Elsevier

Methods in Enzymology

Volume 504, 2012, Pages 255-271
Methods in Enzymology

Chapter thirteen - Visualization of TGN-Endosome Trafficking in Mammalian and Drosophila Cells

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Abstract

Mannose 6-phosphate receptors (MPRs) are known to be shuttled between the trans-Golgi network (TGN) and endosomes, thereby several lysosomal hydrolases are delivered through the endocytic pathway into lysosomes. This interorganellar transport is mediated by transport intermediates, now called transport carriers. Previous studies employing green fluorescent protein (GFP)-based live-cell imaging demonstrated that these transport carriers are pleiomorphic structures composed of tubular and vesicular elements. Introducing a time-axis into light microscopic observations enabled us to identify transport carriers that are derived from or targeted at a distinct organelle. In this study, we describe several methods for the observation of GFP-tagged MPRs. Photobleaching the peripheral region of a cell before a time-lapse observation allows us to monitor TGN-derived transport carriers for longer periods (more than 4 min). Events of their targeting into endosomes can be visualized by dual-color imaging of both GFP-MPRs and fluorescently tagged transferrin that is internalized by cells. By using a technique of fluorescence recovery after photobleaching (FRAP), we can analyze overall cycling kinetics of MPRs in a single cell. Transport of MPRs is regulated by several cytosolic factors like clathrin adaptors, AP1, and GGAs. The adaptors on the TGN membranes are exchanging with their cytosolic pool, which can also be analyzed by FRAP. In addition, the relationships of the MPR-containing transport carriers that left the TGN and the adaptors can be visualized by dual-color imaging. A similar system of membrane transport and its regulation is well documented in drosophila cells. As Drosophila melanogaster has only a single MPR (LERP), AP1, or GGA, it is an ideal model system for the understanding of specific functions of each cytosolic factor. To visualize these molecules in drosophila cells, however, we need to consider that multiple Golgi dots exist scattered in the cytoplasm. Thus, the Golgi dots or endosomes should be identified before live-cell imaging.

Introduction

Targeting of cellular components to their own destination is crucial for cells to maintain homeostasis and to adopt various environmental conditions. Integral membrane proteins and secretory proteins are initially synthesized in the endoplasmic reticulum and delivered through the Golgi compartments to the trans-Golgi network (TGN), where they are sorted for the post-Golgi compartments including plasma membrane, endosomes, and lysosomes. In previous studies, each transport pathway between organelles had been described as being mediated by transport vesicles (Rothman and Wieland, 1996). The introduction of live-cell imaging using GFP (green fluorescent protein)-technology clearly demonstrated that these organelles, in fact, look like pleiomorphic structures composed of vesicular or tubular elements (Bonifacino and Lippincott-Schwartz, 2003, Ghosh et al., 2003). Importantly, observations of such transport carriers along the temporal axis have provided us with the directional information of the moving structures; we can therefore describe them as “x-derived” or “x-targeting” transport carriers.

The following section mainly deals with the trafficking of the mannose 6-phosphate receptors (MPRs). MPRs capture newly synthesized lysosomal enzymes at the TGN and transport them to endosomes for the degradation of endocytic or autophagic materials (Fig. 13.1). After the delivery of enzymes, MPRs can return to the TGN for the next round of transportation, thus cycling between the TGN and endosomes. Moreover, they are known to appear on the cell surface contributing extracellular secretion of lysosomal enzymes. Further, one of the receptors, cation-independent MPR (CIMPR: another is cation-dependent MPR), functions on the cell surface as a scavenger receptor for insulin-like growth factor II (Ghosh et al., 2003). Thus, they cycle between at least three post-Golgi organelles, making the live-cell observations complicated. By fluorescence recovery after photobleaching (FRAP) technique, however, we can investigate the overall transport dynamics of MPRs, which easily tell us that MPRs are in rapid equilibrium between the post-Golgi compartments (Waguri et al., 2006). Another important aspect in MPR trafficking is the regulatory mechanisms by the clathrin adaptor molecules, adaptor protein complex 1 (AP1) and Golgi-localized, gamma ear-containing, Arf-bindig proteins 1–3 (GGA1–3). At the TGN these adaptors connect the MPRs or other cargo proteins to the clathrin triskelions contributing to the incorporation of MPRs into the clathrin-coated vesicles. This segregation and packaging events are thought to increase the efficiency in the correct sorting of MPRs. FRAP analysis also revealed that the association of the adaptors to the TGN membrane is not stable but rapidly exchanging with the cytosolic pool (Kametaka et al., 2005, Puertollano et al., 2001, Wu et al., 2003). Moreover, live-cell imaging of these proteins revealed that they are not only localized on the TGN and endosomes but also on the transport carriers (Puertollano et al., 2001, Puertollano et al., 2003, Waguri et al., 2003). It appears that both adaptors are involved in anterograde- and retrograde-transport, but the sites and modes of function are still under controversy.

Recently, Dennes and coworkers identified LERP (lysosomal enzyme receptor protein), as an ortholog of mammalian CIMPR in the genomic database of fruit fly, Drosophila melanogaster (Dennes et al., 2005). Many of the mammalian proteins that are involved in cargo sorting and transport carrier formation are also conserved in the fly genome, most often as a single protein for each (Boehm and Bonifacino, 2001), indicating that this organism possesses similar mechanisms to those used in mammalian cells. More recently, we (Kametaka et al., 2010) and Hirst et al. (Hirst et al., 2009) showed that transport of LERP involves the function of drosophila AP1 and GGA. As a result, we now know that the sorting system of CIMPR is conserved from the fly to mammals. Mammalian cells contain multiple isoforms for each subunit of AP1 and GGA (Robinson and Bonifacino, 2001), which often caused difficulties in understanding specific functions of each molecule. On the other hand, drosophila cells have only a single set of AP1 subunits or single GGA, being an ideal model system for the analysis of TGN-endosome transport.

In this section, we describe methods for live-cell imaging of TGN-endosome transport by using GFP-tagged MPRs, AP1, and GGA. We especially focus on the detection of TGN-derived or -targeting transport carriers, and some application of FRAP analysis for the overall post-Golgi transport kinetics and exchange kinetics of AP1 and GGAs. We also mention how to observe drosophila cells for live-cell imaging in the final section.

Section snippets

Molecular Tools

There have been several fluorescent proteins (FPs)-tagged tools in previous studies that have described the behavior of molecules involved in the TGN-endosome transport (summarized in Table 13.1). MPRs are type-I transmembrane proteins whose cytoplasmic domains are implicated in the interaction with several cytoplasmic factors such as AP1, GGAs, PACS1, TIP47/rab9, and retromer complex. Thus, FP fused to the cytoplasmic domain of MPRs might be sufficient for mimicking the MPR transport, making

Transfection in HeLa cells

Expression vectors containing FP-fusion constructs are transiently expressed in HeLa cells using a lipofection reagent as follows:

  • 1.

    HeLa cells are grown in α-MEM (Invitrogen) supplemented with 10% FBS (Hyclone Laboratories, Inc.), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2. As a passage procedure, ∼ 10% of cells are transferred to a new dish when they reach a confluency of approximately 80%.

  • 2.

    Two days before the live-cell imaging, HeLa

Live-Cell Imaging in Drosophila Cells

In drosophila Schneider S2 cells (hereafter referred to as S2 cells), Golgi compartments are identified as punctate structures scattering throughout the cytoplasm (Kametaka et al., 2010, Kondylis et al., 2001). This characteristic distribution pattern (Fig. 13.5) makes it hard to tell which dots are Golgi and which are endosomes at a light microscopic level. However, it has recently been shown that clathrin and clathrin adaptor dGGA are mainly localized at the Golgi compartments in S2 cells

Conclusion Remarks

We described practical live-cell imaging techniques for observation of the TGN-endosome transport from several aspects. Using these methods, we can successfully visualize transport carriers for MPRs that move between the TGN and endosomes, together with clathrin adaptors. We can also analyze the overall transport kinetics of MPR in a single cell, or exchange kinetics of clathrin coat-associated molecules between the TGN membrane and cytosolic pool. Moreover, the TGN-endosome transport can be

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