MANY OF THE ADAPTIVE RESPONSES TO STRESS THAT A CELL SHOULD NEED DURING SPACE FLIGHT ARE REGULATED ON EARTH BY SUMO. SUMO IS A SMALL UBIQUITIN-LIKE PROTEIN THAT REGULATES A LARGE NUMBER OF BASIC AND FUNDAMENTAL CELLULAR PROCESSES. SUMO CAN BE ATTACHED TO LYSINE RESIDUES WITHIN TARGET PROTEINS AS A POST-TRANSLATIONAL MODIFICATION ALTERING THEIR FUNCTION AND CELLULAR LOCALIZATION. SUMO CAN ALSO BIND NON-COVALENTLY TO OTHER PROTEINS THROUGH THEIR SUMO-INTERACTING MOTIFS (SIMS). SUMO MEDIATES CELLULAR RESPONSES TO A LARGE NUMBER OF STRESSES INCLUDING HYPOXIA RADIATION THERMO-TOLERANCE AND OXIDATIVE STRESS. HOWEVER A SIGNIFICANT GAP IN KNOWLEDGE EXISTS ABOUT HOW THE SUMO SIGNAL TRANSDUCTION SYSTEM IS USED BY THE CELL TO SENSE AND REGULATE ITS RESPONSE TO STRESSES FOUND IN SPACE AND MICROGRAVITY IN PARTICULAR. THE LONG-TERM GOAL OF THE MILLER LAB IS TO UNDERSTAND HOW SIGNAL TRANSDUCTION CIRCUITS CONTROL MICROTUBULES SO THAT EFFECTIVE AND TARGETED INTERVENTIONS CAN BE DEVELOPED TO MANIPULATE MICROTUBULE FUNCTIONS IN STRESSFUL SITUATIONS LIKE DISEASE AND SPACE TRAVEL. THE MILLER LAB RECENTLY ESTABLISHED THAT THE MICROTUBULE-ASSOCIATED PROTEIN STU2P INTERACTS WITH SUMO. PRELIMINARY DATA FROM THE MILLER LAB SUGGEST THAT THERE ARE TWO MODES OF INTERACTION BETWEEN STU2P AND SUMO: A COVALENT INTERACTION IN WHICH STU2P IS CONJUGATED BY SUMO AND A NON-COVALENT INTERACTION IN WHICH STU2P SIMPLY BINDS TO SUMO. THE OBJECTIVE OF THIS WORK IS TO IDENTIFY NOVEL SIGNAL TRANSDUCTION MECHANISMS THAT REGULATE CYTOSKELETAL NETWORKS IN RESPONSE TO SIMULATED MICROGRAVITY CONDITIONS. THE RATIONALE IS THAT BY ACHIEVING A FULLER UNDERSTANDING OF THE REGULATION OF CYTOSKELETAL POLYMERS UNDER SIMULATED MICROGRAVITY CONDITIONS ON EARTH RESEARCHERS CAN DEVELOP MORE COMPLETE HYPOTHESES OF HOW THE CYTOSKELETON RESPONDS TO GRAVITATIONAL CHANGES IN THE SPACE ENVIRONMENT WHICH CAN BE TESTED IN FUTURE SPACE MISSIONS. THIS KNOWLEDGE MAY BE USED TO DESIGN INTERVENTIONS FOR ADVERSE HEALTH EFFECTS THAT ARE ASSOCIATED WITH SPACE TRAVEL. THE SPECIFIC GOAL OF THIS PROPOSAL IS TO DETERMINE HOW SIMULATED MICROGRAVITY ALTERS THE INTERACTION OF SUMO WITH THE CELLULAR PROTEOME AND MICROTUBULE-ASSOCIATED PROTEINS (MAPS) IN PARTICULAR. THE CENTRAL HYPOTHESIS OF THIS WORK IS THAT MICROGRAVITY ALTERS THE POST-TRANSLATIONAL MODIFICATIONS OF LYSINE RESIDUES OF A WIDE VARIETY OF CYTOSKELETAL PROTEINS. TO TEST THIS HYPOTHESIS THE POST-TRANSLATIONAL MODIFICATIONS ON LYSINES RESIDUES WILL BE ANALYZED ON A PROTEOME-WIDE BASIS BY MASSSPECTROMETRY AND IMMUNOBLOTTING BLOTTING UNDER SIMULATED MICROGRAVITY CONDITIONS. WE WILL ALSO TEST WHETHER THE PROTEOME BINDS DIFFERENTIALLY TO SUMO WHEN IT EXPERIENCES MICROGRAVITY. AN ADDITIONAL EMPHASIS WILL BE PLACED ON THE MAP STU2P. THIS WORK IS SIGNIFICANT BECAUSE IT WILL HELP IDENTIFY SUMOYLATION AS A NOVEL MOLECULAR MECHANISM AT THE BIOCHEMICAL AND MOLECULAR LEVEL WITHIN THE YEAST CELL THAT SENSES AND RESPONDS TO THE ENVIRONMENTAL FACTORS OF SPACE. UNDERSTANDING THESE EFFECTS ON BASIC CELL BIOLOGY MAY LEAD TO A BETTER UNDERSTANDING OF HOW SPACE TRAVEL AFFECTS NORMAL TISSUE FUNCTION IMMUNE FUNCTION STEM CELL FUNCTION TISSUE REGENERATION AND EMBRYOGENESIS IN HUMANS. AS TUBULIN AND THE HUMAN VERSION OF STU2 PLAY IMPORTANT ROLES IN CELL DIVISION THESE STUDIES MAY ALSO SHED LIGHT ON MOLECULAR MECHANISMS THAT ARE IMPORTANT IN CANCER.