Project Grant 2423216

BIOENGINEERING SINGLE CRYSTAL GROWTH -TECHNICAL: THE PROPOSED ACTIVITIES ADDRESS GAPS IN THE UNDERSTANDING OF HOW LIVING ORGANISMS CONTROL CRYSTAL GROWTH PROCESSES, WITH THE LONG-TERM OBJECTIVE OF DEVELOPING BIO-INSPIRED AND BIO-ENABLED MATERIALS. IN PRIOR WORK, THE TEAM DISCOVERED THE ROLE OF VEGF SIGNALING IN THE BRANCHING OF ENDOSKELETAL SINGLE CRYSTALS OF CALCITE THAT ARE DEPOSITED BY PRIMARY MESENCHYME CELLS (PMCS) OF THE SEA URCHIN EMBRYO. GOING FORWARD, THE TEAM WILL USE QUANTITATIVE PROTEOMICS IN COMBINATION WITH TRANSCRIPTOMIC DATA TO IDENTIFY PROTEINS INVOLVED IN CRYSTAL GROWTH CONTROL. SELECTED PROTEINS WILL BE PRODUCED RECOMBINANTLY, AND RECOMBINANT ANTIBODIES (RABS) WILL BE RAISED AGAINST THEM. RABS WILL BE USED TO MAP PROTEINS ACROSS THE SPICULE DEPOSITION VESICLE AND THE SPICULE ITSELF. TO DISSECT THE IMPACT OF NATIVE AND RECOMBINANT PROTEINS ON NUCLEATION, POLYMORPH SELECTION, AND CRYSTAL GROWTH, THE TEAM WILL USE A DROPLET MICROFLUIDIC NUCLEATION RATE ASSAY. IN PARALLEL, THE TEAM WILL USE SERIAL BLOCK-FACE ELECTRON MICROSCOPY, CRYO-FIB/SEM SLICE-AND-VIEW, AND CRYO-ELECTRON TOMOGRAPHY TO STUDY INTERFACIAL PROCESSES AT THE GROWTH FRONT AND ELUCIDATE THE ULTRASTRUCTURE OF THE SPICULE ELONGATION APPARATUS. TAKEN TOGETHER, THE TEAM EXPECTS TO DEVELOP A DETAILED MECHANISTIC UNDERSTANDING OF HOW THE EXPRESSION OF THE RELEVANT PROTEINS, LOCALIZATION IN THE SPICULE MATRIX, AND IMPACT ON NUCLEATION KINETICS AND CRYSTAL GROWTH MAY BE CONNECTED. THIS IS AN IMPORTANT FIRST STEP TOWARDS PORTING KEY MOLECULAR PLAYERS INTO A SYSTEM THAT IS MORE EASILY ENGINEERED AND SCALED UP, ESSENTIALLY USING THE TOOLS OF SYNTHETIC BIOLOGY FOR MATERIALS PROCESSING. THE TEAM WILL THEREBY ADDRESS ALL FOUR CHALLENGES IN HARD MATERIALS IDENTIFIED IN THE REPORT ON THE 2012 NSF BIOMATERIALS WORKSHOP. COMPLEMENTARY TO THE PROPOSED RESEARCH OBJECTIVES, THE TEAM WILL ENGAGE UNDERGRADUATES IN HIGHLY INTERDISCIPLINARY RESEARCH, LEVERAGING THE NSF REU PROGRAM AND THE MATERIALS INITIATIVE FOR COMPREHENSIVE RESEARCH OPPORTUNITY (MICRO) AT NORTHWESTERN UNIVERSITY. FURTHER, THE TEAM WILL INTEGRATE RESEARCH OUTCOMES INTO UNDERGRADUATE LABORATORY MODULES AND DEVELOP NEW EXPERIMENTS FOR OUTREACH ACTIVITIES. NON-TECHNICAL: OPTIMIZED DURING HUNDREDS OF MILLIONS OF YEARS OF EVOLUTION, BIOMINERALIZED TISSUES FREQUENTLY DISPLAY EXTRAORDINARY PERFORMANCE. BONE, FOR EXAMPLE, HAS HIGH TOUGHNESS AT LOW WEIGHT AND IS CAPABLE OF SELF-REPAIR; IN SOME ORGANISMS, TEETH GROW CONTINUOUSLY AND SELF-SHARPEN. DESPITE THE ABUNDANCE OF SUCH MATERIALS IN NATURE, MANY OF THE MECHANISMS THAT ALLOW THE ORGANISM TO CONTROL THEIR FORMATION REMAIN POORLY UNDERSTOOD. SEA URCHIN EMBRYOS CREATE SMOOTHLY CURVING AND BRANCHED, YET SINGLE CRYSTALLINE SPICULES OF CALCIUM CARBONATE (CACO3) FOR THEIR ENDOSKELETON. PREVIOUSLY, THE TEAM DESIGNED AN IN VITRO CULTURE SYSTEM OF SEA URCHIN EMBRYO PRIMARY MESENCHYME CELLS (PMCS) TO CONTROL THE GROWTH OF THESE SPICULES IN THE LABORATORY. THEY DISCOVERED THAT A SIGNALING MOLECULE, VEGF, CONTROLS THE SHAPE OF SPICULES DEPOSITED BY PMCS. HOWEVER, IT REMAINS UNCLEAR WHAT HAPPENS DOWNSTREAM OF THE INTERACTION OF VEGF WITH ITS RECEPTOR. THE GOAL OF THIS PROPOSAL IS TO IDENTIFY AND CHARACTERIZE PROTEINS THAT ARE DIRECTLY INVOLVED IN CONTROLLING CRYSTAL GROWTH. TECHNIQUES INCLUDE PROTEOMICS, IMMUNOHISTOCHEMISTRY USING RECOMBINANT ANTIBODIES, AND MICROFLUIDIC DROPLET ASSAYS OF NUCLEATION AND GROWTH. IN PARALLEL, THE TEAM WILL USE STATE-OF-THE-ART IMAGING, INCLUDING CRYO-ELECTRON TOMOGRAPHY, TO OBSERVE THE CELLULAR APPARATUS THAT ELONGATES THE SPICULE. IN COMBINATION, THESE EXPERIMENTS WILL LEAD TO AN IMPROVED UNDERSTANDING OF HOW THE SEA URCHIN EMBRYO DYNAMICALLY CONTROLS THE SHAPE OF ITS ENDOSKELETON. POISED AT THE INTERSECTION OF MOLECULAR BIOLOGY, MATERIALS SCIENCE, AND BIOENGINEERING, THE PROPOSED 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 APPROACH OF THE PROPOSED ACTIVITIES TO ENGAGE UNDERGRADUATE STUDENTS FROM A BROAD RANGE OF DISCIPLINARY AND SOCIOECONOMIC BACKGROUNDS IN RESEARCH, DRAWING ON THE NSF REU PROGRAM AND THE MATERIALS INITIATIVE FOR COMPREHENSIVE RESEARCH OPPORTUNITY (MICRO) AT NORTHWESTERN UNIVERSITY. ADDITIONALLY, THE TEAM WILL DEVELOP MICROFLUIDIC DEVICES TO BE USED IN AN UNDERGRADUATE LABORATORY CLASS ON PHASE TRANSFORMATIONS AND FOR OUTREACH ACTIVITIES. 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.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.