question in this debate is whether truly free, coated pinocytic vesicles exist. This question can only be answered by electron microscopy of very thin consecutive sections. Serial sectioning was applied to cultured mouse L-929 fibroblasts and human skin fibroblasts in a recent study at our laboratory 3. Three-dimensional reconstructions based on the consecutive sections strongly suggested that free coated pinocytic vesicles exist in these cells. Accordingly, Helenius et al. 1 write that 'recent serial section analysis by van Deurs has unequivocally demonstrated the existence of endocytic coated vesicles as independent organelles'. In an additional serial section study on the protozoon, Tetrahymena, we have similarly shown the existence of free, coated pinocytic vesicles( However, in relation to our work on fibroblasts~ and the work by Fan et al?, Pastan and WiUingham" state that 'it is still not clear that the contrast of tangentially sectioned membranes in such small necks (those connecting coated pits with the cell surface) would be sufficient to allow a clear image of these structures in every case'. We therefore feel that comments on the problems of membrane contrast and section thickness in serial section studies are timely. In serial section electron microscopy the accuracy of the three-dimensional
reconstruction depends, first of all, on the section thickness 6. The thinner the sections the more detailed the reconstructions which can be made. Consequently, the thickness of consecutive
sections must be adjusted to the dimensions of the structure under investigation. Unfortunately, there is no equation which can give optimal section thickness for a given problem; however, as a general rule, the thickness of the individual sections has to be less than the dimensions of the smallest structural detail of interest. In the differentiations between coated pits and free coated pinocytic vesicles this is the neck region of the coated pit - if some of these structures are lost, the analysis will over-estimate the number of free vesicles. The outer diameter of the smallest necks is about 25 nm; we therefore based our analysis 3 on consecutive sections of thicknesses between 15 and 20 nm. Contrast also plays an important role in the analysis of serial sections. In our
ELECTRONBEAM
PROJECTEDIMAGES
I I 1
LO sectionthickness: 8Ohm PROJECTEDIMAGES
ELECTRONBEAM
/
©
\1©
Fig. 1. Schematic drawing o f serial section electron microscopy applied to a coated pit and an adjacent free coated vesicle. (,4) The structures are partially contained in two consecutive 80 nm sections. It is not possible to reconstruct the structures from the projected images. Due to overlapping material the ' neck region will be reduced to some fuzz near the surface membrane in section 1 and at the edge of the coated pit in section 2. The coated pit appears as a free vesicle and its relation to the free coated vesicle cannot be determined. Thus, the example shows that the number of free (or apparently free) coated vesicles tends to be overestimated in a series o f such thkk sections. (B) The critical parts of the structures are contained in five consecutive 15 nm sections. The projected images clearly illustrate that these much thinner sections yield a much better topographical resolution. The free coated vesicle is easily differentiated from the coated pit and the neck region o f the coated pit can be reconstructed unequivocally.
TIBS - November
401
1983
analysis the contrast had to be sufficient to allow the membranes to be well defined, but at the same time we had to keep the general contrast level in the sections as low as possible, because we used cationized ferritin molecules as an exogenous marker of the coated pits and vesicles (necessary to document that a given profile is endocytic3). In the very thin sections, ferritin molecules can only be identified with certainty in uncontaminated sections of low contrast. This 'contrast problem' was solved by treating the cells with an intense staining en b l o c and by avoiding post-staining of the sections. After this treatment the general contrast level in sections of conventional thickness (40-50 nm) was normal 7 while in the very thin sections (15-20 nm) the general contrast w a s l o w 3. However, perpendicularly sectioned membranes were well defined. Also, membranes sectioned at angles which deviate moderately from perpendicular could be identified. This turned out to be sufficient to identify the narrowest necks. Typically, the individual coated pits were contained in six to eight sections while necks with an outer diameter of about 25
nm could be identified in only one or two sections. Thus, it was not a problem to identify the necks. The suggestion by Pastan and Willingham" that all the coated vesicles which were registered as free in our analysis~* are, in reality, provided with an unidentified neck, is therefore not justified. Criticism could be directed instead towards studies which are based on much thicker consecutive sections of high contrast. With increasing thickness the topographical resolution decreases and when the section thickness exceeds 70 nm it is questionable whether meaningful reconstructions of the coated structures can be made. This is illustrated in Fig. 1 by a schematic example in which the topographical resolution of 80 nm thick and 15 nm thick consecutive sections are compared. In conclusion, our demonstration that coated pits are not permanently surfaceassociated structures makes the 'receptosome concept' unlikely and favours the 'endosome-concept'. * 10 out of 97 profiles in L-cells, and 29 out of 81 in skin fibroblasts.
References
1 Helenius, A., Mellman, I., Wall, D. and Hubbard, A. (1983) Trends Biochem. Sci. 8, 245 2 Pastan, I. and Willingham, M. C. (1983) Trends Biochem. Sci. 8, 250 3 Petersen, O. W. and van Deurs, B. (1983) J. Cell Biol. 96, 277
4 Nilsson, J. R. and van Deurs, B. J. Cell Sci. (in press) 5 Fan, J. Y., Carpentier, J.-L., Gordon, P., Obberghen, E. V., Blackett, N. M., Grunfeld, C. and Orci, L. (1982) Proc. Nat/ Acad. Sci. USA 79, 7788 6 Bundgaard, M. (1983) Fed. Proc. 42, 2425 7 van Deurs, B. and Nilausen, K. (1982)J. Cell Biol. 94, 279 n o V A N D E U R S , O L E W. P E T E R S E N AND MAGNUS BUNDGAARD
Departments of Anatomy and Physiology, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.