Project Grant F32GM165037

BIOPHYSICAL MECHANISMS GOVERNING THE MATERIAL PROPERTIES OF MITOCHONDRIAL NUCLEOID CONDENSATES - PROJECT SUMMARY / ABSTRACT BIOMOLECULAR CONDENSATES ARE EMERGING DRIVERS OF FUNCTIONAL ORGANIZATION IN THE CELL. THESE CONDENSATES ARE MATERIALS THAT FORM SPONTANEOUSLY BY ATTRACTIVE INTERACTIONS BETWEEN CONSTITUENT BIOMACROMOLECULES. CONDENSATE MECHANICAL PROPERTIES ARE ENCODED BY MACROMOLECULAR SEQUENCES, AND ARE TUNED BY THE CONDENSATE'S CHEMICAL MICROENVIRONMENT. THESE DISTINCT MECHANICAL PROPERTIES THEN ENABLE DISTINCT BIOLOGICAL FUNCTIONS. FOR EXAMPLE, LIQUID-LIKE VISCOSITY MIGHT ENHANCE ENZYMATIC REACTION RATES, WHILE SOLID-LIKE ELASTICITY MIGHT ENABLE A CONDENSATE TO BEND A MEMBRANE. RECENTLY, MITOCHONDRIAL NUCLEOIDS HAVE BEEN FOUND TO BE BIOMOLECULAR CONDENSATES. MITOCHONDRIAL NUCLEOIDS ARE NUCLEOPROTEIN COMPLEXES INSIDE MITOCHONDRIA WHOSE FUNCTION IS TO PACKAGE AND TRANSCRIBE MITOCHONDRIAL DNA (MTDNA) WITH TFAM, A PACKAGING PROTEIN AND TRANSCRIPTION FACTOR. MITOCHONDRIAL NUCLEOIDS ARE CRUCIAL FOR METABOLIC FUNCTION. HOWEVER, VERY LITTLE IS KNOWN ABOUT THEIR SEQUENCE-ENCODED MECHANICAL PROPERTIES, OR HOW THESE PROPERTIES MIGHT RESPOND TO THE DYNAMIC CHEMICAL MICROENVIRONMENT IN THE MITOCHONDRIAL MATRIX. THIS KNOWLEDGE COULD SHED MECHANISTIC LIGHT ON PRIOR OBSERVATIONS OF NUCLEOID-DRIVEN MEMBRANE BENDING OR NUCLEOID CLUSTERING IN RESPONSE TO CELL STRESSES. THIS RESEARCH PLAN TESTS THE HYPOTHESIS THAT SPECIFIC SEQUENCES IN TFAM AND MTDNA ENCODE A CONDENSATE THAT IS MECHANICALLY RESPONSIVE TO ITS CHEMICAL MICROENVIRONMENT. THIS PLAN USES IN VITRO RECONSTITUTION OF TFAM:DNA CONDENSATES, AND EMPLOYS METHODS INCLUDING TARGETED PROTEIN SEQUENCE PERTURBATIONS, QUANTITATIVE CONFOCAL MICROSCOPY TECHNIQUES, PARTICLE-TRACKING MICRORHEOLOGY, AND ATOMIC FORCE MICROSCOPY. AIM 1 PROBES HOW SEQUENCE FEATURES IN TFAM AND MTDNA ENCODE NUCLEOID VISCOELASTICITY, INTERFACIAL PROPERTIES, AND MEMBRANE BENDING. AIM 2 TESTS THE HYPOTHESIS THAT PHYSIOLOGICALLY-RELEVANT CHEMICAL COMPONENTS MODULATE MECHANICAL PROPERTIES AND MTDNA TRANSCRIPTION IN THE NUCLEOID. THE APPLICANT WILL BE ADVISED BY A MENTORING TEAM COMPRISING A BIOPHYSICIST SPECIALIZING IN THE MECHANICAL AND INTERFACIAL PROPERTIES OF BIOMOLECULAR CONDENSATES; AND A PROTEIN BIOCHEMIST EXPERT IN THE TRANSCRIPTIONAL MECHANISMS OF TFAM AND MTDNA. THE TRAINING ENVIRONMENT INCLUDES OPPORTUNITIES FOR MENTORSHIP, PROFESSIONAL DEVELOPMENT PROGRAMMING, DISCUSSION AND COLLABORATION WITH LEADING RESEARCHERS IN CONDENSATE BIOPHYSICS, AND UNIQUE TRAINING AT THE INTERSECTION OF MITOCHONDRIAL NUCLEOID BIOLOGY AND BIOMOLECULAR CONDENSATE MECHANICS. OVERALL, THE PROPOSED WORK REPRESENTS BASIC RESEARCH ADVANCING OUR BIOPHYSICAL UNDERSTANDING OF THE MITOCHONDRIAL NUCLEOID, A STRUCTURE RELEVANT TO MANY PATHOLOGIES CHARACTERIZED BY MITOCHONDRIAL DYSFUNCTION SUCH AS CANCERS AND METABOLIC SYNDROME. IN ADDITION, THE BIOPHYSICAL MECHANISMS UNCOVERED IN THIS SYSTEM MAY SHED LIGHT ON THE MECHANICAL REGULATION OF OTHER BIOMOLECULAR CONDENSATES IMPORTANT TO HUMAN PHYSIOLOGY.