Rajesh Ramachandran, Ph.D. Associate Professor Department of Physiology and Biophysics 10900 Euclid Avenue Cleveland, Ohio 44106-4970 Phone: 216.368.2513 Fax: 216.368.5586 E-Mail: rajesh.ramachandran@case.edu Apr 29, 2020 Center for Scientific Review National Institutes of Health Bethesda, MD Application for Supplement for NIGMS research grant (R01) - NOT-GM-20-013 To whom it may concern: I am pleased to submit our application for an Administrative Supplement (Instrumentation) to our NIH R01 grant award (5 R01 GM121583 03) entitled ?Molecular Mechanisms of dynamin-related protein 1-mediated mitochondrial fission? for consideration. We expect the unobligated balance for GM121583 to be below 25% at the end of the current year's budget period. Thank you very much for your consideration. Sincerely, Rajesh Ramachandran, Ph.D. Associate Professor, Department of Physiology and Biophysics Case Western Reserve University School of Medicine PROJECT SUMMARY Mitochondria are dynamic organelles that undergo continuous fission and fusion. Mitochondrial dynamics are essential for cell survival, as well as for mitochondrial quality control, transport, distribution and inheritance. Defects in mitochondrial dynamics are implicated in various neurological disorders including Alzheimer?s, Parkinson?s and Huntington?s diseases, as well as in cardiovascular disease and cancer. The molecular mechanisms that accomplish mitochondrial membrane fission and fusion are poorly understood, as are the roles of the molecules involved in these processes. The long-term goal of this proposal is to address such issues. The mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1) is the master regulator of mitochondrial fission. Cytosolic Drp1 initiates mitochondrial fission via interactions with adaptor proteins, Mff, MiD49/51, or Fis1 localized at the mitochondrial surface. Subsequent Drp1 polymerization as ?helical scaffolds? around pre-destined mitochondrial division sites and GTP hydrolysis-driven scaffold constriction catalyzes mitochondrial fission. Exciting new studies have also necessitated a cooperative role for direct Drp1-phospholipid interactions, specifically with the mitochondrial lipid, cardiolipin (CL), in mitochondrial fission. However, very little is known about the cooperativity of Drp1-adaptor and Drp1-CL interactions, either in space or in time, during this process. Several unknown fundamental issues essential for understanding Drp1-mediated mitochondrial fission will be addressed in this application. These include 1) the mechanisms underlying Drp1 CL recognition, and the identity of Drp1 residues involved in specific phospholipid interactions, 2) the mechanism of the Drp1 variable domain (VD) in CL reorganization and nonbilayer phase transition, 3) the domain-specific topography of Drp1 on the membrane surface and conformational rearrangements that ensue upon specific adaptor and CL interactions, and 4) the cooperativity of CL and adaptor interactions in effecting mitochondrial fission. The proposed experiments will test the overarching hypothesis that cooperative Drp1 interactions with protein adaptors and CL promote the formation of a productive ?fission complex? that is localized in CL-rich micro-environments and drives membrane remodeling and fission through a Drp1 GTP hydrolysis-dependent CL bilayer-to-nonbilayer phase transition mechanism. We will use a tailor-made array of innovative fluorescence spectroscopic and microscopic approaches, coupled to solution and solid state NMR, to address these issues. These include the use of a novel variation of the FRET approach to determine domain-specific Drp1-membrane distances, collisional quenching of fluorescence to determine and measure Drp1 VD membrane insertion, and fluorescence imaging on model GUVs to visualize adaptor- and CL-regulated, Drp1-mediated membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying mitochondrial fission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate mitochondrial dynamics under various disease states.
Research Plan: Summary of the parent grant (R01GM121583): Mitochondria are dynamic organelles that undergo continuous fission and fusion. Mitochondrial dynamics are essential for cell survival, as well as for mitochondrial quality control, transport, distribution and inheritance. Defects in mitochondrial dynamics are implicated in various neurological disorders including Alzheimer?s, Parkinson?s and Huntington?s diseases, as well as in cardiovascular disease and cancer. The molecular mechanisms that accomplish mitochondrial membrane fission and fusion are poorly understood, as are the roles of the molecules involved in these processes. The long-term goal of this proposal is to address such issues. The mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1) is the master regulator of mitochondrial fission. Cytosolic Drp1 initiates mitochondrial fission via interactions with adaptor proteins, Mff, MiD49/51, or Fis1 localized at the mitochondrial surface. Subsequent Drp1 polymerization as ?helical scaffolds? around pre-destined mitochondrial division sites and GTP hydrolysis-driven scaffold constriction catalyzes mitochondrial fission. Exciting new studies have also necessitated a cooperative role for direct Drp1-phospholipid interactions, specifically with the mitochondrial lipid, cardiolipin (CL), in mitochondrial fission. However, very little is known about the cooperativity of Drp1-adaptor and Drp1-CL interactions, either in space or in time, during this process. Several unknown fundamental issues essential for understanding Drp1- mediated mitochondrial fission will be addressed in this application. These include 1) the mechanisms underlying Drp1 CL recognition, and the identity of Drp1 residues involved in specific phospholipid interactions, 2) the mechanism of the Drp1 variable domain (VD) in CL reorganization and nonbilayer phase transition, 3) the domain-specific topography of Drp1 on the membrane surface and conformational rearrangements that ensue upon specific adaptor and CL interactions, and 4) the cooperativity of CL and adaptor interactions in effecting mitochondrial fission. The proposed experiments will test the overarching hypothesis that cooperative Drp1 interactions with protein adaptors and CL promote the formation of a productive ?fission complex? that is localized in CL-rich micro-environments and drives membrane remodeling and fission through a Drp1 GTP hydrolysis- dependent CL bilayer-to-nonbilayer phase transition mechanism. We will use a tailor-made array of innovative fluorescence spectroscopic and microscopic approaches, coupled to solution and solid state NMR, to address these issues. These include the use of a novel variation of the FRET approach to determine domain-specific Drp1-membrane distances, collisional quenching of fluorescence to determine and measure Drp1 VD membrane insertion, and fluorescence imaging on model GUVs to visualize adaptor- and CL-regulated, Drp1-mediated membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying mitochondrial fission, and (ii) a molecular foundation for the design of drugs. Scientific Justification for the requested equipment: Drp1?s mechanoenzymatic activity in mitochondrial fission is contingent upon its coincident and cooperative interactions with protein adaptors Mff, MiD49/51, or Fis1 anchored in the mitochondrial outer membrane (MOM), as well as with MOM-localized cardiolipin (CL) externalized at mitochondrial division sites (1-4). However, no information is currently available on the topography (orientation) of these complexes on the membrane surface, as are their effects on CL sequestration and nonbilayer phase transition essential for localized, leak-free membrane fission, which are both stated goals of our parent grant proposal. We have successfully employed multiple independent fluorescence spectroscopic techniques, including Frster Resonance Energy Transfer (FRET) and the application of environmentally sensitive fluorescent dyes such as NBD, recently to investigate Drp1-membrane interactions and Drp1-catalyzed CL nonbilayer phase transition in isolation (5-8). However, these results were obtained painstakingly, one measurement-at-a-time, using a cumbersome, cuvette-based, spectrofluorometer setup utilizing relatively large sample volumes (400 ?L minimum per sample), and in a time- consuming and cost-ineffective manner. Given the next step is to investigate Drp1 topography and membrane effects by FRET and environmentally sensitive fluorophores in the presence of low-yield, detergent-solubilized, and membrane-reconstituted transmembrane domain (TMD)-anchored adaptors, it is imperative that the sample volumes be kept to a minimum and experiments be run in parallel with controls to avoid time-dependent changes in sample behavior. A monochromator-based, temperature-controlled, high sensitivity multimode fluorescence plate reader such as the Tecan Spark requested here provides the ideal solution to overcome these problems. This system would allow us to measure 96-or-more samples in parallel and will utilize as low as 25 ?L sample volume per well. Therefore, we request funds for the purchase of a new, state-of-the-art ?TECAN Spark? multimode fluorescence microplate reader with superior capabilities in terms of sensitivity, flexibility, and efficiency. Based on the above justification, I am requesting the supplement fund in the amount of $54,504.35 to purchase a ?Tecan Spark? system with temperature control (Te-Cool) from the industry leader, TECAN (quote included). References 1. Ramachandran, R. (2018) Mitochondrial dynamics: The dynamin superfamily and execution by collusion. Semin Cell Dev Biol 76, 201-212 2. Chu, C. T., Ji, J., Dagda, R. K., Jiang, J. F., Tyurina, Y. Y., Kapralov, A. A., Tyurin, V. A., Yanamala, N., Shrivastava, I. H., Mohammadyani, D., Wang, K. Z. Q., Zhu, J., Klein-Seetharaman, J., Balasubramanian, K., Amoscato, A. A., Borisenko, G., Huang, Z., Gusdon, A. M., Cheikhi, A., Steer, E. K., Wang, R., Baty, C., Watkins, S., Bahar, I., Bayir, H., and Kagan, V. E. (2013) Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells. Nat Cell Biol 15, 1197-1205 3. Agrawal, A., and Ramachandran, R. (2019) Exploring the links between lipid geometry and mitochondrial fission: Emerging concepts. Mitochondrion 49, 305-313 4. Adachi, Y., Itoh, K., Yamada, T., Cerveny, K. L., Suzuki, T. L., Macdonald, P., Frohman, M. A., Ramachandran, R., Iijima, M., and Sesaki, H. (2016) Coincident Phosphatidic Acid Interaction Restrains Drp1 in Mitochondrial Division. Mol Cell 63, 1034-1043 5. Macdonald, P. J., Stepanyants, N., Mehrotra, N., Mears, J. A., Qi, X., Sesaki, H., and Ramachandran, R. (2014) A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission. Mol Biol Cell 25, 1905-1915 6. Stepanyants, N., Macdonald, P. J., Francy, C. A., Mears, J. A., Qi, X., and Ramachandran, R. (2015) Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission. Mol Biol Cell 26, 3104-3116 7. Macdonald, P. J., Francy, C. A., Stepanyants, N., Lehman, L., Baglio, A., Mears, J. A., Qi, X., and Ramachandran, R. (2016) Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity. J Biol Chem 291, 493-507 8. Lu, B., Kennedy, B., Clinton, R. W., Wang, E. J., McHugh, D., Stepanyants, N., Macdonald, P. J., Mears, J. A., Qi, X., and Ramachandran, R. (2018) Steric interference from intrinsically disordered regions controls dynamin-related protein 1 self-assembly during mitochondrial fission. Sci Rep 8, 10879
| Lu, Bin; Kennedy, Bridget; Clinton, Ryan W et al. (2018) Steric interference from intrinsically disordered regions controls dynamin-related protein 1 self-assembly during mitochondrial fission. Sci Rep 8:10879 |
| Ramachandran, Rajesh; Schmid, Sandra L (2018) The dynamin superfamily. Curr Biol 28:R411-R416 |
| Ramachandran, Rajesh (2018) Mitochondrial dynamics: The dynamin superfamily and execution by collusion. Semin Cell Dev Biol 76:201-212 |
