The space between organels in a living cell called cytosol is a medium crowded with molecules. It is estimated that 30% of its volume is taken up by macromolecules, proteins and nucleic acids. While a small subset of these molecules can be interacting biochemically with a particular enzyme, the vast majority is chemically inert and is just occupying volume around the enzyme. The consequences of crowding are that more compact structures are favored in crowded media since they free up volume for the crowding molecules to occupy, thus increasing the overall entropy of the system. While this qualitative picture is simple and has borne out in some experiments, a more quantitative understanding of the consequences of crowding on protein function remains scanty, and more so in relation to molecular machines. This project will bring better quantitative understanding of crowding in cells using an experimental system that is amenable to precision measurements and manipulation of molecular motors. Recent studies have linked the down regulation of motor activity to various diseases. The rationale for the proposed studies is that the identification of the mechanisms by which multiple motors and motor co-factors cooperate to haul cargoes in the complex environment of the cell will lead to a fundamental framework for eventual understanding of how failure of these mechanisms is linked to disease. With the latter in mind, the impact of the project will in part be achieved by taking part in training a new generation of graduate and undergraduate students who are proficient in working in the interdisciplinary environment of the proposed activity and who will advance the field as they proceed in their careers. The principal investigator has, and will continue to disseminate the findings of his research program among the general public through high school student internships in his laboratory and through lectures to the students, their parents and their teachers in area high schools.
The PI will use two such machines, the molecular motor proteins kinein-1 and cytoplasmic dynein, to investigate the effects of macromolecular crowding on the function of these enzymes. Motor proteins bind and shuttle cargo inside the cell by stepping along the cytoskeletal filaments. The motors choreographed mechano-chemical cycle has been well characterized in dilute buffer over the past three decades. A single step of a motor involves multiple events of ligand-substrate binding and conformational changes all of which can be modulated by crowders. Indeed, preliminary findings in the PI's lab show that kinesin motility is altered in the presence of crowders. The aims of the proposed work seek to determine the details of this altered function by probing and manipulating motors down to the single molecule level, both in vitro and in vivo. Moreover, the consequences of the altered function of motors on emergent properties of multiple motors hauling cargo within the cell will be established. This work will also address the need to quantify crowding within the heterogeneous cellular makeup. To that end the PI will develop and calibrate a molecular sensor that can report on the local crowding state within the cell. The function of motors will be studied in their native environment as a function of crowding. A crowding sensor is a general tool that will be a crucial leap towards quantitative understanding of the role crowding plays in biological processes.
