The Biochemical Basis for the Mechanics of Cytokinesis

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PROJECT SUMMARY Cytokinesis, the division of a cell into two daughter cells, serves as an elegant cell behavior that highlights the biomechanical systems required for many cell shape change processes. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cytokinesis furrow ingression. We identified key molecular pathways that control these properties and found that the circuitry is wired like a control system complete with feedback loops that allows mechanical and chemical signals to tune the accumulation of the contractile machinery. In this proposal, we will build upon our understanding of cytokinesis and this mechano-responsive contractility network. We use a suite of techniques, including genetics, proteomics, Single Molecule Pulldown (SiMPull), and Fluorescence Correlation and Cross- Correlation Spectroscopy (FCS/FCCS) to study this network. We discovered that many of the proteins in the mechano-responsive contractility network are organized into complexes in the cytoplasm, forming Contractility Kits (CKs). Several CK components have unknown functions in the context of cell contractility and are the subject of this proposal. Among these, the lectin discoidin 1A, traditionally viewed as a secreted protein, assembles with the CKs in the cytoplasm and is necessary for a key protein, the actin crosslinker cortexillin I, to localize fully to the cortex. Moreover, discoidin 1A has a complex genetic relationship with cortexillin I and its binding partner and regulator IQGAP2. We will determine how discoidin 1 operates in the CKs and promotes cortical assembly. Next, we are studying two ribonucleoproteins, RNP1A and RNP1B. Both proteins contain predicted RNA- recognition motifs. RNP1A is also required for normal cortexillin I mRNA levels. We originally identified RNP1A over-expression as a genetic suppressor of the microtubule-destabilizer nocodazole (same study that gave rise to the RacE-14-3-3-myosin II pathway that we discovered). We have now found that RNP1A is required for normal microtubule length. Given the changes in mRNA levels of cortexillin I, we conducted RNAseq analysis and found that several CK proteins have altered gene expression in rnp1A knockdown cells. To identify RNAs that the RNP1s might bind, we are using CLIP-Seq and have already found in a preliminary study that RNP1B may bind to 14-3-3 mRNA. Here, we will flesh out how the RNP1s impact CK assembly and expression. Finally, we discovered that the adenine nucleotide translocase (ANT, encoded by ancA in Dictyostelium) interacts genetically with myosin II and racE. Intriguingly, we are finding that CK protein nulls (e.g., cortexillin I) have reduced metabolic activity, leading to reduced ATP production and a lower energetic state. We will probe how increasing energy production through ANT can bypass some of the cellular functions of the CK proteins. Overall, these studies will seek to decipher how the CK network operates and integrates with other processes, such as mRNA levels and metabolism, leading to a deeper understanding of the cell shape change process more generally.