Cell Biology Protocols

154FRACTIONATION OF SUBCELLULAR MEMBRANES

Protocol 5.14 Analysis of early and recycling endosomes in preformed

Introduction

In conjunction with confocal microscopy, density gradients are often used to analyse processes that might be broadly defined as membrane trafficking and cell signalling. They are relevant to a diverse range of cellular events such as secretion, biosynthesis, endocytosis and virus processing. They involve the use of gradients to fractionate not only the principal subcellular organelles such as smooth and rough endoplasmic reticulum (ER) and the Golgi, but also subcompartments such as the trans-Golgi network (TGN), the ER-Golgi Intermediate Compartment (ERGIC), early and late endosomes, transport and secretory vesicles and plasma membrane domains such as caveolae and lipid rafts. This rich area of cell biology research also involves studies into the transfer of proteins from the cytosol to a membrane, which might occur for example during cell differentiation.

Methods available

Frequently, a simple post-nuclear supernatant is used as a gradient input rather than a partially purified fraction such as a light mitochondrial pellet or microsomes. Since the gradient is often used not so much to purify one particular type of membrane (see Chapter 4), but rather to analyse the translocation of proteins or other molecules from compartment to compartment, it is often considered more important that none of the potentially involved compartments gets lost in some pre-gradient treatment. Such a simple treatment of the homogenate also reduces the experimentation time and number of manipulations to a minimum.

For many years, sucrose gradients were the principal vehicle for analysing these membrane compartments and a number of the most well-established protocols using this solute are presented in this chapter. Increasingly, however, the use of iodinated density gradient media, notably Nycodenz and iodixanol, are being used for this purpose and a number of examples of the use of these gradient solutes are given. They have several advantages over sucrose; they are commercially available as dense solutions, making gradient solutions easy to prepare, and their lower osmolality compared to sucrose solutions of similar density (particularly iodixanol solutions) often results in a greater resolving power than that of sucrose.

The viscosity of Nycodenz and iodixanol solutions is also lower than that of sucrose solutions of similar density and there are many examples that take advantage of the consequent more rapid movement of membrane particles through the gradient. Gradients of Nycodenz and iodixanol are frequently run for 2–4 h, rather than overnight, which

is the norm for sucrose gradients. However, there is a school of thought that maintains that to get true equilibrium density banding it is necessary to centrifuge for long periods (> 12 h) at relatively low centrifugation speeds, irrespective of the gradient medium. A number of iodixanol gradient methods support this contention (see Protocol 5.8). There are also some examples of the use of very short centrifugation times, which tend to separate particles principally on the basis of sedimentation velocity (see Protocol 5. 9).

One property of iodixanol that is not available for sucrose is its ability to form selfgenerated gradients, given a sufficiently high g-force and the right sort of rotor (vertical or near-vertical) [1]. There are several advantages of using self-generated gradients over the more standard preformed gradients: they are very easy to prepare (the sample is simply adjusted to a certain iodixanol concentration); they are very reproducible and very shallow gradients may be generated (under the appropriate centrifugation conditions) – such gradients are not easily preformed. Protocol 5.15 is an example of the use a self-generated gradient.

Thus, the protocols in this chapter are generally not aimed at the isolation of an individual membrane compartment; rather they describe the use of a variety of gradient systems that might be used for analysing some aspect of membrane trafficking or cell signalling. An exception is the group of protocols at the start of this chapter that describes the isolation of various types of plasma membrane domain. Because plasma membrane domains have a unique composition and function, they are of particular importance in studies on the means by which the cell directs and controls the flow of molecules to and from the surface.

Plasma membrane domains

Bile canalicular and basolateral domains of rat liver plasma membrane are prepared from the plasma membrane sheets, isolated in Protocol 4.26, after vesiculation, which is usually produced either by sonication [2, 3] or liquid shear [4–6]. The two domains are then separated in a continuous or discontinuous sucrose gradient (Protocol 5.1). Sometimes Nycodenz gradients are used to provide additional purification [7, 8]. The sinusoidal domain is rather less easy to purify by density gradients and it is more usually obtained by density perturbation with protein A-sepharose beads (Protocol 5.2 ). The beads are bound to an antibody to the polymeric IgA receptor, often called the secretory component (SC), which is a specific marker for the sinusoidal plasma membrane domain [9]. Once bound to the beads, the sinusoidal membrane vesicles can be harvested in a microcentrifuge. The methodology has been widely reviewed [10, 11]. Protocols 5.3 and 5.4 provide methods for the fractionation of apical and basolateral domains from polarized cells such Caco-2 [12] and MDCK cells [13, 14] in sucrose and iodixanol gradients respectively.

Lipid rafts, the specialized cholesterol-, sphingolipidand caveolin-rich microdomains at the surface of a variety of cell types, are involved in an important range of signaltransduction events, virus processing and lipid transport. They are routinely isolated as detergent-resistant membranes (DRMs) by flotation through a discontinuous iodixanol gradient (Protocol 5.5). The method was initially worked out for a line of mouse mammary epithelial cells [15] and for MDCK cells [16], but has since been extended to a huge range of cell types and the details of the gradient centrifugation have been varied

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considerably. In contrast, the flotation gradient for the purification of caveolae (Protocol 5.6), which has also been extended from human skin fibroblasts [17] to the use of a vast range of tissue and cell types, has hardly been modified at all [18]. It relies on the sonication of a purified plasma membrane preparation, followed by two iodixanol flotation gradients: the first being a purification in a continuous gradient, the second being a concentration in a discontinuous gradient [17, 18].

Analysis of membrane compartments in the endoplasmic reticulum–Golgi–plasma membrane pathway

Sucrose–D2O gradients

Before iodinated density gradient media became widely used for analysis of membrane trafficking, one particular strategy to reduce the concentration of sucrose solutions necessary to band subcompartments of the ER and Golgi membrane systems was to dissolve the sucrose in D2O (Protocol 5.7). This allowed gradients of lower osmolality and viscosity to be used [19]. The method was used primarily to study the transfer of proteins from the rough endoplasmic reticulum to the cis-Golgi. The gradient showed that this intercompartmental transport involved a low-density vesicle quite distinct from its source and destination membranes. Part of the resolution of such gradients is achieved by collection of the gradients in small fraction volumes (12 ml gradients in 32 fractions). The gradients are also capable of a high degree of resolution of ER-Golgi-trans Golgi network (TGN) events [19, 20] and subfractionation of the Golgi membranes in the analysis of N-linked oligosaccharide processing [21].

Buoyant density iodixanol gradients (preformed)

Continuous iodixanol gradients were first used by Yang et al. [22] and Zhang et al. [23] to fractionate the ER and Golgi from COS-7 and CHO cells (Protocol 5.8 ). The density of these membranes generally decreases in the order ER > Golgi > PM. Most centrifugations are carried out for approx. 3 h, but the separation of ER and Golgi is enhanced by longer periods (16 h) and relatively low g-forces (<100 000g). This strategy has permitted the clear resolution of ERGIC in the middle of the gradient between the Golgi and the ER [24]. Long period centrifugations have also permitted the resolution of perinuclear ER from that ER more further from the nuclear region [25]. Most gradients are in the region of 1–25% iodixanol, and depending on the cell type, other compartments such as early and late endosomes and TGN may also be at least partially resolved (see Table 5.2 in Protocol 5.8 for a summary of some of the variations).

Continuous linear gradients may be constructed by diffusion of the solute between layers of the same volume, whose density increases regularly from one layer to the other. Thus a 10–30% iodixanol gradient might be formed from equal volumes of 10, 20 and 30% iodixanol. Sometimes increased resolution may be obtained in convex or concave gradients. Although there are devices to create such gradients [26] they are not particularly common, nor are they easy to use; an alternative is to construct the gradient from steps of increasing (or decreasing) volume and/or increasing (or decreasing) concentration interval. Table 5.3 in Protocol 5.8 describes some of the variants and their use.

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vesicle budding. The strategy for separating membranes and cytosol, which involves flotation of the membranes through an iodixanol barrier from a dense sample, has been developed for both eukaryotes and bacteria (Protocol 5.13). This strategy allows maximum resolution of the two components, since the proteins, which are much denser than the membranes, tend to sediment rather than float. It removes any ambiguity in the results from top-loaded sucrose gradients, in which the membranes sediment and the proteins move more slowly in the same direction due to sedimentation and diffusion. The difference in density between the proteins and the membrane vesicles is also much greater in iodixanol than in sucrose, primarily due to the much lower osmolality of the iodixanol gradients. Similar methods are used for the separation of liposomes from denser proteoliposomes.

Endocytosis

Ligand labelling

In most instances the endocytic process is followed experimentally by using a ligand that can be identified by (i) a radiolabel, (ii) a covalently bound enzyme, subsequently detected by provision of a substrate, or (iii) colloidal gold. Although such modifications to the ligand may modulate its behaviour, unless the ligand itself can be easily identified, they are difficult to avoid. The basic strategy is to present the labelled ligand to the tissue or cells in short pulse, after which it is chased through the various intracellular compartments.

Tissue systems

Establishment of a perfused rat liver the system requires an anaesthetized animal; it must be performed by a trained and licensed person. After allowing the isolated perfused liver to equilibrate with the perfusion medium, using a continuous recycling system, the labelled ligand is presented in a single-pass perfusion for 1 min, followed by unlabelled medium for varying times. The process is arrested by perfusion with ice-cold homogenization medium. The perfused liver provides a situation as close to that in vivo as possible, but it is difficult to use with very short chase times or when studying large numbers of variables.

Cell systems

A cell monolayer culture provides the most easily managed system for investigating incubation conditions and exposure to perturbants or use of more than one ligand; cultured hepatocytes or MDCK cells are popular choices. The ligand is allowed to bind to the cells at 0 ◦C; the ligand-containing medium is then removed and the monolayer washed with culture medium. Endocytosis is then allowed to proceed at 37 ◦C (in the presence or absence of perturbants) and then rapidly cooled to 0 ◦C (with ice-cold homogenization medium) to arrest further ligand processing. Detailed information on strategies for studying endocytosis has been published elsewhere [31].

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Analysis

Sucrose gradients can be used to separate the endocytic compartments on the basis of density [32] but unless the ligand is attached to a marker that can perturb the density of the endosomes, the resolving power is not great. The two most commonly employed methods use either colloidal gold [33] or horseradish peroxidase (HRP) [34]. In the latter case the density perturbation is achieved prior to gradient by incubation of the membrane fraction with H2O2 and benzamidine, which is polymerized in the HRPcontaining vesicles. There are, however, several gradient techniques which do not require these sometimes inconvenient perturbation techniques and they have been reviewed in ref. 31. Three examples are provided: a preformed iodixanol gradient for analysing early and recycling endosomes (Protocol 5.14), a self-generated iodixanol gradient for analysing the processing of clathrin-coated vesicles (Protocol 5.15) and a polysucroseNycodenz gradient for analysis of late endosomes/lysosome events (Protocol 5.16).