OPA1 is a 120kDa large GTPase belonging to the dynamin superfamily. It is the only known mitochondrial inner membrane fusion protein, mediating fusion of the mitochondrial inner membranes following outer membrane fusion. Additionally, OPA1 also regulates cristae morphology and maintains respiratory chain function.
OPA1 has two forms—inner-membrane-anchored long forms (l-OPA1) and cleaved inter-membrane-space only short forms (s-OPA1). L-OPA1 are proteolytically processed by two mitochondrial proteases—OMA1 and YME1L, acting at cleavage sites S1 and S2 respectively, to produce s-OPA1.
In both mice and human, half of the mRNA splice forms of Opa1 are constitutively processed post translation to yield exclusively s-OPA1. However, the specific function of s-OPA1 in mitochondrial fusion has been debated—under basal conditions, s-OPA1 are needed to maintain optimal fusion activity, but in certain stress conditions, s-OPA1 is dispensable for fusion. By constructing cells in which the Opa1 locus no longer produces transcripts with S2 cleavage sites using CRISPR-Cas9, we generated a simplified system to identify the novel YME1L-dependent site S3 that mediates constitutive and complete cleavage of OPA1. We found that S3 site locates within the C-terminal leucine string of Opa1 exon4b, slightly upstream of the well-established S1. We show that mitochondrial morphology is highly sensitive to the ratio of l-OPA1 to s-OPA1, indicating that s-OPA1 fine tunes mitochondrial fusion.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/1mes-yw82, author = {Varuzhanyan, Grigor}, title = {Mitochondrial Dynamics and Mitophagy during Male Germline Development}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/1mes-yw82}, url = {https://resolver.caltech.edu/CaltechTHESIS:09232020-201548312}, abstract = {Mitochondrial fusion and fission (mitochondrial dynamics) and mitophagy are well-established mitochondrial quality control mechanisms that safeguard cellular homeostasis. However, their role during development remains poorly understood. In this thesis, we establish the role of mitochondrial dynamics and mitophagy during the development of the male germline (spermatogenesis).
Spermatogenesis is one of biology’s most complex and lengthy differentiation processes, transforming spermatogonial stem cells into highly specialized sperm cells capable of fertilization. This elaborate differentiation program requires multiple transitions in mitochondrial morphology and extensive degradation of mitochondria, making it an attractive model system for investigating mitochondrial dynamics and mitophagy in vivo. Indeed, the field of mitochondrial dynamics has a long history with spermatogenesis. The first mitochondrial dynamics gene, Fuzzy onions (Fzo), was discovered in 1997 to mediate mitochondrial fusion during Drosophila spermatogenesis. However, the role of mitochondrial dynamics during mammalian spermatogenesis remained unknown for nearly two decades after discovery of Fzo. To address this gap in knowledge, we investigate mitochondrial dynamics and mitophagy during mammalian spermatogenesis. We uncover essential roles for mitochondrial fusion (Chapter 2), mitochondrial fission (Chapter 3), and mitophagy (Chapter 4) during spermatogenesis and show that each of these mitochondrial quality control mechanisms regulates a distinct stage of germ cell development. Our analyses reveal requirements for mitochondrial fusion, fission, and mitophagy that correspond to the mitochondrial and metabolic needs of the developing germ cells.
We also investigate the role of mitochondrial fusion and fission in regulating subcellular mitochondrial domains upon fusion of a skeletal muscle stem cell with a myofiber (Chapter 5). Thus, the work presented in this thesis characterizes the in vivo role of mitochondrial dynamics in two systems: male germline development and skeletal muscle regeneration. However, we focus on the role of mitochondrial dynamics and mitophagy during male germline development.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/Z99Z92ZF, author = {Liu, Raymond}, title = {Mechanisms of Drp1 Recruitment to Mitochondria}, school = {California Institute of Technology}, year = {2017}, doi = {10.7907/Z99Z92ZF}, url = {https://resolver.caltech.edu/CaltechTHESIS:05312017-190514797}, abstract = {Dynamin-related protein 1 (Drp1) is a GTPase of the dynamin superfamily that catalyzes mitochondrial fission in the cell. Cytosolic Drp1 is recruited to mitochondria by receptors anchored to the outer mitochondrial membrane. Once there, Drp1 assembles into a complex around the mitochondrial circumference to drive division via a GTP-dependent constriction process. The four known receptors of Drp1 are Fis1, Mff, MiD51, and MiD49, but stable interactions between Drp1 and these proteins have not been established. In addition, though mounting evidence suggests these receptors have non-redundant roles in their interaction with Drp1, mechanistic details explaining these distinctions are lacking. Here we address these questions, and show that the Insert B domain of Drp1 inhibits its interaction with Mff. Removal of this domain stabilizes a complex of Drp1 and Mff in vitro. In addition, we show that Drp1 oligomerization is a requirement for Mff recruitment, but not for MiD51 or MiD49-mediated recruitment. Together the results suggest a model in which Drp1 recruitment to mitochondria is regulated by the oligomeric state of Drp1, such that Mff, MiD51, and MiD49 recruit different subpopulations of Drp1 from the cytosol.
With this model as a framework, we analyze the effect that a Drp1 R403C mutant, identified in several human patients presenting with encephalopathy and refractory epilepsy, has on mitochondrial morphology in cultured cells. We find that the loss of Drp1 oligomerization in these mutants impedes its ability to be recruited by Mff, leading to abnormal elongation of the mitochondrial population.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/Z96Q1V75, author = {Rojansky, Rebecca Bloom}, title = {A Core Mitophagic Machinery Promotes Selective Degradation of Paternal Mitochondria in Mouse Embryos and MEF Cells}, school = {California Institute of Technology}, year = {2016}, doi = {10.7907/Z96Q1V75}, url = {https://resolver.caltech.edu/CaltechTHESIS:04292016-193643737}, abstract = {The maternal mode of mitochondrial inheritance is conserved across mammalian species; however, little is known about how mitochondria from the sperm are eliminated from early mammalian embryos. Mitophagy, the regulated degradation of mitochondria in the lysosome, has been proposed as a possible mechanism. Mitophagy is an important means by which the cell responds to changes in mitochondrial fitness, and has been observed under a number of physiological and non-physiological circumstances, including, but not limited to, hypoxia, mitochondrial depolarization, mitochondrial fission, and erythrocyte differentiation.
Here we examine the core component of mitophagy proteins involved in three physiological states: respiration-induced mitophagy in cultured mouse fibroblasts, mitophagy of dysfunctional mitochondria in the absence of mitochondrial fusion, and degradation of paternal mitochondria in pre-implantation mouse embryos. We find that a common pathway is used for elimination of mitochondria, involving mitochondrial depolarization, and the E3 ubiquitin ligases PARKIN and MUL1. We find that PARKIN and MUL1 play partially redundant roles in elimination of paternal mitochondria that is also dependent on PINK1 kinase, the fission factor, FIS1, and the autophagy receptor, p62. We find that p62 is specifically recruited to defective mitochondria in fusion deficient cells by a mechanism independent of ubiquitin binding. Our results elucidate the molecular mechanism of strict maternal transmission of mitochondria and uncover a collaboration between MUL1 and PARKIN in mitophagy.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/1MN9-DR40, author = {Wang, Yun Elisabeth}, title = {Characterizing the Regulation of Mitochondrial Nucleoids}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/1MN9-DR40}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282014-004119763}, abstract = {Mitochondria contain a 16.6 kb circular genome encoding 13 proteins as well as mitochondrial tRNAs and rRNAs. Copies of the genome are organized into nucleoids containing both DNA and proteins, including the machinery required for mtDNA replication and transcription. Although mtDNA integrity is essential for cellular and organismal viability, regulation of proliferation of the mitochondrial genome is poorly understood. To elucidate the mechanisms behind this, we chose to study the interplay between mtDNA copy number and the proteins involved in mitochondrial fusion, another required function in cells. Strikingly, we found that mouse embryonic fibroblasts lacking fusion also had a mtDNA copy number deficit. To understand this phenomenon further, we analyzed the binding of mitochondrial transcription factor A, whose role in transcription, replication, and packaging of the genome is well-established and crucial for cellular maintenance. Using ChIP-seq, we were able to detect largely uniform, non-specific binding across the genome, with no occupancy in the known specific binding sites in the regulatory region. We did detect a single binding site directly upstream of a known origin of replication, suggesting that TFAM may play a direct role in replication. Finally, although TFAM has been previously shown to localize to the nuclear genome, we found no evidence for such binding sites in our system.
To further understand the regulation of mtDNA by other proteins, we analyzed publicly available ChIP-seq datasets from ENCODE, modENCODE, and mouseENCODE for evidence of nuclear transcription factor binding to the mitochondrial genome. We identified eight human transcription factors and three mouse transcription factors that demonstrated binding events with the classical strand asymmetrical morphology of classical binding sites. ChIP-seq is a powerful tool for understanding the interactions between proteins and the mitochondrial genome, and future studies promise to further the understanding of how mtDNA is regulated within the nucleoid.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, month = {July}, advisor = {Chan, David C.}, } @phdthesis{10.7907/J23G-KQ18, author = {Loson, Oliver Calvin}, title = {Regulation of Mitochondrial Division by the Drp1 Receptors}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/J23G-KQ18}, url = {https://resolver.caltech.edu/CaltechTHESIS:05072014-160207088}, abstract = {Mitochondria can remodel their membranes by fusing or dividing. These processes are required for the proper development and viability of multicellular organisms. At the cellular level, fusion is important for mitochondrial Ca2+ homeostasis, mitochondrial DNA maintenance, mitochondrial membrane potential, and respiration. Mitochondrial division, which is better known as fission, is important for apoptosis, mitophagy, and for the proper allocation of mitochondria to daughter cells during cellular division.
The functions of proteins involved in fission have been best characterized in the yeast model organism Sarccharomyces cerevisiae. Mitochondrial fission in mammals has some similarities. In both systems, a cytosolic dynamin-like protein, called Dnm1 in yeast and Drp1 in mammals, must be recruited to the mitochondrial surface and polymerized to promote membrane division. Recruitment of yeast Dnm1 requires only one mitochondrial outer membrane protein, named Fis1. Fis1 is conserved in mammals, but its importance for Drp1 recruitment is minor. In mammals, three other receptor proteins—Mff, MiD49, and MiD51—play a major role in recruiting Drp1 to mitochondria. Why mammals require three additional receptors, and whether they function together or separately, are fundamental questions for understanding the mechanism of mitochondrial fission in mammals.
We have determined that Mff, MiD49, or MiD51 can function independently of one another to recruit Drp1 to mitochondria. Fis1 plays a minor role in Drp1 recruitment, suggesting that the emergence of these additional receptors has replaced the system used by yeast. Additionally, we found that Fis1/Mff and the MiDs regulate Drp1 activity differentially. Fis1 and Mff promote constitutive mitochondrial fission, whereas the MiDs activate recruited Drp1 only during loss of respiration.
To better understand the function of the MiDs, we have determined the atomic structure of the cytoplasmic domain of MiD51, and performed a structure-function analysis of MiD49 based on its homology to MiD51. MiD51 adopts a nucleotidyl transferase fold, and binds ADP as a co-factor that is essential for its function. Both MiDs contain a loop segment that is not present in other nucleotidyl transferase proteins, and this loop is used to interact with Drp1 and to recruit it to mitochondria.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/E61Z-9C26, author = {Pham, Anh Hoang}, title = {Tools for Assessing Mitochondrial Dynamics in Mouse Tissues and Neurodegenerative Models}, school = {California Institute of Technology}, year = {2013}, doi = {10.7907/E61Z-9C26}, url = {https://resolver.caltech.edu/CaltechTHESIS:09012012-181931478}, abstract = {Mitochondria are dynamic organelles that undergo membrane fusion and fission and transport. The dynamic properties of mitochondria are important for regulating mitochondrial function. Defects in mitochondrial dynamics are linked to neurodegenerative diseases and affect the development of many tissues. To investigate the role of mitochondrial dynamics in diseases, versatile tools are needed to explore the physiology of these dynamic organelles in multiple tissues. Current tools for monitoring mitochondrial dynamics have been limited to studies in cell culture, which may be inadequate model systems for exploring the network of tissues. Here, we have generated mouse models for monitoring mitochondrial dynamics in a broad spectrum of tissues and cell types. The photoactivatable mitochondria (PhAMfloxed) line enables Cre-inducible expression of a mitochondrial targeted photoconvertible protein, Dendra2 (mito-Dendra2). In the PhAMexcised line, mito-Dendra2 is ubiquitously expressed to facilitate broad analysis of mitochondria at various developmental processes.
We have utilized these models to study mitochondrial dynamics in the nigrostriatal circuit of Parkinson’s disease (PD) and in the development of skeletal muscles. Increasing evidences implicate aberrant regulation of mitochondrial fusion and fission in models of PD. To assess the function of mitochondrial dynamics in the nigrostriatal circuit, we utilized transgenic techniques to abrogate mitochondrial fusion. We show that deletion of the Mfn2 leads to the degeneration of dopaminergic neurons and Parkinson’s-like features in mice. To elucidate the dynamic properties of mitochondria during muscle development, we established a platform for examining mitochondrial compartmentalization in skeletal muscles. This model system may yield clues to the role of mitochondrial dynamics in mitochondrial myopathies.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/7MXP-WS63, author = {Detmer, Scott A.}, title = {The Role of Mitofusin Proteins in Mitochondrial Fusion and Disease}, school = {California Institute of Technology}, year = {2007}, doi = {10.7907/7MXP-WS63}, url = {https://resolver.caltech.edu/CaltechETD:etd-04132007-181115}, abstract = {We have investigated the role of mitofusin proteins in mitochondrial fusion and Charcot-Marie-Tooth disease Type 2A (CMT2A). Mitofusins (Mfn1 and Mfn2) are required for mammalian mitochondrial fusion. In structure-function analysis, we have identified loss-of-function mutations in mitofusin GTPase and heptad-repeat domains that disrupt homotypic and heterotypic domain interactions. Mutations in Mfn2 cause CMT2A, a progressive peripheral neuropathy. We have functionally characterized Mfn2 disease mutations and find that wild-type Mfn1, but not Mfn2, can efficiently complement nonfunctional CMT2A alleles to restore mitochondrial fusion. This finding demonstrates the importance of Mfn1-Mfn2 heterooligomers and suggests that Mfn1 expression is important in determining the cell-type specificity of CMT2A. To study the consequences of an Mfn2 CMT2A allele in vivo, we generated transgenic mice that express Mfn2 T105M in motor neurons. These animals demonstrate gait impairments due to distal muscle loss, axonopathy and altered mitochondrial morphology and distribution in motor neurons. In a second approach, we have generated CMT2A knock-in mice by replacing the endogenous genomic Mfn2 with Mfn2 alleles L76P or R94Q. Preliminary characterizations suggest that heterozygous animals have no disease symptoms, but homozygous Mfn2 R94Q animals are severely affected. Together, these mouse models provide means to assess the pathology of Mfn2 CMT2A alleles and the role of mitochondrial dynamics in vivo.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/902F-8M09, author = {Griffin, Erik Edmund}, title = {Mechanisms of Mitochondrial Fusion and Fission}, school = {California Institute of Technology}, year = {2006}, doi = {10.7907/902F-8M09}, url = {https://resolver.caltech.edu/CaltechETD:etd-05242006-134647}, abstract = {We have studied the mechanisms of mitochondrial fusion and fission in S. cerevisiae. Using a proteomics-based approach, we have identified the WD domain protein Caf4p as a Fis1p binding partner that, along with its paralog Mdv1p, functions as a molecular adaptor between Fis1p and Dnm1p. This work defines a role for Mdv1p and Caf4p in the recruitment of Dnm1p to mitochondrial fission complexes. In a separate study, we focus on the role of Fzo1p during mitochondrial fusion. Fzo1p and its mammalian homologs, Mfn1 and Mfn2, are conserved transmembrane GTPases that are required for mitochondrial fusion. A structure/function analysis has established an essential role for three Fzo1p heptad repeat regions during mitochondrial fusion. Furthermore, we show that Fzo1p functions as an oligomer and forms critical interactions between the HRN/GTPase and HR1/HR2 regions. Finally, we have identified Om14p as a novel regulator of mitochondrial morphology. Om14p interacts with Fzo1p and Ugo1p and may be the first inhibitor of mitochondrial fusio}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Chan, David C.}, } @phdthesis{10.7907/EF19-GE34, author = {Murayama, Stanley Tetsu}, title = {I. Synthesis of dl-cis-Tetrahydroeremophilone. II. Studies on Systems Free from Angular Strain}, school = {California Institute of Technology}, year = {1969}, doi = {10.7907/EF19-GE34}, url = {https://resolver.caltech.edu/CaltechTHESIS:03232017-140452739}, abstract = {I. The total synthesis of racemic cis-tetrahydroeremophilone (17) has been achieved. The key feature of the synthesis was a solvolytic cyclization that proceeded through a symmetrical cation 41 and gave rise to two major products, one of which had the stereochemistry required for the synthesis of tetrahydroeremophilone.