Characterization of Mid1 domains for targeting and scaffolding in fission yeast cytokinesis

Abstract

SummaryDivision-site selection and contractile-ring assembly are two crucial steps in cytokinesis. In fission yeast, the anillin-like Mid1 protein specifies the division site at the cell equator by assembling cortical nodes, the precursors of the contractile ring. Thus, Mid1 is essential for linking the positional cues for the cleavage site to contractile-ring formation. However, how Mid1 domains cooperate to regulate cytokinesis is poorly understood. Here we unravel the functions of different Mid1 domains (motifs) by a series of truncations. We report that the conserved PH domain stabilizes Mid1 in nodes by binding to lipids and is required for Mid1 cortical localization during interphase in the absence of Cdr2 kinase. Mid1 lacking an internal region that is approximately one third of the full-length protein has higher nuclear and cortical concentration and suppresses the division-site positioning defects in cells with a deletion of the dualspecificity tyrosine-regulated kinase Pom1. The N-terminus of Mid1 physically interacts with cytokinesis node proteins. When fused to cortical node protein Cdr2, Mid1(1-100) is sufficient to assemble cytokinesis nodes and the contractile ring. Collectively, our study recognizes domains regulating Mid1 cortical localization and reveals domains sufficient for contractile-ring assembly.

Contractile Ring Stability in S. pombe Depends on F-BAR Protein Cdc15p and Bgs1p Transport from the Golgi Complex

“…One possible mechanism is that Cdc15p is a physical link between the plasma membrane and the contractile ring, given that it interacts with contractile ring proteins (Carnahan and Gould, 2003; Laporte et al, 2011; Lee and Wu, 2012; Roberts-Galbraith et al, 2009) as well as membrane lipids (Takeda et al, 2004). While this may be true, our observations on cells with mutated or depleted Cdc15p revealed that the underlying defect responsible for contractile ring sliding is slow transfer of β-glucan synthetase Bgs1p (and perhaps other proteins) from the Golgi apparatus to the plasma membrane.…”

Sbg1 Is a Novel Regulator for the Localization of the β-Glucan Synthase Bgs1 in Fission Yeast

“…We carried out IP assays and Western blotting as previously described [63,71–73]. Briefly, GFP-tagged protein expressed at its endogenous level was pulled down from S .…”

Counting protein molecules using quantitative fluorescence microscopy

“…If the standard can be distinguished from the protein of interest, it is desirable to include cells that express the standard and experimental fusion proteins on the same slide to ensure comparable illumination. If the standard is not distinguishable, images can be taken consecutively or another marker can be imaged separately to distinguish the control cells [27,34] as long as the two fluorophores are sufficiently distant to eliminate Förster resonance energy transfer (FRET). One advantage of this method is that a much greater number of protein molecules can be counted than with the photobleaching method.…”