**8. Conclusions**

18 Crosstalk and Integration of Membrane Trafficking Pathways

of certain proteins in mitotic cells might be masked. Our semi-intact cell assay is suitable for investigating the biochemical requirements of specific processes, which might be masked by the orchestrated physiological reactions. For example, the ER disassembly assay revealed that a p97/p47-mediated fusion process plays a crucial role in the maintenance of the ER network when microtubules are disrupted by nocodazole. In another case, the ER reformation assay revealed that reformation of the ER is accomplished even in nocodazoletreated semi-intact cells. When microtubules are intact, the contribution of the fusion process to the maintenance or reformation of the ER network appears to be masked. Our transport assay also revealed that, even in the presence of mitotic cytosol, the retrograde transport of GT-GFP occurs normally when microtubules remain intact, but ERES are disassembled easily under these conditions (Fig.14). The findings suggest that mitotic cytosol can facilitate retrograde transport as long as microtubule integrity is maintained, but anterograde transport ceases rapidly in the presence of mitotic cytosol. Thus, the ability to manipulate the cytoskeleton easily in semi-intact cell systems will be useful in elucidating the role of the cytoskeleton in the process of morphological change in organelles or

Many *in vitro* reconstitution assays have been developed to investigate the biochemical requirements for the maintenance or mitotic alteration of Golgi or ER morphology, and a variety of key molecules have been identified using these methods (Acharya et al.,1998; Lowe et al., 1998,2000; Hetzer et al., 2001). Our semi-intact cell assays will be useful for confirming the precise role of these molecules in the maintenance or alteration of morphology under conditions in which the configuration between organelles and the cytoskeleton is almost the same as in living cells. Thus, our assays will provide additional spatial information about where the molecules function or where the biological reactions occur in cells. By applying the Golgi disassembly assay in semi-intact cells, we found that punctate Golgi structures (Fig. 3, stage II Golgi), which are produced mainly by MEK1 from cisternal Golgi and are referred as to Golgi mini-stacks, are found mainly on the apical side of the nucleus and are associated with apical microtubules. Given that the spatial configuration of the cell is virtually unaffected in semi-intact cells, the semi-intact cell assays are superior to *in vitro* reconstitution assays for investigating the anterograde or retrograde

There are some differences between *in vitro* reconstitution systems and our semi-intact cell system. For example, an *in vitro* ER formation assay developed by Dreier and Rapoport (2000) revealed that the characteristic polygonal structure of the ER was formed from microsomal membranes. However, the *in vitro* network produced in their assay appeared to be slightly different from the ER network formed in CHO-HSP cells. The length of one side of the three-way junctions was approximately 5 μm in their reconstituted network, compared to 1–1.5 μm in our intact or semi-intact CHO-HSP cells. We have frequently observed that this length varies with the cellular conditions. For example, following serum

Collectively, our semi-intact cell assays are superior to *in vitro* reconstitution assays in terms of obtaining morphological or spatial information, but *in vitro* assays are more appropriate for determining biochemical requirements than semi-intact cell assays. Using both assays together will enable us to identify the key molecules involved in morphological changes, which might be masked by the orchestrated processes that occur

starvation, the length appears to be greater than 5 μm (F. K., unpublished data).

membrane trafficking during mitosis.

transport between the Golgi and the ER.

The mechanisms that regulate the cell cycle-dependent changes in Golgi morphology in mammalian cells have been studied extensively (see reviews, Wei & Seemann, 2009). In terms of the relationship between Golgi morphology and membrane trafficking, the size and morphology of the Golgi are thought to be determined mainly by the membrane influx/efflux ratio. Thus, the characteristic features of Golgi morphology could depend on the stage of the cell cycle, cell type or intracellular conditions (Sengupta & Linstedt, 2011). In contrast, many aspects of the regulation of the morphology of the ER network remain poorly understood. The ratio of membrane influx/efflux at the ER seems to affect ER morphology less than the ratio at the Golgi affects Golgi morphology because a large amount of membrane is retained in ER structures and this could have a buffering effect on ER morphology. Unlike the case of the Golgi, a variety of ER stress responses might be induced by the aberrant accumulation of secreted proteins in the ER by the inhibition of anterograde transport, and these responses might cause not only ER dysfunction but also the change in its morphology. Furthermore, accumulating evidence suggests that the communication between the early secretory organelles and plasma membrane exists. For example, signaling by growth factors (e.g. MAPK/ERK) at plasma membranes affects the early secretory pathway (anterograde transport) via the ERES (Farhan et al., 2010). Thus, it is important to investigate the overall balance of membrane trafficking between the relevant organelles, as well as the plasma membrane, to elucidate the changes in Golgi and ER morphology that occur during mitosis more fully. The quantitative analysis of membrane trafficking while the spatial configuration of cells is maintained will be of increasing significance. Therefore, our semi-intact cell assays will provide one suitable tool for studying the regulatory mechanisms of membrane trafficking, not only during mitosis, but also under other cellular conditions, for example, disease conditions.

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**2** 

*Italy* 

**Morphogenesis and Dynamics of** 

Roman S. Polishchuk1,\* and Elena V. Polishchuk1,2

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

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

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

composition became the subjects of significant interest over the last decade.

**1. Introduction** 

which they belong.

Corresponding Author

 \*

**Post-Golgi Transport Carriers** 

*1Telethon Institute of Genetics and Medicine, Naples* 

*2Institute of Protein Biochemistry, Naples* 

Zaal, K.J.M.; Smith, C.L.; Polishchuk, R.S.; Altan, N.; Cole, N.B.; Ellenberg, J.; Hirschberg, K.; 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.*  99, pp. 589–601.
