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19 Electron Microscopy

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19 Electron Microscopy, Page 1 of 2

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Abstract:

Pathogens have evolved a wide variety of strategies to circumvent the host microbicidal activities and to use the cellular machinery to their own advantage. This chapter is devoted to electron microscopy, which is the approach of choice for optimal resolution and precision. A wide variety of cytochemical and immunoelectron microscopy methods can be used to characterize pathogens, analyze the intracellular compartment in which they reside, and localize bacterial virulence factors or cell components involved in their survival. Some of the major morphological methods, recent and less recent, of special interest to host-pathogen interplay are reviewed in this chapter. Phagosomes that retain intermingling characteristics of early endosomes are considered to be immature; those that become mature lose their ability to fuse with early endosomes and fuse with lysosomes to become phagolysosomes. Endocytosis and phagocytosis both involve an extensive transfer of membrane in both directions between the cell surface and intracellular membrane compartments. Given the large amounts of membrane required during uptake of particles and also during replication of endoparasites within their phagocytic vacuole, one of the questions that have been raised recently was whether other cell compartments, and more especially the endoplasmic reticulum (ER), could be an additional source of membrane for forming and/or dividing phagosomes.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19

Key Concept Ranking

Endoplasmic Reticulum
0.52973086
Plasma Membrane
0.44814906
Electron Microscopy
0.44425714
Immunoelectron Microscopy
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0.52973086
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Figures

Image of Figure 19.1
Figure 19.1

Morphological appearance of bacteria in conventional electron microscopy. Macrophages were infected with live LO-28 for 45 min. At 2 h after infection, the bacteria were found within phagosomes , in which they were morphologically intact or damaged , or had escaped from the phagosome after lysis of the membrane (arrowheads). In the cytoplasm, the bacteria are surrounded by a thick network of actin filaments (arrows in ). Some bacteria are being extruded from the cell . Bar, 0.5 μm.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.2
Figure 19.2

Morphological appearance of early endosomes and lysosomes (and prelysosomes) . Macrophages were given HRP (25 μg/ml) for 30 min, fixed, and stained for the endocytic content marker, HRP. In early endosomes (E), the reaction product lines only the inner face of the membrane, whereas in lysosomes (L), it entirely fills the lumen . Bar, 0.25 μm.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.3
Figure 19.3

Fusion of phagosomes with early endosomes or lysosomes stained with HRP, as observed by electron microscopy. The cells underwent phagocytosis of different types of latex beads. At 2 h after phagocytic uptake, cells were given HRP as an endocytic content marker. They were then fixed and stained for HRP. The 1-μm-diameter hydrophobic bead-containing phagosomes fuse with early endosomes (E) (arrow in ) but not with lysosomes (L) . The 1-μm-diameter hydrophilic beadcontaining phagosomes have matured and fuse with lysosomes . Bar, 0.5 μm.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.4
Figure 19.4

Acquisition of lysosomal marker by phagosomes. Macrophages were incubated for 30 min with BSA tagged with gold, washed, and incubated for 2 h in medium devoid of BSA tagged with gold to chase the marker to the lysosomes (L). The cells were then incubated in the presence of different types of latex beads. Phagosomes containing 1-μm-diameter hydrophobic beads do not fuse with lysosomes , but phagosomes containing smaller beads (0.1–0.5-μm diameter) do Bar, 0.5 μm.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.5
Figure 19.5

Examples of enzyme cytochemistry. Staining of -infected macrophages for the hydrolytic enzyme acid phosphatase. Bone marrow-derived macrophages from BCG-susceptible mice (BALB/c) were infected with TMC 724 (Ma). Seven days later, the cells were fixed, stained for acid phosphatase, and processed for electron microscopy. Lysosomes (L) were strongly labeled, but most phagosomes were not stained. Only a few of them displayed small deposits (arrowhead). Staining of (Ba)-infected macrophages for glucose- 6-phosphatase (arrowheads), an enzyme specific to the endoplasmic reticulum. Although the ER has been recruited in the vicinity of the two phagosomes, it has not yet fused with them. As a result, the two phagosomes are not stained for the enzyme. Bar, 0.5 μm.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.6
Figure 19.6

Morphological assessment of phagosome acidification. Macrophages were infected for 45 min with , washed, and reincubated in fresh medium. One hour later, DAMP (30 min, 60 μM) was added for passive accumulation in acidic organelles. The cells were fixed and embedded in Lowicryl. The probe was localized by a postembedding labeling method as follows. Lowicryl thin sections were sequentially incubated with rabbit anti- DNP (dinitrophenol) antibodies and with protein A coupled to 10-nm-diameter gold particles. A high abundance of gold particles was observed within lysosomes (L) and phagosomes (P), thereby indicating that they are acidic. Intracytoplasmic bacteria (arrow) were not labeled. Bar, 0.5 μm. From such pictures, one can estimate the intraphagosomal pH by the method described by Orci.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.7
Figure 19.7

Schematic depiction of methods used for immunoelectron microscopy. See the text for details.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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Image of Figure 19.8
Figure 19.8

Localization of constituents by immunolabeling of ultrathin cryosections. Human macrophages were infected with HIV-1. Fourteen days later, they were fixed and processed for immunolabeling of ultrathin cryosections. Sections were double-immunogold labeled for p24 (gold particles 15 nm in diameter) to localize HIV-1 and for MHC II (major histocompatibility class II antigens) (gold particles 10 nm in diameter). Note the accumulation of HIV particles positive for p24 in enlarged compartments. These compartments contain MHCII. In addition, MHCII also colocalizes with the p24-positive virions. Bar, 0.5 μm. From Raposo et al. (2002), reprinted with permission.

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19
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References

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1. Anderson, R. G. W.,, J. R. Falck,, J. L. Goldstein,, and M. S. Brown. 1984. Visualization of acidic organelles in intact cells by electron microscopy. Proc. Natl. Acad. Sci. USA 81:48384842.
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6. de Chastellier, C.,, and L. Thilo. 2002. Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection. Eur. J. Cell Biol. 81:1725. This paper describes and emphasizes the use of autoradiography and morphometry methods as discussed in Methods To Study Intersection with the Endocytic Pathway to study membrane trafficking and to analyze the phagosome membrane composition in terms of plasma-membrane-derived glycoconjugates. These methods were used to show how mycobacteria remodel the phagosome membrane in the course of infection.
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9. Dubochet, J.,, and N. Sartori Blanc. 2001. The cell in absence of aggregation artefacts. Micron 32:9199. This paper compares and discusses different EM methods, i.e., (i) conventional resin-embedding and sectioning, (ii) low-temperature embedding and sectioning of freeze-substituted samples, and (iii) cryosections of vitrified samples, in terms of cell structure and organization.
10. Gagnon, E.,, S. Duclos,, C. Rondeau,, E. Chevet,, P. H. Cameron,, O. Steele-Mortimer,, P. Paiement,, J. M. Bergeron,, and M. Desjardins. 2002. Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110:119131.
11. Griffiths, G. (ed.). 1993. Fine Structure Immunocytochemistry. Springer-Verlag, Berlin, Germany. Excellent book about immunoelectron microscopy methods.
12. Griffiths, G.,, J. M. Lucocq,, and T. M. Mayhew. 2001. Electron microscopy applications for quantitative cellular microbiology. Cell. Microbiol. 3:659668. This paper emphasizes the power of EM approaches in conjunction with stereology approaches to analyze intracellular membrane trafficking.
13. Griffiths, G.,, P. Quinn,, and G. Warren. 1983. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki forest virus. J. Cell Biol. 96:835850. This paper indicates how to stain cells for glucose-6-phosphatase.
14. Harding, C. V.,, and H. J. Geuze. 1992. Class II MHC molecules are present in macrophage lysosomes and phagolysosomes that function in the phagocytic processing of Listeria monocytogenes for presentation to T cells. J. Cell Biol. 119:531542.
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19. Ozawa, H.,, R. Picart,, A. Barret,, and C. Tougard. 1994 Heterogeneity in the pattern of distribution of the specific hormonal product and secretogranins within the secretory granules of rat prolactin cells. J. Histochem. Cytochem. 42:10971107. This paper describes and illustrates postembedding immunolabelings on LR White thin sections.
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22. Raposo, G.,, M. Moore,, D. Innes,, R. Leijendekker,, A. Leigh-Brown,, P. Benroch,, and H. Geuze. 2002. Human macrophages accumulate HIV-1 particles in MHC II compartments. Traffic 3:718729.
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24. Roth, J.,, J. M. Lucocq,, and D. J. Taatjes. 1988. Light and electron microscopical detection of sugar residues in tissue sections by gold labeled lectins and glycoproteins. I. Methodological aspects. Acta Histochem. Suppl. 36:8199.
25. Russell, D. G.,, J. Dant,, and S. Sturgill-Koszycki. 1996. Mycobacterium avium and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma. J. Immunol. 156:47644773.
26. Slot, J. W.,, and H. J. Geuze. 1985. A new method of preparing gold probes for multiple-labelling cytochemistry. Eur. J. Cell Biol. 38:8793.
27. Slot, J. W.,, H. J. Geuze,, and A. J. Weerkamp. 1988. Localization of macromolecular components by application of the immunogold technique on cryosectioned bacteria. Methods Microbiol. 20:211236.
28. Studer, D.,, W. Grabber,, A. Al-Amoudi,, and P. Eggli. 2001. A new approach for cryofixation by high-pressure freezing. J. Microsc. 203:285294.
29. Tilney, L. G.,, D. J. DeRosier,, and M. S. Tilney. 1992. How Listeria exploits host cell actin to form its own cytoskeleton. I. Formation of a tail and how that tail might be involved in movement. J. Cell Biol. 118:7181.
30. Tougard, C.,, and R. Picart. 1986. Use of pre-embedding ultrastructural immunocytochemistry in the localization of a secretory product and membrane proteins in cultured prolactin cells. Am. J. Anat. 175:161177. This article gives an excellent and detailed account of preembedding immunolabeling methods (from sample fixation to immunolabeling) and discusses some of the pitfalls/ problems that might be encountered.

Tables

Generic image for table
Table 19.1

Membrane or content markers used to identify phagosomes as resembling a given endocytic compartment or the ER

Citation: De Chastellier C. 2004. 19 Electron Microscopy, p 451-471. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch19

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