© 2017. Published by The Company of Biologists Ltd | Journal of Cell Science (2017) 130, 853-867 doi:10.1242/jcs.199182 RESEARCH ARTICLE Defining the dynamin-based ring organizing center on the peroxisome-dividing machinery isolated from Cyanidioschyzon merolae Yuuta Imoto1, Yuichi Abe1, Kanji Okumoto2, Masanori Honsho1, Haruko Kuroiwa3,4, Tsuneyoshi Kuroiwa3,4 and Yukio Fujiki1,* ABSTRACT elastic stress and requires external force to accomplish it. Numerous Organelle division is executed through contraction of a ring-shaped molecular genetic studies have thus determined fission factors – supramolecular dividing machinery. A core component of the involved in the fission process of many membranes these include machinery is the dynamin-based ring conserved during the division a member of the GTPase dynamin family in the division of of mitochondrion, plastid and peroxisome. Here, using isolated mitochondrion, plastid and peroxisome (Bleazard et al., 1999; peroxisome-dividing (POD) machinery from a unicellular red algae, Miyagishima et al., 2003; Koch et al., 2003) and in synaptic vesicle Cyanidioschyzon merolae, we identified a dynamin-based ring endocytosis (Obar et al., 1999; Chen et al., 1991; van der Bliek organizing center (DOC) that acts as an initiation point for formation and Meyerowrtz, 1991). Despite their biological importance, of the dynamin-based ring. C. merolae contains a single peroxisome, the ultrastructure and molecular mechanism of supramolecular the division of which can be highly synchronized by light–dark division machinery remain enigmatic because isolation and direct in vivo stimulation; thus, intact POD machinery can be isolated in bulk. observation of the machinery is difficult as most of the model Dynamin-based ring homeostasis is maintained by the turnover of the organism contains numerous organelles and vesicles that are shaped GTP-bound form of the dynamin-related protein Dnm1 between the irregularly and divide randomly. To conquer these problems, – cytosol and division machinery via the DOC. A single DOC is formed light dark-stimulated organelle division system in simple model Cyanidioschyzon on the POD machinery with a diameter of 500–700 nm, and the organisms, such as the unicellular red alga merolae C. merolae dynamin-based ring is unidirectionally elongated from the DOC in a , has recently been used. contains a minimum – manner that is dependent on GTP concentration. During the later step set of organelles i.e. only one plastid, a mitochondrion and a of membrane fission, the second DOC is formed and constructs the peroxisome (Miyagishima et al., 1999a; Matsuzaki et al., 2004; double dynamin-based ring to make the machinery thicker. These Imoto et al., 2010). Division of the organelles takes place once per – findings provide new insights to define fundamental mechanisms cell cycle and is synchronized by light dark circadian rhythm underlying the dynamin-based membrane fission in eukaryotic cells. stimulation (Suzuki et al., 1994; Miyagishima et al., 2014). Thus, organelle divisions can be observed in a strictly sequential order KEY WORDS: DOC, Dnm1, GTP, Dynamin-based ring, through the cell cycle (Miyagishima et al., 1999b; Imoto et al., Organelle-dividing machinery, Peroxisome 2011). These unique biological features make C. merolae an ideal model system in which to study organelle division, as illustrated by INTRODUCTION the first identification and recent successful isolation of the electron- Organelle membranes constantly reshape for distinct types of dense organelle division apparatus – the mitochondrion-dividing biological events, including organelle division and the transport of machinery, the plastid-dividing machinery (Kuroiwa et al., 1993; materials by endocytosis or budding organelles. For example, Yoshida et al., 2006, 2010, 2013) and the peroxisome-dividing organelles such as mitochondria, plastids and peroxisomes contract (POD) machinery (Imoto et al., 2012, 2013). POD machinery that a part of their membrane for division. For homeostatic maintenance sustains in vivo structures can be purified to a high degree from cells, of plasma membrane proteins, the plasma membrane invaginates and ultrastructural analysis shows that the machinery consists of a and forms endocytotic vesicles. Likewise, transport vesicles pinch closed-ring structure comprising an outer string (dynamin-based off from the organelle membrane. As the membrane reshapes during ring) and an inner skeletal filament (filamentous ring). Hence, POD organelle division and vesicle generation, the membrane must close machinery is a typical and ideal model system to further at the last step through membrane fission. This process leads to understanding of the ultrastructure and molecular mechanisms underlying membrane fission machinery. The motive force for contraction is generated by the dynamin-based ring, which includes 1Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, the GTPase dynamin-like protein Dnm1 (Imoto et al., 2013). Dnm1 Fukuoka 812-8582, Japan. 2Department of Biology, Faculty of Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395, Japan. 3Department of is responsible for the division of the peroxisome and mitochondrion Chemical and Biological Science, Faculty of Science, Japan Women’s University, that is mediated by GTPase activity and is highly conserved in plant, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan. 4Core Research for Evolutional yeast and mammalian cells (Bleazard et al., 1999; Smirnova et al., Science and Technology, Japan Science and Technology Agency, Gobancho, Chiyoda-ku, Tokyo 102-0076, Japan. 2001; Sesaki and Jensen, 1999; Koch et al., 2003; Li and Gould, 2003; Mano et al., 2004; Tanaka et al., 2006; Motley and Hettema, *Author for correspondence ([email protected]) 2007). Importantly, Dnm1-associated proteins such as fission1 Y.F., 0000-0002-8138-6376 (Fis1) (Mozdy et al., 2000), mitochondrial division (Mdv1) (Tieu and Nunnari, 2000) and mitochondrial fission factor (Mff) (Gandre- Received 1 November 2016; Accepted 5 January 2017 Babbe and van der Bliek, 2008) are also used in both divisions of Journal of Cell Science 853 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 853-867 doi:10.1242/jcs.199182 peroxisomes and mitochondria (Motley and Hettema, 2007; Motley Isolation of cytosolic Dnm1 patches et al., 2008). Moreover, Dnm1 is phylogenetically linked to Dnm2/ To compare the characteristics of Dnm1 localized at the peroxisome DRP5B, which is involved in plastid division (Miyagishima et al., division plane with those of Dnm1 in the cytosolic patches, we 2003; 2008), and dynamin-1, which is involved in synaptic vesicle attempted to determine the nucleotide state of Dnm1 in these two endocytosis (Hinsaw and Schmid, 1995). Thus, the dynamin-based pools. First, an isolation method for cytosolic Dnm1 patches was ring is likely to be a core structure of the membrane fission developed. Upon cell homogenization and Percoll density gradient machineries. However, the mechanism by which the dynamin- ultracentrifugation, Dnm1 concentrated in the organelle fractions based ring is organized and formed around the membrane fission and not in the cytosolic fraction – even in synchronized cells plane is unknown. POD machinery, including the dynamin-based collected during the G1 phase, at which stage the majority of Dnm1 ring, is specifically present during the late M phase (Imoto et al., is localized to the cytosolic Dnm1 patches (Fig. 2A). This indicated 2013), although Dnm1 is continuously expressed during the cell that the cytosolic Dnm1 patches are not freely soluble in the cytosol. cycle (Nishida et al., 2003). Dnm1 resides in the cytosol and After ultracentrifugation on an Opti-prep density gradient, a couple relocates to the cytosolic side of the division plane of mitochondrion of Dnm1-containing fractions (3.0-4.0 ml from the bottom) were during the early M phase (Nishida et al., 2003) and to that of the shown to be completely free of mitochondrion, peroxisome, Golgi peroxisome during late M phases (Imoto et al., 2013). Hence, the body, ER and lysosome contamination (Fig. 2B, square boxed). molecular dynamics and assembly of Dnm1 surrounding the Immunofluorescence microscopy showed that Dnm1 in the division plane at the cytosolic side is apparently important. cytosolic patches isolated from this fraction was localized to a Understanding how Dnm1 molecules assemble into a ring-shaped vesicle-like dense structure, which was distinct from the pattern in supramolecular nanomachinery should provide new insights into the POD machinery fraction, and the Dnm1-positive isolated the function and molecular mechanics of the membrane fission vesicle-like structures were almost the same size as Dnm1 patches machinery. observed in whole cells (Fig. 2C). Thus, we termed these fractions Herein,wesuccessfullyisolatedDnm1inthecytosoland as cytosolic Dnm1 patch fractions. determined the GTP–GDP state of Dnm1 in comparison to that of isolated POD machineries. We provide evidence that turnover between The GTP-bound form of Dnm1 is important for Dnm1 the GTP-bound Dnm1 in the cytosol and GTP-hydrolyzed Dnm1 in assembly around the peroxisomal division plane the dynamin-based ring is important for maintaining dynamin-based To investigate the biochemical features of the isolated cytosolic ring homeostasis under ‘semi-in-vivo’ conditions. Furthermore, a Dnm1 patches, we determined the nucleotide state of the cytosolic semi-in-vivo assay was developed for dynamin-based