Cytokinesis of animal and fungi cells depends crucially on the anillin scaffold proteins. Fission yeast anillin-related Mid1 anchors cytokinetic ring precursor nodes to the membrane. However, it is unclear if both of its Pleckstrin Homology (PH) and C2 C-terminal domains bind to the membrane as monomers or dimers, and if one domain plays a dominant role. We studied Mid1 membrane binding with all-atom molecular dynamics near a membrane with yeast-like lipid composition. In simulations with the full C terminal region started away from the membrane, Mid1 binds through the disordered L3 loop of C2 in a vertical orientation, with the PH away from the membrane. However, a configuration with both C2 and PH initially bound to the membrane remains associated with the membrane. Simulations of C2-PH dimers show extensive asymmetric membrane contacts. These multiple modes of binding may reflect Mid1\'s multiple interactions with membranes, node proteins, and ability to sustain mechanical forces.

The fission yeast Schizosaccharomyces pombe, an ascomycete fungus, is an established model organism for studying eukaryotic molecular and cellular events such as the cell cycle due to its powerful genetics, a sequenced genome, and the ease of molecular manipulation (Wood et al., Nature 415:871-880, 2002; Hoffman et al., Genetics 201:403-423, 2015). This chapter describes genetic and cytological methods to study endosomal sorting complex required for transport (ESCRT) function during the cell cycle in fission yeast. These include tetrad analysis to allow the creation of double mutants to test for genetic interactions by synthetic phenotype characterization, such as cellular growth and the analysis of division septa by calcofluor-white staining.

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries.