High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

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Abstract

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.

Introduction

Fine structural and functional analyses of complex and dynamic cell compartments, such as those of the biosynthetic and endocytic systems, require methods that allow a high temporal resolution together with an excellent fine structural preservation, which in turn guarantees high spatial resolution. In the electron microscope, dynamic processes can be analyzed only by series of static snapshots obtained by appropriate preparation procedures (Plattner, 1989). In order to generate copies of in vivo-processes, and to prevent the occurrence of supravital artifacts, physical fixation methods, in particular fast freezing techniques, have been set up as methods of choice. Since the introduction of pressure-freezing leading to vitrification of the samples and avoidance of ice crystal formations (Moor, 1986, Moor, 1987), appropriate cryo-fixation and substitution procedures have been established for a variety of different types of samples (Dahl and Staehelin, 1989, Dubochet, 2009, Edelmann, 1991, Hawes et al., 2007, Hohenberg et al., 1994, Humbel, 2009, Jiménez et al., 2006, Kaech, 2009, Robards and Sleytr, 1985, Steinbrecht and Müller, 1987, Studer et al., 1989). Main advantages of fast freezing techniques are the avoidance of artifacts produced by chemicals and freezing damages, and the rapid immobilization of the cellular life processes permitting the imaging of dynamic processes with high temporal resolution. For the success of the preparation, and the quality of the final structural appearance both the freezing quality and the substitution protocol are equally important (Bohrmann and Kellenberger, 2001, Giddings, 2003, Hawes et al., 2007, Matsko and Mueller, 2005, Monaghan et al., 1998, Schwarz and Humbel, 2007, Studer et al., 2008, Vanhecke and Studer, 2009).

In mammalian cells, complex membrane compartments characterize the transition zones at the interfaces of the biosynthetic and endocytic cellular pathways at the trans-Golgi side (Ladinsky et al., 1999, Marsh, 2005, Marsh et al., 2001, Mogelsvang et al., 2004, Vetterlein et al., 2002). These include trans-Golgi cisternae, multiple heterogeneous and polymorphous endocytic compartments, multivesiculated bodies, and the endocytic trans-Golgi network (endocytic TGN; Pavelka et al., 1998, Pavelka et al., 2008, Vetterlein et al., 2002). These organelles are dynamic, change shapes rapidly, interact with each other, and establish continuities with neighboring compartments. Previous studies proved the importance of a rapid vitrification of cells, e.g. by high-pressure freezing (HPF), for a detailed insight into architectures and organization of such complex and dynamic organelles (Bouchet-Marchis et al., 2008, Geerts et al., 2009, Hess et al., 2000, Knoll et al., 1987, Ladinsky et al., 1999, Leis et al., 2008, Lucic´ et al., 2008, Marsh, 2005, Marsh et al., 2001, Mogelsvang et al., 2004, Murk et al., 2003, Plitzko et al., 2002, Robinson and Karnovsky, 1991, Verkade, 2008). However, there exist limitations mainly concerning concurrent cytochemical analyses: in cryo-immobilized cells, the differentiation of endocytic compartments by a common, widely used cytochemical method, which localizes internalized peroxidase-labeled molecules (Graham and Karnovsky, 1966), is only possible with limitations. The use of peroxidase-labeled substances and their localizations by means of diaminobenzidine (DAB) precipitations provides a widely used approach for the analyses of endocytic routes well known since many years (Gonatas et al., 1977, Pavelka, 1987, van Deurs et al., 1986). We prefer the peroxidase system in studies that deal with complex organelle architectures made up of branched membrane systems containing fine bridges and tiny membrane continuities. In these cases, the peroxidase method has advantages over other techniques, since the DAB-staining produces clearly labeled structures, which can easily be traced and hence are especially useful for EM-tomography and the modeling of 3D-architectures.

Retrograde routes from the plasma membrane to intracellular destinations involve complex and dynamic compartments (Bonifacino and Rojas, 2006, Johannes and Popoff, 2008, Maxfield and McGraw, 2004, Pavelka and Ellinger, 2008, Pavelka et al., 2008, van Deurs et al., 1986), which are visited physiologically but also misused by toxins, and used for drug targeting to the interior of cells (Sandvig and Van Deurs, 2005, Tarrago-Trani and Storrie, 2007, Weissenböck et al., 2004). In previous work, we characterized changes of the Golgi apparatus and TGN during endocytosis using internalized peroxidase-conjugated wheat germ agglutinin (WGA; Pavelka et al., 1998, Pavelka et al., 2008, Vetterlein et al., 2002, Vetterlein et al., 2003). With the goal to combine this method with the advantages of fast freezing, we adapted a technique for in vivo-staining of endocytic organelles prior to fixation. In this method that originally was used for cell fractionation (Courtoy et al., 1984), for immunoelectron microscopic examinations of endosomes in nonsectioned cells by whole-mount electron microscopy (Stoorvogel et al., 1996), and for inactivation of endosomes, lysosomes, and Golgi apparatus (Brachet et al., 1999, Futter et al., 1996, Jollivet et al., 2007, Pond and Watts, 1999), the DAB-reaction is performed in the living cells, which subsequently are cryo-immobilized, freeze-substituted, and embedded in resin.

The main steps include uptake of horseradish peroxidase (HRP)-labeled molecules, in vivo-staining of the involved compartments and their immobilization from the luminal side by generation of DAB precipitates via oxidation by HRP, vitrification of the samples by means of HPF, freeze-substitution, and embedding in Epon. The results are compared with the findings obtained by HPF without cytochemistry, and by DAB-staining prior to and after chemical fixation. The novel technique proved to be well suitable for morpho-functional studies and 3D-explorations of the complex and dynamic compartments of the endocytosis system.

Section snippets

Cell cultures

All cell culture reagents were purchased from Sigma (Sigma Chemicals, St. Louis, MO). Cells were cultured according to standard procedures; briefly, human HepG2 hepatoma cells (American Type Culture Collection, Rockville, MD) were cultured in modified Eagle’s medium (MEM), supplemented with 10% fetal bovine serum, 2 mM l-glutamine (PAA Laboratories, Linz, Austria) at 37 °C in a humified atmosphere of 95% air, 5% CO2. Cells were grown in monolayers on 13 mm ∅ round glass coverslips or 3 mm ∅ round

Cell cultures

The HepG2 hepatoma cells are well characterized, and multiple details of the secretory and endocytic pathways have been analyzed in previous studies (Pavelka et al., 1998, Pavelka et al., 2008, Vetterlein et al., 2002, Vetterlein et al., 2003). Nevertheless, the general suitability of this culture system for HPF and in vivo cytochemical staining procedures had to be tested; in initial experiments, several parameters were checked, such as the adherence capacity of the HepG2 cells on the sapphire

Discussion

Quick freezing methods including high-pressure freezing, leading to vitrification of the cell samples, are state of the art for analyses of cell and tissue structures close their natural properties at high spatial and temporal resolutions. However, the practicability of HPF is limited in cases, which require a combination with cytochemical methods, in particular with techniques using peroxidase-catalyzed DAB-oxidation. DAB-cytochemistry introduced by Graham and Karnovsky (1966) is widely

Acknowledgments

The authors gratefully acknowledge the skilful technical assistance of Mrs. Barbara Kornprat, Mrs. Beatrix Mallinger, and Mr. Peter Auinger, and thank Mr. Ulrich Kaindl for his valuable help with the artwork and the 3D-models.

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