Background

Development of live cell imaging by state-of-the-art light microscopy has greatly contributed to address many problems remaining in cell biology. For example, to visualize the events in secretory pathway, in which small membrane carriers are rapidly moving around in the cytoplasm, requires high speed and three-dimensional (3D) image acquisition. Super-resolution confocal live imaging microscopy (SCLIM), which we have developed (Kurokawa et al., 2013), can achieve high speed visualization of dynamic behavior of 3D structures tagged with green and red fluorescence proteins in living cells with spatial resolution beyond the diffraction limit of light (Matsuura-Tokita et al., 2006; Ito et al., 2012; Okamoto et al., 2012; Suda et al., 2013; Uemura et al., 2014; Kurokawa et al., 2014; Iwai et al., 2016; Ishii et al., 2016 and 2019; Kurokawa et al., 2016). We can now use 3-color version of SCLIM, which enable us to conduct simultaneous 3-color (green, red, and infrared) observation in living yeast, Drosophila, plant, and mammalian cells at high spatial and time resolution (Ito et al., 2018; Kurokawa et al., 2019; Maeda et al., 2019; Tojima et al., 2019; Fujii et al., 2020). We have been working on the molecular mechanisms underlying protein transport and sorting in the Golgi apparatus and have utilized the cutting-edge performance of our technology to tackle a big question in this field. The purpose of this article is to introduce to readers our approaches by super-resolution and high-speed live cell imaging and provide basic protocols.

The Golgi apparatus is the central station of membrane traffic. One third of proteins in eukaryotic cells are newly synthesized in the endoplasmic reticulum (ER) and then delivered to the Golgi. In the Golgi, they are processed and glycosylated before being sorted to their final destinations (Emr et al., 2009). The Golgi is usually in the form of ordered stacks of several cisternae. Secretory cargo travels across the stack from the cis side to the trans side of cisternae and then to the trans-Golgi network (TGN) (Griffiths and Simons, 1986). The budding yeast Saccharomyces cerevisiae presents a unique example of the Golgi, in which individual cisternae, cis, medial and trans, do not stack but scatter in the cytoplasm (Matsuura-Tokita et al., 2006; Okamoto et al., 2012). Three major models of secretory cargo traffic within the Golgi have been proposed: 1) traffic via cisternal maturation, 2) traffic by anterograde vesicular carriers, and 3) traffic via interconnected continuity of cisternae (Glick and Nakano, 2009; Nakano and Luini, 2010; Pfeffer, 2010; Glick and Luini, 2011). Among these models, the cisternal maturation model has been favored to explain the live imaging observation in budding yeast that Golgi cisternae mature from early to late cisternae (Losev et al., 2006; Matsuura-Tokita et al., 2006; Rivera-Molina and Novick, 2009) and the movement of large cargo through the Golgi stacks without leaving cisternae in mammals (Bonfanti et al., 1998; Lanoix et al., 2001; Martinez-Menarguez et al., 2001; Mironov et al., 2001).The lacking proof of this mechanism was to visualize the delivery of cargo remaining in the maturing Golgi cisterna.

Electron microscopy shows detailed snap information where secretory cargo and Golgi resident proteins localize in the individual Golgi cisternae, but cannot directly visualize how secretory cargo traverse between these cisternae. Live imaging by using conventional epi- and confocal fluorescence microscopies visualize behavior of secretory cargo in vivo, but cannot provide detailed information where secretory cargo and Golgi resident proteins locate within the individual Golgi cisternae because of low spatial resolution. Super-resolution microscopy methods, for example STED, PALM/STORM, and SIM overcoming the diffraction limit of light, provide greater spatial resolution (Schermelleh et al., 2010). However, these methods are not high in temporal resolution, because time and space resolutions usually trade off each other. By using SCLIM technology, we have shown that secretory cargo is delivered within the Golgi by cisternal maturation. Maturing cisterna involves segregated zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation (Kurokawa et al., 2019).