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ROLE OF ORGANIC MATRIX MOLECULES IN THE FORMATION OF VERY HIGH MAGNESIUM CALCITE -NON-TECHNICAL ABSTRACT: MINERALIZED TISSUES ARE SOPHISTICATED ORGANIC-INORGANIC COMPOSITES THAT ARE ASSEMBLED BOTTOM-UP AND EXHIBIT HIERARCHICAL ARCHITECTURE. HIGHLY EVOLVED DESIGN ENABLES FEATURES SUCH AS HIGH BONE TOUGHNESS AT LOW WEIGHT, SELF-SHARPENING TEETH, AND CONTINUOUS ADAPTIVE REMODELING/SELF-REPAIR. THE ARRANGEMENT OF THE CELLULAR COMPONENTS OF THE SEA URCHIN TOOTH ALLOWS CONTINUOUS GROWTH AND REGENERATION, IN RESPONSE TO TOOTH WEAR AND ABRASION DURING FEEDING. FROM A MATERIALS PERSPECTIVE, THE SEA URCHIN TOOTH IS HIGHLY UNUSUAL IN THAT IT IS THE ONLY KNOWN INSTANCE OF VERY HIGH MAGNESIUM CALCITE (VHMC) IN THE COLUMNS THAT CONNECT THE PRIMARY AND SECONDARY PLATES TO EACH OTHER. WITH UP TO 33% MG, VHMC IS FAR FROM EQUILIBRIUM, MUCH HARDER THAN CALCITE, AND CANNOT BE MADE IN THE LAB. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO ELUCIDATE THE MECHANISM OF FORMATION OF VHMC IN THE SEA URCHIN TOOTH. THE PI PREVIOUSLY ISOLATED A SET OF FOUR PROLINE-ALANINE RICH ACIDIC PHOSPHOPROTEINS (PARPS) THAT ARE UNIQUE TO THE TOOTH BIOMINERAL. THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT PARPS ARE INVOLVED IN VHMC FORMATION. THE MAIN OBJECTIVE OF THE PROPOSED WORK IS TO TEST THIS HYPOTHESIS BY A) MAPPING THE DISTRIBUTION OF PARPS IN THE TOOTH IN RELATION TO THE COLUMNS USING IMMUNOHISTOCHEMISTRY; B) QUANTIFYING THE IMPACT OF RECOMBINANT PARPS ON NUCLEATION, GROWTH, AND FINAL COMPOSITION OF CRYSTALLINE CALCIUM CARBONATE IN THE PRESENCE OF MAGNESIUM IN VITRO; AND FINALLY, BY C) DETERMINING THE IMPACT OF KNOCK DOWN OF THE MOST PROMISING CANDIDATE IN VIVO. POISED AT THE INTERSECTION OF MOLECULAR BIOLOGY, MATERIALS SCIENCE, AND BIOENGINEERING, THIS RESEARCH HAS THE POTENTIAL TO INFORM A WEALTH OF NEW TECHNOLOGIES, FROM BIO-INSPIRED AND BIO-ENABLED MATERIALS TO MATERIALS FOR CARBON DIOXIDE SEQUESTRATION. THE TEAM WILL LEVERAGE THE INTERDISCIPLINARY POTENTIAL OF THIS RESEARCH TO TRAIN HIGH SCHOOL, UNDERGRADUATE, MASTERS AND A GRADUATE STUDENT FROM A BROAD RANGE OF BACKGROUNDS. TECHNICAL ABSTRACT: THE PROPOSED ACTIVITIES ADDRESS GAPS IN THE UNDERSTANDING OF HOW LIVING ORGANISMS CONTROL CRYSTAL GROWTH PROCESSES, WITH THE LONG-TERM OBJECTIVE TO DEVELOP BIO-INSPIRED AND BIO-ENABLED MATERIALS. THE ARRANGEMENT OF THE CELLULAR COMPONENTS OF THE SEA URCHIN TOOTH ALLOWS CONTINUOUS GROWTH AND REGENERATION, IN RESPONSE TO TOOTH WEAR AND ABRASION DURING FEEDING. AN INTRIGUING ASPECT OF THE TOOTH ARCHITECTURE IS THE USE OF BOTH HIGH MAGNESIUM CALCITE IN THE PLATES (HMC, CA1-XMGXCO3, WHERE X ~ 0.13) AND VERY HIGH MAGNESIUM CALCITE (VHMC, X~0.33) IN THE INTER-PLATE COLUMNS. WHILE OTHER MINERALIZED TISSUES IN THE SEA URCHIN ARE COMPRISED OF HMC, VHMC IS UNIQUE TO THE TOOTH. VHMC COMPOSITION IS FAR FROM EQUILIBRIUM AND THERE IS NO KNOWN SYNTHESIS. THE PI PREVIOUSLY IDENTIFIED A SET OF PROLINE-ALANINE RICH ACIDIC PHOSPHOPROTEINS (PARPS) THAT ARE UNIQUE TO THE TOOTH BIOMINERAL. THE CENTRAL HYPOTHESIS OF THIS PROPOSAL IS THAT PARPS ARE INVOLVED IN VHMC FORMATION. TO TEST THIS HYPOTHESIS, ALL 4 PARPS WILL BE RECOMBINANTLY EXPRESSED IN A SYSTEM THAT WILL ENSURE APPROPRIATE POST-TRANSLATIONAL MODIFICATION (PHOSPHORYLATION). ANTIBODIES WILL BE RAISED AGAINST ALL RECOMBINANT PARPS TO DETERMINE, USING IMMUNOHISTOCHEMISTRY, WHICH OF THEM, IF ANY, CO-LOCALIZE WITH THE INTER-PLATE COLUMNS, BUT NOT THE PLATES. THE TWO MOST PROMISING CANDIDATES WILL BE CHOSEN FOR IN-VITRO EXPERIMENTS TO DETERMINE THEIR IMPACT ON NUCLEATION, GROWTH AND COMPOSITION OF CALCIUM CARBONATE IN THE PRESENCE OF MG. BOTH FULLY AND UN-PHOSPHORYLATED FORMS WILL BE TESTED TO ESTABLISH THE FUNCTIONAL ROLE OF PHOSPHORYLATION. FINALLY, BASED ON THE IN-VITRO CRYSTALLIZATION RESULTS, THE MOST PROMISING CANDIDATE WILL BE KNOCKED DOWN IN THE ADULT SEA URCHIN (USING IN-VIVO MORPHOLINOS) AND ITS EFFECT ON THE COMPOSITION OF THE INTER-PLATE COLUMNS DETERMINED. THE RESULTS SHOULD PROVIDE A MECHANISTIC BASIS FOR BIOINSPIRED SYNTHESIS OF VHMC, AND HOW THE SEA URCHIN SYNTHESIZES CALCITE WITH A COMPOSITION VERY FAR FROM EQUILIBRIUM, THEREBY IMPROVING THE PROPERTIES OF THE MATERIAL, AND THE PERFORMANCE OF THE TOOTH DRAMATICALLY. ELUCIDATING THE MECHANISM OF VHMC FORMATION WILL FEED BACK INTO THE FIELDS OF CELL AND DEVELOPMENT BIOLOGY. COMPLEMENTARY TO THE PROPOSED RESEARCH OBJECTIVES, THE TEAM WILL ENGAGE UNDERGRADUATES, A MASTERS AND A GRADUATE STUDENT IN RESEARCH, AND HOST A HIGH SCHOOL INTERN THROUGH NORTHWESTERN UNIVERSITY'S SCIENCE IN SOCIETY PROGRAM. 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.
$75.0k 7/21/22