**Semi-Intact Cell Systems – Application to the Analysis of Membrane Trafficking Between the Endoplasmic Reticulum and the Golgi Apparatus and of Cell Cycle-Dependent Changes in the Morphology of These Organelles**

Masayuki Murata and Fumi Kano *The University of Tokyo Japan* 

#### **1. Introduction**

The endoplasmic reticulum (ER) and the Golgi apparatus both maintain their specific morphology, composition, and function in spite of the exchange of proteins and lipids between the two organelles through membrane trafficking. The morphology of the Golgi apparatus is closely linked to the balance between anterograde (ER-to-Golgi) and retrograde (Golgi-to-ER) transport. It has been reported that the inhibition of anterograde transport leads to the redistribution of Golgi components to the cytoplasm or the ER (Storrie et al., 1998; Ward et al., 2001; Miles et al., 2001). Inhibition of anterograde transport at the onset of mitosis also results in the relocation of Golgi enzymes to the ER (Zaal et al., 1999; Altran-Bonnet et al., 2006), although it is controversial as to whether the Golgi becomes integrated with the ER or whether they remain separate throughout mitosis (Lowe and Barr, 2007). Thus, in living cells, the rates of anterograde and retrograde transport between the two organelles appear to have a substantial effect on the morphology of the Golgi. The effect of the balance between anterograde and retrograde transport on the morphology of the ER remains to be explored. Recently, increasing evidence has suggested that alteration in Golgi morphology during mitosis is not a passive process but rather an active one, which is highly coordinated with entry into mitosis and its progression. On the basis of the concept that cell cycle-dependent membrane trafficking between the ER and Golgi during mitosis should be tightly coupled with the changes in their morphology, it will be important to elucidate the cell cycle-dependent regulation of membrane trafficking to understand the morphological changes in more detail.

However, in general, investigation of morphological changes in the Golgi and ER during mitosis is hampered by the fact that it is difficult to observe the precise morphology of organelles in mammalian cells during mitosis by light microscopy due to the round shape of the cells. The perturbation of the distinct characteristics of the two organelles during mitosis makes it difficult to analyze the efficiency of membrane trafficking between the organelles using quantitative microscopic methods, such as fluorescence recovery after photobleaching

Semi-Intact Cell Systems – Application to the Analysis of Membrane

insensitive to temperature.

Trafficking and of Cell Cycle Dependent Changes in the Morphology of the Organelles 3

difficult to avoid damage to intracellular structures when cells are permeabilized with digitonin, a well-known pore-forming toxin, because digitonin-induced permeabilization is

By exchanging cytoplasmic proteins with exogenously added proteins, antibodies or cytosol that has been prepared from cells at distinct stages of the cell cycle or differentiation, or from disease states, we can modulate the intracellular environment and reconstitute various physiological phenomena in semi-intact cells. The semi-intact cell method was originally established by Dr. Simons' group to study polarized vesicular trafficking in Madin-Darby canine kidney (MDCK) cells (Ikonen et al., 1995). We have refined the method by coupling it with GFP-visualization techniques and have established many types of assay for cell cycledependent changes in organelle morphology and membrane trafficking. Using our analytical system, we can manipulate intracellular conditions and then observe the resulting morphological changes in GFP-tagged organelles by fluorescence microscopy. In addition, we are able to dissect complex reaction processes in cells on a morphological basis and to investigate the biochemical requirements and kinetics of each process, for example the vesicular transport between the ER and the Golgi during mitosis. In particular, the maintenance of the integrity of the organelles and their configuration in the semi-intact cells

enables us to analyze membrane trafficking in as intrinsic environment as possible.

typical of Golgi membranes in living mitotic MDCK-GT cells.

**3. Reconstitution of Golgi disassembly by mitotic cytosol in semi-intact cells**  Disassembly of the Golgi during mitosis is a dynamic and highly regulated process, and is required for an equal partitioning of Golgi membranes into the two daughter cells. To investigate the biochemical requirements and kinetics of Golgi disassembly during mitosis, we reconstituted the process by adding mitotic cytosol prepared from *Xenopus* eggs to semiintact cells and visualized it with GFP-tagged proteins (Kano et al., 2000). To this end, first, we produced the stable transfectant MDCK-GT, which continuously expresses mouse galactosyltransferase (GT) fused with GFP (GT-GFP). GT-GFP has been used to study Golgi membrane dynamics in living cells and has been characterized in detail (Cole et al., 1996). Next, we prepared semi-intact MDCK cells, which had been grown on polycarbonate membranes, incubated them with various types of cytosol, and observed the resulting changes in Golgi morphology. In semi-intact cells incubated with *Xenopus* interphase extracts, the Golgi apparatus forms perinuclear, tubular structures, which are typical of the Golgi apparatus in MDCK-GT cells. By contrast, incubation with *Xenopus* egg (M phase) extracts causes the Golgi to disassemble and the fluorescence of GT-GFP diffuses completely throughout the cytoplasm (see Fig. 2A, stage III). The diffuse staining pattern of GT-GFP that is observed, and which corresponds to small heterogeneous vesicular structures, is

Fluorescence microscopic observation of Golgi disassembly induced by mitotic cytosol in single MDCK-GT cells revealed that the disassembly process can be divided into three stages: stage I (intact), II (punctate), and III (dispersed) (Fig. 2). During stage I (intact), the tubular and stacked structures of a typical intact Golgi apparatus are observed in the perinuclear region through the apical to the middle part of the cells. During stage II (punctate), punctate Golgi structures are observed on mainly the apical side of the nucleus (Fig. 2A, stage II). These punctate structures are seldom seen in the basolateral cytoplasm. During stage III (dispersed), a diffuse staining pattern is observed throughout the cytoplasm

(FRAP), etc. In addition, the fact that concerted morphological changes occur in the organelles simultaneously and transiently in a single cell during mitosis makes it extremely difficult to dissect these changes morphologically and biochemically. The analysis is complicated further by the asynchronous progression of the cell cycle in individual cells.

Herein, we describe a novel method that addresses the above-mentioned problems, namely, a semi-intact cell assay coupled with green fluorescence protein (GFP)-visualization techniques. By using the semi-intact cell system, we can observe the morphological changes that occur in "preexisting" organelles during mitosis more easily, and, at the same time, can investigate the effects of exogenously added antibodies, drugs, and recombinant proteins on the process. By reconstituting cell cycle-dependent morphological changes in organelles using interphase or mitotic phase cytosol, we can dissect processes that occur in an orchestrated manner in the cells, morphologically and biochemically, into elementary reactions, and investigate the biochemical requirements for each reaction.

### **2. Semi-intact cell assays**

Semi-intact cells are cells whose plasma membrane has been permeabilized with detergent or toxins, and can be referred to as "cell-type test tubes" (Fig. 1). We use a bacterial poreforming toxin, streptolysin O (SLO), to permeabilize the cells. At 4℃, SLO binds to cholesterol in plasma membranes. At warmer temperatures, SLO assembles to form amphiphilic oligomers, which results in the generation of small, stable transmembrane pores (Bhakdi et al., 1985). SLO-induced pores are approximately 30 nm in diameter, which is sufficiently large to allow immunoglobulin to enter into the cells (immunoglobulin G: 150 kDa). Protein complexes that are larger than immunoglobulin, such as homo- or heterooligomers, can enter the cells through the pores as individual subunits, and the complexes are reconstituted inside the semi-intact cells. After permeabilization, almost ~80% of the cytosol flows out through the pores into the medium. However, despite this loss of cytosol, the relative intracellular configuration of the cytoskeleton and organelles or between independent organelles can be maintained because damage to the membranes of the intracellular organelles caused by entry of SLO into the semi-intact cells can be minimized by washing away any excess SLO at 4℃ before pore formation is initiated. In contrast, it is

Fig. 1. A scheme of semi-intact cell assay

(FRAP), etc. In addition, the fact that concerted morphological changes occur in the organelles simultaneously and transiently in a single cell during mitosis makes it extremely difficult to dissect these changes morphologically and biochemically. The analysis is complicated further by the asynchronous progression of the cell cycle in individual cells.

Herein, we describe a novel method that addresses the above-mentioned problems, namely, a semi-intact cell assay coupled with green fluorescence protein (GFP)-visualization techniques. By using the semi-intact cell system, we can observe the morphological changes that occur in "preexisting" organelles during mitosis more easily, and, at the same time, can investigate the effects of exogenously added antibodies, drugs, and recombinant proteins on the process. By reconstituting cell cycle-dependent morphological changes in organelles using interphase or mitotic phase cytosol, we can dissect processes that occur in an orchestrated manner in the cells, morphologically and biochemically, into elementary

Semi-intact cells are cells whose plasma membrane has been permeabilized with detergent or toxins, and can be referred to as "cell-type test tubes" (Fig. 1). We use a bacterial poreforming toxin, streptolysin O (SLO), to permeabilize the cells. At 4℃, SLO binds to cholesterol in plasma membranes. At warmer temperatures, SLO assembles to form amphiphilic oligomers, which results in the generation of small, stable transmembrane pores (Bhakdi et al., 1985). SLO-induced pores are approximately 30 nm in diameter, which is sufficiently large to allow immunoglobulin to enter into the cells (immunoglobulin G: 150 kDa). Protein complexes that are larger than immunoglobulin, such as homo- or heterooligomers, can enter the cells through the pores as individual subunits, and the complexes are reconstituted inside the semi-intact cells. After permeabilization, almost ~80% of the cytosol flows out through the pores into the medium. However, despite this loss of cytosol, the relative intracellular configuration of the cytoskeleton and organelles or between independent organelles can be maintained because damage to the membranes of the intracellular organelles caused by entry of SLO into the semi-intact cells can be minimized by washing away any excess SLO at 4℃ before pore formation is initiated. In contrast, it is

reactions, and investigate the biochemical requirements for each reaction.

**2. Semi-intact cell assays** 

Fig. 1. A scheme of semi-intact cell assay

difficult to avoid damage to intracellular structures when cells are permeabilized with digitonin, a well-known pore-forming toxin, because digitonin-induced permeabilization is insensitive to temperature.

By exchanging cytoplasmic proteins with exogenously added proteins, antibodies or cytosol that has been prepared from cells at distinct stages of the cell cycle or differentiation, or from disease states, we can modulate the intracellular environment and reconstitute various physiological phenomena in semi-intact cells. The semi-intact cell method was originally established by Dr. Simons' group to study polarized vesicular trafficking in Madin-Darby canine kidney (MDCK) cells (Ikonen et al., 1995). We have refined the method by coupling it with GFP-visualization techniques and have established many types of assay for cell cycledependent changes in organelle morphology and membrane trafficking. Using our analytical system, we can manipulate intracellular conditions and then observe the resulting morphological changes in GFP-tagged organelles by fluorescence microscopy. In addition, we are able to dissect complex reaction processes in cells on a morphological basis and to investigate the biochemical requirements and kinetics of each process, for example the vesicular transport between the ER and the Golgi during mitosis. In particular, the maintenance of the integrity of the organelles and their configuration in the semi-intact cells enables us to analyze membrane trafficking in as intrinsic environment as possible.
