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Ultrastructural Imaging of Endocytic Sites in Saccharomyces cerevisiae by Transmission Electron Microscopy and Immunolabeling

  • Christopher Buser (a1) and David G. Drubin (a1)
Abstract
Abstract

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein–tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

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*Corresponding author. E-mail: drubin@berkeley.edu
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A.E. Adams & J.R. Pringle (1984). Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol 98, 934945.

D.R. Boettner , R.J. Chi & S.K. Lemmon (2012). Lessons from yeast for clathrin-mediated endocytosis. Nat Cell Biol 14, 210.

E. Boucrot , S. Saffarian , R. Massol , T. Kirchhausen & M. Ehrlich (2006). Role of lipids and actin in the formation of clathrin-coated pits. Exp Cell Res 312, 40364048.

S. Boulant , C. Kural , J.C. Zeeh , F. Ubelmann & T. Kirchhausen (2011). Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat Cell Biol 13, 11241131.

C. Buser & K. McDonald (2010). Correlative GFP-immunoelectron microscopy in yeast. Methods Enzymol 470, 603618.

C. Buser & P. Walther (2008). Freeze-substitution: The addition of water to polar solvents enhances the retention of structure and acts at temperatures around −60 degrees C. J Microsc 230, 268277.

E. Carlemalm , R.M. Garavito & W. Villiger (1982). Resin development for electron-microscopy and an analysis of embedding at low-temperature. J Microsc-Oxford 126, 123143.

A. Collins , A. Warrington , K.A. Taylor & T. Svitkina (2011). Structural organization of the actin cytoskeleton at sites of clathrin-mediated endocytosis. Curr Biol 21, 11671175.

K.M. Collins , N.L. Thorngren , R.A. Fratti & W.T. Wickner (2005). Sec17p and HOPS, in distinct SNARE complexes, mediate SNARE complex disruption or assembly for fusion. EMBO J 24, 17751786.

T.J. Collins (2007). ImageJ for microscopy. Biotechniques 43, 2530.

G.J. Doherty & H.T. McMahon (2009). Mechanisms of endocytosis. Annu Rev Biochem 78, 857902.

A.E. Engqvist-Goldstein & D.G. Drubin (2003). Actin assembly and endocytosis: From yeast to mammals. Annu Rev Cell Dev Biol 19, 287332.

B. Fazi , M.J. Cope , A. Douangamath , S. Ferracuti , K. Schirwitz , A. Zucconi , D.G. Drubin , M. Wilmanns , G. Cesareni & L. Castagnoli (2002). Unusual binding properties of the SH3 domain of the yeast actin-binding protein Abp1: Structural and functional analysis. J Biol Chem 277, 52905298.

B.L. Goode , A.A. Rodal , G. Barnes & D.G. Drubin (2001). Activation of the Arp2/3 complex by the actin filament binding protein Abp1p. J Cell Biol 153, 627634.

W.M. Henne , E. Boucrot , M. Meinecke , E. Evergren , Y. Vallis , R. Mittal & H.T. McMahon (2010). FCHo proteins are nucleators of clathrin-mediated endocytosis. Science 328, 12811284.

F.Z. Idrissi , A. Blasco , A. Espinal & M.I. Geli (2012). Ultrastructural dynamics of proteins involved in endocytic budding. Proc Natl Acad Sci USA 109(39), E2589–2594.

F.Z. Idrissi , H. Grotsch , I.M. Fernandez-Golbano , C. Presciatto-Baschong , H. Riezman & M.I. Geli (2008). Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane. J Cell Biol 180, 12191232.

M. Kaksonen , Y. Sun & D.G. Drubin (2003). A pathway for association of receptors, adaptors, and actin during endocytic internalization. Cell 115, 475487.

M. Kaksonen , C.P. Toret & D.G. Drubin (2005). A modular design for the clathrin- and actin-mediated endocytosis machinery. Cell 123, 305320.

M. Kaksonen , C.P. Toret & D.G. Drubin (2006). Harnessing actin dynamics for clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 7, 404414.

J.V. Kilmartin & A.E. Adams (1984). Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol 98, 922933.

K. Kim , B.J. Galletta , K.O. Schmidt , F.S. Chang , K.J. Blumer & J.A. Cooper (2006). Actin-based motility during endocytosis in budding yeast. Mol Biol Cell 17, 13541363.

T. Kishimoto , Y. Sun , C. Buser , J. Liu , A. Michelot & D.G. Drubin (2011). Determinants of endocytic membrane geometry, stability, and scission. Proc Natl Acad Sci USA 108, E979–988.

S.A. Koestler , S. Auinger , M. Vinzenz , K. Rottner & J.V. Small (2008). Differentially oriented populations of actin filaments generated in lamellipodia collaborate in pushing and pausing at the cell front. Nat Cell Biol 10, 306313.

J.R. Kremer , D.N. Mastronarde & J.R. McIntosh (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 7176.

W. Kukulski , M. Schorb , M. Kaksonen & J.A. Briggs (2012). Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150, 508520.

W. Kukulski , M. Schorb , S. Welsch , A. Picco , M. Kaksonen & J.A. Briggs (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J Cell Biol 192, 111119.

J. Liu , M. Kaksonen , D.G. Drubin & G. Oster (2006). Endocytic vesicle scission by lipid phase boundary forces. Proc Natl Acad Sci USA 103, 1027710282.

J. Liu , Y. Sun , D.G. Drubin & G.F. Oster (2009). The mechanochemistry of endocytosis. PLoS Biol 7, e1000204.

D.N. Mastronarde (1997). Dual-axis tomography: An approach with alignment methods that preserve resolution. J Struct Biol 120, 343352.

D.N. Mastronarde (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 3651.

H.T. McMahon & E. Boucrot (2011). Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 12, 517533.

C.P. Mercogliano & D.J. DeRosier (2007). Concatenated metallothionein as a clonable gold label for electron microscopy. J Struct Biol 160, 7082.

C.J. Merrifield , M.E. Feldman , L. Wan & W. Almers (2002). Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits. Nat Cell Biol 4, 691698.

K.E. Moreira , T.C. Walther , P.S. Aguilar & P. Walter (2009). Pil1 controls eisosome biogenesis. Mol Biol Cell 20, 809818.

J. Mulholland , D. Preuss , A. Moon , A. Wong , D. Drubin & D. Botstein (1994). Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J Cell Biol 125, 381391.

J.L. Murk , G. Posthuma , A.J. Koster , H.J. Geuze , A.J. Verkleij , M.J. Kleijmeer & B.M. Humbel (2003). Influence of aldehyde fixation on the morphology of endosomes and lysosomes: Quantitative analysis and electron tomography. J Microsc 212, 8190.

T.M. Newpher , R.P. Smith , V. Lemmon & S.K. Lemmon (2005). In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast. Dev Cell 9, 8798.

S.J. Nixon , R.I. Webb , M. Floetenmeyer , N. Schieber , H.P. Lo & R.G. Parton (2009). A single method for cryofixation and correlative light, electron microscopy and tomography of zebrafish embryos. Traffic 10, 131136.

M. Seedorf , M. Damelin , J. Kahana , T. Taura & P.A. Silver (1999). Interactions between a nuclear transporter and a subset of nuclear pore complex proteins depend on Ran GTPase. Mol Cell Biol 19, 15471557.

P.A. Sims & J.D. Hardin (2007). Fluorescence-integrated transmission electron microscopy images: Integrating fluorescence microscopy with transmission electron microscopy. Methods Mol Biol 369, 291308.

I. Smaczynska-de Rooij , E.G. Allwood , S. Aghamohammadzadeh , E.H. Hettema , M.W. Goldberg & K.R. Ayscough (2010). A role for the dynamin-like protein Vps1 during endocytosis in yeast. J Cell Sci 123, 34963506.

E. Smythe & K.R. Ayscough (2006). Actin regulation in endocytosis. J Cell Sci 119, 45894598.

C.J. Stefan , S.M. Padilla , A. Audhya & S.D. Emr (2005). The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis. Mol Cell Biol 25, 29102923.

V. Stradalova , M. Blazikova , G. Grossmann , M. Opekarova , W. Tanner & J. Malinsky (2012). Distribution of cortical endoplasmic reticulum determines positioning of endocytic events in yeast plasma membrane. PLoS One 7, e35132.

V. Stradalova , W. Stahlschmidt , G. Grossmann , M. Blazikova , R. Rachel , W. Tanner & J. Malinsky (2009). Furrow-like invaginations of the yeast plasma membrane correspond to membrane compartment of Can1. J Cell Sci 122, 28872894.

Y. Sun , A.C. Martin & D.G. Drubin (2006). Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell 11, 3346.

T.M. Svitkina & G.G. Borisy (1999). Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J Cell Biol 145, 10091026.

K. Takeo , M. Shigeta & Y. Takagi (1976). Plasma membrane ultrastructural differences between the exponential and stationary phases of Saccharomyces cerevisiae as revealed by freeze-etching. J Gen Microbiol 97, 323329.

K.T. Tokuyasu (1973). A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57, 551565.

C.P. Toret , L. Lee , M. Sekiya-Kawasaki & D.G. Drubin (2008). Multiple pathways regulate endocytic coat disassembly in Saccharomyces cerevisiae for optimal downstream trafficking. Traffic 9, 848859.

P. Walther (2008). High-resolution cryo-SEM allows direct identification of F-actin at the inner nuclear membrane of Xenopus oocytes by virtue of its structural features. J Microsc 232, 379385.

P. Walther , M. Muller & M.E. Schweingruber (1984). The ultrastructure of the cell-surface and plasma-membrane of exponential and stationary phase cells of Schizosaccharomyces-Pombe, grown in different media. Arch Microbiol 137, 128134.

P. Walther & A. Ziegler (2002). Freeze substitution of high-pressure frozen samples: The visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208, 310.

T.C. Walther , J.H. Brickner , P.S. Aguilar , S. Bernales , C. Pantoja & P. Walter (2006). Eisosomes mark static sites of endocytosis. Nature 439, 9981003.

Q. Wang , C.P. Mercogliano & J. Lowe (2011). A ferritin-based label for cellular electron cryotomography. Structure 19, 147154.

J. Weinberg & D.G. Drubin (2012). Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol 22, 113.

D. Yarar , C.M. Waterman-Storer & S.L. Schmid (2005). A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis. Mol Biol Cell 16, 964975.

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Microscopy and Microanalysis
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Supplementary Materials

Buser Supplementary Material
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