Characterization of two types of interphase nodes that merge to form cytokinetic nodes for contractile ring assembly in fission yeast

During cytokinesis in fission yeast, a contractile ring made of actin and myosin assembles from a broad band of ~100 discrete multiprotein complexes at the cell equator called cytokinetic nodes. Nodes originate during interphase and sequentially accumulate cytokinesis proteins during mitosis, but the assembly process of these interphase nodes had not been investigated. Understanding the spatiotemporal pathway of node assembly during interphase was necessary to understand the contributions interphase node proteins make to cytokinesis.;I used confocal microscopy to image fission yeast cells endogenously expressing fluorescent fusion proteins marking interphase nodes, and discovered that fission yeast have two different types of nodes during interphase. The two types of nodes are compositionally distinct and form separately. Type 1 nodes containing kinase Cdr1p, kinase Cdr2p and anillin Mid1p form in the cell cortex around the nucleus early in interphase. Type 2 nodes containing protein Blt1p, GTP exchange factor Gef2p and kinesin Klp8p emerge from the disassembling contractile ring as it constricts. I used particle tracking and stochastic simulations of a computational model to show that these two types of nodes come together by a diffuse-and-capture mechanism: Type 2 nodes diffuse to the cell equator and are captured by stationary Type 1 nodes. During mitosis cytokinetic nodes with Mid1p and all of the Type 2 node markers incorporate into the contractile ring whereas Type 1 nodes with Cdr1p and Cdr2p follow the separating nuclei before dispersing into the cytoplasm.;Based on the cellular distribution of Type 1 nodes dispersing into the cytoplasm during mitosis, I hypothesized that a mitotic signaling network called the Septation Initiation Network (SIN) disperses the Type 1 nodes in preparation for cytokinesis. Undergraduate Kai-Ming Pu and I used cells with temperature sensitive mutations in SIN proteins to turn the SIN on and off at different stages of the cell cycle and established that the SIN is necessary and sufficient to disperse Type 1 node markers Cdr2p and Mid1p into the cytoplasm.;With graduate student Chad McCormick I compared the lab's method of counting molecules in cells to orthogonal quantitation methods and found that the methods differ by ≤30%. Quantitative measurements of the SIN kinase Sid2p at the two separating mitotic spindle pole bodies showed that in late anaphase there are fewer Sid2p molecules at the spindle pole body with active SIN, concurrent with its translocation to the contractile ring, supporting the model that this SIN effector kinase leaves the active spindle pole body for the contractile ring to trigger cytokinesis.;The results in this dissertation demonstrate that in fission yeast, two types of interphase nodes follow parallel branches of the pathway to prepare nodes for cytokinesis. We propose a node cycle that accounts for the organization, regulation by the SIN, and movement of the constituent proteins across the entire cell cycle.