Centrosomes and nuclei are the two most fundamental organelles in eukaryotic cells, as they provide for the basic properties of life: motility and inheritance. Centrosomes organize the microtubule cytoskeleton that is required for cell structure and organelle movement. Nuclei maintain the genetic material that provides instruction for nearly every cell action. Interactions between these organelles play important roles in cell polarity, motility, development, and division. While genetic and molecular analyses reveal a complex framework of proteins that interconnect these two organelles, how these interactions are established and maintained remains poorly understood. Moreover, virtually nothing is known about how these physical interactions influence the replicative cycles of centrosomes and nuclei. The long-term goal of this project is to understand the mechanisms that coordinate centrosomal and nuclear cycles. Through the deletion of a kinesin molecular motor, previous work has developed a novel means to uncouple these two organelles and yet maintain cell viability. This deletion leads to supernumerary centrosome accumulation and to errors in cell division that ultimately impact cell growth. The project will utilize combinations of light and electron microscopy, and laser ablation of cell organelles to determine how centrosome replication is governed by nuclear attachment. Efforts will establish whether multiple attachment mechanisms are at play and distinguish between their functional roles, address nuclear vs. cytoplasmic requirements for spindle assembly in a closed mitotic system, and determine how the kinesin motor participates in the attachment process. Overall the objectives will lead to an understanding of the mechanisms by which centrosomes engage nuclei and how these two organelles coordinate their functions to maintain cell viability and genomic stability.
Broader Impact The research projects capitalize on state of the art live cell microscopy, coupled with analyses by electron microscopy. Undergraduate students from two local top-notch teaching colleges (Siena and Russell-Sage) will be exposed to this technology, in a laboratory environment that significantly extends their coursework. Students will be taught to assemble molecular constructs with various fluorescent tags and how to image these molecules inside live cells. The methods will constitute a fundamental set of skills for many different cell biology applications and are designed to stimulate continued interest in biology as a long-term career. Students will be supervised but also responsible for all aspects of data collection and analysis, with the driving goal of producing publication quality results. Students will be provided with opportunities to present their results at local and national meetings. The PI will also continue an active role in community service to lecture at local colleges, primary and secondary education institutions.
