Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy

Abstract

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem.

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

“…Images of cells coexpressing Spc72-GFP and Spc42-CFP (as reference) were acquired with a Nikon Eclipse E800 with a CFI Plan Apochromat 100x, NA 1.4 objective, a Chroma Technology CFP/YFP filter set and a Coolsnap-HQ CCD camera (Roper Scientific) ( Guo and Segal, 2017 ) as five-plane Z-stacks of fluorescence images at a distance of 0.8 μm between planes with 2 × 2 binning, paired to a DIC image at the middle focal plane ( Juanes et al, 2013 ). Stacks were processed into 2-color overlays of maximal intensity 2-D projections and analyzed with MetaMorph software (Molecular Devices).…”

Orderly assembly underpinning built-in asymmetry in the yeast centrosome duplication cycle requires cyclin-dependent kinase

“…Chroma Technology CFP/YFP filter set and a Coolsnap-HQ CCD camera (Roper 54 Scientific) (Guo and Segal, 2017) as five-plane Z-stacks of fluorescence images at a 55 distance of 0.8 μm between planes with 2 x 2 binning, paired to a DIC image at the middle 56 focal plane (Juanes et al, 2013). Stacks were processed into 2-color overlays of maximal 57 intensity 2-D projections and analyzed with MetaMorph software (Molecular Devices).…”

Mitotic-Spindle Organizing Protein MztA Mediates Septation Signaling by Suppressing the Regulatory Subunit of Protein Phosphatase 2A-ParA in Aspergillus nidulans

“…Although organisms of different kingdoms have developed unique mechanisms to execute cytokinesis, signals that trigger the onset of cytokinesis are evolutionarily conserved (Wolkow et al, 1996 ; Krapp and Simanis, 2008 ; Seiler and Justa-Schuch, 2010 ). Many lines of evidence have identified that the conserved mitotic exit network (MEN) components, which are tightly connected with cytokinesis, exist in the budding yeast Saccharomyces cerevisiae and mammalian systems (Foltman and Sanchez-Diaz, 2017 ; Guo and Segal, 2017 ; Renicke et al, 2017 ; Scarfone and Piatti, 2017 ). A pathway homologous to the MEN, termed the septation initiation network (SIN), has also been found to couple mitotic exit with cytokinesis in the fission yeast, Schizosaccharomyces pombe and the model filamentous fungus A. nidulans (Barr and Gruneberg, 2007 ; Csikasz-Nagy et al, 2007 ; Zhong et al, 2012 ; Simanis, 2015 ).…”