**1. Introduction**

22 Crosstalk and Integration of Membrane Trafficking Pathways

Zaal, K.J.M.; Smith, C.L.; Polishchuk, R.S.; Altan, N.; Cole, N.B.; Ellenberg, J.; Hirschberg, K.;

99, pp. 589–601.

Presley, J.F.; Roberts, T.H.; Siggia, E.;Phair, A,D. & Lippincott-Schwartz, J. (1999). Golgi membranes are absorbed into and reemerge from the ER during mitosis. *Cell.* 

> The identities of many intracellular organelles and of specific domains of the cell surface rely on the delivery of proteins and lipids through biosynthetic or/and endocytic pathways to the sites of their specific activities. The Golgi complex serves as a central station in the biosynthetic pathway, from where proteins are sorted towards their different destinations, such as various domains of the cell surface or the endosomal-lysosomal system. To be delivered from the Golgi complex to their target compartments, cargo proteins are incorporated into dynamic membrane-bound organelles that are generally known as 'post-Golgi carriers'. Given that these post-Golgi carriers have such an important role in the process of intracellular transport their morphology, living dynamics and molecular composition became the subjects of significant interest over the last decade.

> Post-Golgi carriers (PGCs) were originally discovered and described as a result of the development of green fluorescent protein (GFP) technology and live-cell imaging (Lippincott-Schwartz et al., 2000). The first few fluorescently tagged cargo proteins observed in living cells revealed a new world of highly dynamic structures traveling from the Golgi complex to the plasma membrane (Hirschberg et al., 1998; Nakata et al., 1998). With time, the list of molecules that could be visualized *in vivo* expanded greatly, to expose the unexpected complexity of the post-Golgi transport pathways. However, in mammalian cells, most of PGSs have several common features that are independent of the pathway(s) to which they belong.

> PGCs form from membrane domains of the Golgi complex that lack resident Golgi enzymes, and there are known as 'PGC precursors' (Hirschberg et al., 1998; Keller et al., 2001; Polishchuk et al., 2003; Puertollano et al., 2003). The shapes and sizes of PGCs that can even carry the same cargo vary across a relatively wide range. Most that were seen under light microscopy were clearly larger that plasma membrane (PM)-associated clathrin vesicles and 100-nm-diameter fluorescent beads (Lippincott-Schwartz et al., 2000). Indeed, while the smaller PGSs can usually have an extension of 300 nm to 400 nm, some large carriers can reach dozens of microns in length. Video microscopy has revealed that many of these

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Morphogenesis and Dynamics of Post-Golgi Transport Carriers 25

The first step of PGC formation coincides with the segregation of the cargo proteins from the Golgi-resident enzymes. This appears to be a common process for proteins that exit the Golgi complex to head towards different post-Golgi compartments, such as the basolateral PM (Hirschberg et al., 1998; Polishchuk et al., 2003), the apical PM (Keller et al., 2001), and the endosomal-lysosomal system (Puertollano et al., 2003). These enzyme-free Golgi domains usually exhibit a tubular structure and contain *bona-fide* TGN markers (Polishchuk et al., 2003; Puertollano et al., 2003). At the EM level, this segregation of the cargo proteins from the Golgiresident enzymes corresponds to the transition of the cargo-containing compartments from

This thus raises the question of the mechanism by which the originally flat Golgi membranes are converted into highly bent, tubular-reticular TGN structures. A long time ago, Rambourg and Clermont (Rambourg and Clermont, 1990) noted that the cisternae in the middle of a Golgi stack appear quite 'solid' and contain just a few small fenestrae (Fig. 2A). During progression toward the *trans* face of the Golgi complex, both the number and size of these fenestrae increase (Rambourg and Clermont, 1990). Thus the *trans*-most Golgi cisternae generally look like flat tubular webs (Fig. 2A, B), which frequently 'peel off' from the Golgi stack (Rambourg and Clermont, 1990) (Fig. 2C, D), and these can then be easily transformed into TGN membranes by a few fission events (see Fig. 2B). A similar conversion of the Golgi compartments was seen to

The mechanisms behind this transformation are not yet completely clear. The cisterna-like morphology of the Golgi compartments can be stabilized through the formation of large polymers formed by the Golgi enzymes (Nilsson et al., 1996). Indeed, truncation of the protein domains responsible for enzyme oligomerization results in a loss of the regular Golgi morphology (Nilsson et al., 1996). Thus, a gradual reduction in Golgi enzyme polymers in the *trans*-Golgi compartments would favor transformation of cargo-containing cisternae into networks of tubular membranes. This process can be further assisted by changes in the lipid composition of the *trans*-Golgi membranes, which include the input of material from the endocytic system (Pavelka et al., 1998) and the local activities of TGNspecific lipid-modifying enzymes or lipid-transfer proteins (De Matteis and Luini, 2008). Therefore, the membranes become thicker in the TGN and they thus fail to provide a favorable environment for the short transmembrane domains of the Golgi enzymes (Munro, 1995). As a result, the Golgi enzymes get pushed out of the tubular TGN back towards the

The bending of the flat cisternae membranes into TGN tubules might be also facilitated by the action of specific proteins. Various roles of structural proteins in membrane deformation/ tubulation have been widely recognized (McMahon and Gallop, 2005). These proteins act either by forcing membrane curvature or by sensing and stabilizing it. As an example of the former, coat proteins (such as clathrin) polymerize into curved structures that can bend membrane domains (Antonny, 2006). Alternatively, such TGN proteins as the FAPPs can insert their amphipathic helices into the outer leaflet of a lipid bilayer, thereby increasing the positive membrane curvature (Lenoir et al., 2010). Other rigid curved proteins, or protein modules (such as the BAR domain), can bind to curved membranes and stabilize them by electrostatic interactions (Cullen, 2008; McMahon and Gallop, 2005).

**2.1 Morphogenesis of PGC precursors at the TGN** 

cisterna-like into a tubular network morphology (Polishchuk et al., 2003).

occur along the Golgi stack in the yeast *Pichia pastoris* (Mogelsvang et al., 2003).

core Golgi regions that are composed of the stacked cisternae.

carriers are globular in appearance, although they are frequently stretched into tubular shapes during their translocation through the cytosol. Thus, PGCs have been frequently referred to as 'pleiomorphic' structures. PGCs use microtubules to move towards particular locations within the cell. Although carriers can form and support post-Golgi transport without association with microtubules, the correct targeting of cargo proteins is usually compromised under such conditions (Kreitzer et al., 2003; Rindler et al., 1987)

The life cycle of a PGC consists of three stages: (i) formation; (ii) transition through the cytosol; and (iii) docking and fusion with the target membrane (Fig. 1) (Polishchuk et al., 2000). In this chapter we take you on a journey with newly born PGCs, to follow them through all of the stages of their life cycle.

**A.** An example of PGCs dynamics, as taken in the area (dashed box) of a living cell expressing VSVG-GFP upon release of this chimeric protein from the Golgi complex. **B.** Representative images extracted from a time-lapse sequence (corresponding to the area outlined by the dashed box in panel A) show the dynamics of PGC formation from the Golgi complex (arrows). **C.** Transition of the same newly formed PGC through the cytoplasm (arrows). **D.** Fusion of the same PGC (panels B, C) with the target membrane, as shown in these three subsequent time-lapse images by arrows.

Fig. 1. The life cycle of a PGC.
