Histograms of fluorescence intensity per node show that small, dim nodes predominate, although the eye of the observer is drawn to larger, more intense spots ( Akamatsu et al., 2017). Rather than appearing as a uniform population of spots, the fluorescence from node markers ranges from tiny dim spots to much larger and brighter particles both during interphase ( Moseley et al., 2009) and mitosis ( Vavylonis et al., 2008). In spite of their central role in the cell cycle, quantitative analysis of nodes has been challenging, largely due to their heterogeneity observed by fluorescence microscopy. Interphase nodes are thought to sense the cell surface area to control cell size at the time of division ( Facchetti et al., 2019a Pan et al., 2014). During this period, the interphase node components also double in number ( Allard et al., 2018 Pan et al., 2014). Interphase cells grow linearly at their tips ( Fantes and Nurse, 1977 Moreno et al., 1989) until they reach a threshold size of twice the initial cell length ( Gu and Oliferenko, 2019 Neumann and Nurse, 2007). In addition to being the precursors for cytokinesis nodes, interphase nodes are critical for timing cell division. Interestingly, 10–12 min after SPB separation, high SIN activity disperses the Cdr2p-mEGFP from the cytokinesis nodes into the cytoplasm ( Akamatsu et al., 2014 Martin and Berthelot-Grosjean, 2009 Morrell et al., 2004 Moseley et al., 2009). Interactions between actin filaments from one node and myosin-II in adjacent nodes pull the nodes together into a contractile ring ( Vavylonis et al., 2008). At the time of SPB separation, smaller numbers of formin Cdc12p join each node, where they nucleate and elongate actin filaments ( Laporte et al., 2011 Wu et al., 2006). During 10 min prior to spindle pole body (SPB) separation, cytokinesis nodes mature by sequentially accumulating stoichiometric ratios of myosin-II (Myo2 heavy chain and two light chains), IQGAP Rng2p, and F-BAR protein Cdc15p ( Vavylonis et al., 2008 Wu et al., 2006). During the G 2 phase of the cell cycle, type 1 nodes capture diffusing type 2 nodes to form cytokinesis nodes ( Akamatsu et al., 2014). Type 2 nodes containing the proteins Blt1p, Klp8p, Gef2p, and Nod1p ( Jourdain et al., 2013 Martin and Berthelot-Grosjean, 2009 Morrell et al., 2004 Moseley et al., 2009 Ye et al., 2012) originate from the constricting contractile ring from the previous cell cycle and diffuse along the plasma membrane from the new ends of the daughter cells. At the end of cytokinesis, type 1 nodes containing kinases Cdr1 and Cdr2 appear in a broad band around the middle of the daughter cells as SIN (septation initiation network) activity drops ( Pu et al., 2015) and remain stationary throughout the interphase ( Akamatsu et al., 2014 Martin and Berthelot-Grosjean, 2009 Morrell et al., 2004 Moseley et al., 2009). In fission yeast, cytokinesis nodes form from two distinct types of interphase nodes ( Akamatsu et al., 2014). Similar assemblies containing myosin-II may form contractile rings in other cells ( Beach et al., 2014 Henson et al., 2017 Hickson and O’Farrell, 2008 Werner et al., 2007). Editor's evaluationĬytokinesis nodes are stoichiometric assemblies of multiple proteins, which associate with the plasma membrane around the middle of fission yeast cells and polymerize actin filaments that form the cytokinetic contractile ring ( Vavylonis et al., 2008). However, when the concentrations of either kinase Pom1 or kinase Cdr2 were varied with the nmt1 promoter, the numbers of cytokinesis nodes increased above a baseline of about ~190 with the total cellular concentration of either kinase. The Pom1 kinase restricts cytokinesis nodes from the ends of cells, but the surface density of Pom1 on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. The number of cytokinesis nodes scales with cell size in four strains tested, although large diameter rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. Most, but not all of these nodes condense into a contractile ring. The ratio of the total Blt1-mEGFP fluorescence in the broad band of cytokinesis nodes to the average fluorescence of a single node gives about 190 single cytokinesis nodes in wild-type fission yeast cells early in mitosis. Here, we used the ~140 nm resolution of Airyscan super-resolution microscopy to measure the fluorescence intensity of small, single cytokinesis nodes marked with Blt1-mEGFP in live fission yeast cells early in mitosis. The total number of nodes is uncertain, because of the limitations of the methods used previously. Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast.
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