Functional of Rng2p through reverse genetics

Abstract

Cytokinesis is the process where a single cell divides physically into two daughter cells. Schizosaccharomyces pombe is an attractive model for studying cytokinesis as it divides using an actomyosin ring present in a number of eukaryotes. Assembly and positioning of actomyosin contractile ring is a tightly regulated and co-ordinated process. Early in mitosis, many of the actomyosin ring proteins form nodes in the medial cortical region of the cell. Subsequently, these nodes coalesce to form a compact ring which constricts during cytokinesis. Rng2p is one such essential protein whose role is not completely understood. Rng2p is a multidomain protein with an N-terminal CHD (Calponin-Homology Domain) domain, IQ domains, a C-terminal GRD (RAS-GAP Related Domain) domain and a Ras- GAP (GTPase Activating Protein) domain. The goal of the study was to study the role of specific domains. In order to achieve that, mutants were generated using ?Marker Reconstitution Mutagenesis?. Among the mutants generated, the rng2-M1 mutant had a phenotype similar to ring positioning mutants which indicated a role of Rng2p in ring positioning. Characterizing the phenotype of the mutant helped draw similarities between rng2-M1 and the already characterized mid1-18 mutant. Mid1p is the first protein to localize as nodes and is essential for marking the centre of the cell. It gives a positional cue for the placement of the cytokinesis machinery. By studying the genetic interactions of rng2-M1 with other positional mutants, it became evident that Rng2p functions in the Mid1p epistasis group. In rng2-M1, the stability of Mid1p was compromised and the nodes failed to remain at the medial cell cortex. The cellular content of Mid1p was also significantly reduced in rng2-M1. The other node-component proteins like Rng2p, Cdc15p, Cdc12p, Cdc4p, Myo2p and Rlc1p also failed to localize to nodes. Instead, the ring was formed by an alternative pathway from a single dot at random positions in the cell. In rng2-null mutant cells, though Mid1p formed nodes, all other node components failed to localize to nodes. Also, in cells lacking the essential light chain of myosinII (Cdc4p) which is known to interact with Rng2p, none of the node components, including Rng2p formed nodes. Thus, Rng2p was essential for the node formation and stability and there exists a specific hierarchical order in which the node components got localized to nodes. From this study, it is evident that Rng2p plays a role in coordinating both placement and formation of a proper actomyosin ring. The working model of node formation shows that Mid1p comes to the nodes and recruits Rng2p, which in turn is essential for the stable maintenance of Mid1p at nodes and for recruiting other ring components. In conclusion, the precise order of a hierarchical mode of node formation was established which in turn ensures proper placement of division site.