14-3-3 Protein Rad24 and its Effect on Mid1 Localization and Contractile Acto- Myosin Ring Assembly During Cytokinesis in S. pombe

Abstract

In animal and yeast cells, cell division (cytokinesis) is facilitated by the formation of a contractile acto-myosin ring (CAR). Proper CAR formation and constriction is heavily reliant on the temporal regulation, phosphorylation, and localization of key proteins. In the fission yeast S. pombe, Mid1 is an important dimeric CAR scaffolding protein that connects the contractile apparatus to the plasma membrane at the right place and time during cytokinesis. Mid1 is confined to both the nucleus and protein assemblies called nodes during interphase, and transitions to the cell cortex at mitotic entry as nodes mature and coalesce into the CAR. Rad24 is a 14-3-3 protein involved in cell cycle checkpoints known to interact with CAR proteins and some of their regulators. 14-3-3 proteins bind to a conserved consensus phosphorylation motif, RXXpS, which is targeted by Sid2 and other NDR-kinases. The Septation Initiation Network (SIN) is a conserved signaling pathway to facilitate separation of two new daughter cells. Sid2, the terminal kinase of the SIN, has numerous targets in the CAR, including Mid1. Removal of Rad24 has distinct consequences on the timing of major cytokinetic events. The goal of this thesis is to use fission yeast to characterize the interaction between Rad24 and Mid1, which in turn organizes important CAR components F-actin and Myosin II. Live cell video microscopy in rad24Δ cells shows that fluorescently tagged Mid1-NeoGreen remains nodal during interphase and returns to the nucleus early after CAR constriction. rad24Δ cells also show a delay of F-actin (LifeAct-GFP) and myosin-II (Rlc1-tomato) recruitment to the CAR and deferred CAR constriction. In vitro binding assays show that Mid1 and Rad24 interact directly and computational 6 structural speculation concludes that this interaction occurs most likely while Mid1 is in a monomeric form, after or before nuclear export or import. SIN-dependent phosphorylation of Mid1 has definitive consequences on cell division in fission yeast, and the conserved nature of protein interactions during cytokinesis in S. pombe suggests broader implications for the study of cell division and cancer in higher animals.