Project Grant 2313725

COLLABORATIVE RESEARCH: UNDERSTANDING AND CONTROLLING FORCE GENERATION BY A CENTRIN-BASED CONTRACTILE SYSTEM -FORCE GENERATION UNDERLIES MANY OF THE PROCESSES MOST ASSOCIATED WITH LIFE: MOVEMENT, GROWTH, AND REPRODUCTION. THE ULTRAFAST CONTRACTION OF THE CILIATE SPIROSTOMUM AMBIGUUM REPRESENTS THE MOST POWERFUL BIOLOGICAL FORCE GENERATION IN NATURE. THIS EXTREME MOVEMENT IS THOUGHT TO BE DRIVEN BY A CONTRACTILE PROTEIN-BASED NETWORK KNOWN AS MYONEMES, WHICH IS POORLY UNDERSTOOD. THE PROJECT WILL COMBINE EXPERIMENTS AND COMPUTATIONAL MODELING TO ELUCIDATE THE MECHANISM OF MYONEME CONTRACTION WITH A VIEW TOWARD REVEALING NEW PRINCIPLES OF BIOLOGICAL FORCE GENERATION. THE FINDINGS WILL SET THE STAGE TO ENGINEER FORCE-GENERATING SYSTEMS FOR SYNTHETIC CELLS, FOR EXAMPLE TO CONTROL CELL SHAPE AND MOVEMENT. THE TEAM WAS ESTABLISHED AT A 2019 NSF IDEAS LAB ON BUILDING SYNTHETIC CELLS AS PART OF THE RULES OF LIFE INITIATIVE. THE BROADER IMPACT OF THE WORK INCLUDES ITS INTRINSIC NATURE IN REVEALING THE MECHANISTIC DETAILS OF WHAT MAY BE THE MOST POWERFUL BIOLOGIC MOTOR KNOWN. ADDITIONAL ACTIVITIES WILL INCLUDE THE MULTIDISCIPLINARY TRAINING OF HIGH SCHOOL, UNDERGRADUATE, GRADUATE STUDENTS, AND POST-DOCTORAL SCHOLARS. A PERMANENT EXHIBIT ON ?SEEING CELLS? AT THE MARINE BIOLOGICAL LABORATORY (MBL), WHERE 1000+ SCIENTISTS AND MEMBERS OF THE PUBLIC VISIT EACH YEAR, WILL ALSO BE EXPANDED UPON. A THEME OF THE EXHIBIT IS THAT A GIVEN FUNCTION IN A CELL CAN BE ACCOMPLISHED THROUGH DIFFERENT MECHANISMS IN DIFFERENT TYPES OF CELLS. THE PROJECT WILL CATALYZE NEW DISCUSSIONS ON THIS THEME, THE DESIGN OF SYNTHETIC CELLS, AND THEIR POSSIBLE IMPACT ON SOCIETY, AND IDEAS FROM THESE DISCUSSIONS WILL BE INCORPORATED INTO THE EXHIBIT AND TRANSLATED TO AN ONLINE FORMAT TO REACH A WIDE AUDIENCE. MYONEME CONTRACTION IS TRIGGERED BY CALCIUM, AND MYONEMES ARE COMPOSED OF CENTRIN EF-HAND PROTEINS AND SFI1 SCAFFOLD PROTEINS. IN CONTRAST TO THE WELL-STUDIED ATP-DRIVEN ACTOMYOSIN CONTRACTILE SYSTEM, LITTLE IS KNOWN ABOUT HOW MYONEMES GENERATE FORCE. STUDIES AT MULTIPLE SCALES WILL PRODUCE QUANTITATIVE INTEGRATIVE MODELS THAT EXPLAIN HOW MOLECULAR CONFORMATIONAL CHANGES PRODUCE FORCE IN THE WHOLE ORGANISM. A KEY ADVANCE ENABLING THE STUDIES IS THE TEAM?S RECONSTITUTION OF CALCIUM-INDUCED CONTRACTION BY FILAMENTS COMPOSED OF ONLY CENTRIN AND SFI1 IN VITRO. THE PROJECT WILL TEST THE HYPOTHESIS THAT SPECIFIC CONFORMATIONAL CHANGES OF MYONEME PROTEINS AT THE MOLECULAR LEVEL ARE TRIGGERED BY CALCIUM TO DRIVE THE ULTRAFAST CONTRACTION AT THE MILLIMETER SCALE IN THIS ORGANISM. THE AIMS ARE TO 1) DETERMINE THE FACTORS THAT MODULATE ASSEMBLY AND FORCE GENERATION IN VITRO, 2) ELUCIDATE THE STRUCTURAL BASES OF CONTRACTION AT THE MOLECULAR LEVEL, AND 3) DETERMINE HOW THE INTERPLAY OF THE MYONEME NETWORK, CALCIUM DYNAMICS, MICROTUBULES, AND THEIR SURROUNDINGS PRODUCE ULTRAFAST CONTRACTION OF THE WHOLE ORGANISM. IN THE LONG TERM, THIS WORK WILL ENABLE NOVEL UNDERSTANDING OF AN INDEPENDENT BIOLOGICAL MECHANISM FOR ULTRAFAST FORCE GENERATION, WHICH CAN BE HARNESSED TO MANIPULATE BIOLOGICAL MATERIALS, BOTH IN VITRO AND IN VIVO. THIS PROJECT WAS CO-FUNDED BY THE MOLECULAR BIOPHYSICS AND THE SYSTEMS AND SYNTHETIC BIOLOGY PROGRAMS IN THE DIVISION OF MOLECULAR AND CELLULAR BIOSCIENCES. THIS AWARD REFLECTS NSF'S STATUTORY MISSION AND HAS BEEN DEEMED WORTHY OF SUPPORT THROUGH EVALUATION USING THE FOUNDATION'S INTELLECTUAL MERIT AND BROADER IMPACTS REVIEW CRITERIA.