The Biochemical Basis for the Mechanics of Cytokinesis

Johns Hopkins University, Baltimore MD

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Abstract

PROJECT SUMMARY GM66817-18 PARENT GRANT Cell shape change is fundamental for cell division, migration, and tissue formation, and defects in cell shape change are one of the early hallmarks of disease. In our research, we study cell shape change, focusing on the biomechanical systems, and we utilize cytokinesis as an elegant model process. Over the life of this grant, we have demonstrated how an interplay of active force production, cortical tension, surface curvature, and viscoelasticity drive cell shape change, including 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 cell shape change and the 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 partners, IQGAP1 and 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, cell adhesion, and cortical mechanics. 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 have used CLIP-Seq and identified several RNAs as interactors of RNP1A and RNP1B. One of particular interest is the dynamin-like protein 1, which localizes to the cleavage furrow cortex where it assists in actin and myosin II organization. Others are involved in macropinocytosis, another essential cell shape change event that also draws upon much of the CK machinery. Here, we will flesh out how the RNP1s impact CK assembly, expression, and function. Overall, these studies will decipher how the CK network operates and integrates with other cellular systems, leading to a deeper understanding of cell shape change processes more generally.