Automatic morphological subtyping reveals new roles of caspases in mitochondrial dynamics.
ABSTRACT: Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology.
Project description:Mitochondria constantly fuse and divide to adapt organellar morphology to the cell's ever-changing physiological conditions. Little is known about the molecular mechanisms regulating mitochondrial dynamics. F-box proteins are subunits of both Skp1-Cullin-F-box (SCF) ubiquitin ligases and non-SCF complexes that regulate a large number of cellular processes. Here, we analyzed the roles of two yeast F-box proteins, Mfb1 and Mdm30, in mitochondrial dynamics. Mfb1 is a novel mitochondria-associated F-box protein. Mitochondria in mutants lacking Mfb1 are fusion competent, but they form aberrant aggregates of interconnected tubules. In contrast, mitochondria in mutants lacking Mdm30 are highly fragmented due to a defect in mitochondrial fusion. Fragmented mitochondria are docked but nonfused in Deltamdm30 cells. Mitochondrial fusion is also blocked during sporulation of homozygous diploid mutants lacking Mdm30, leading to a mitochondrial inheritance defect in ascospores. Mfb1 and Mdm30 exert nonredundant functions and likely have different target proteins. Because defects in F-box protein mutants could not be mimicked by depletion of SCF complex and proteasome core subunits, additional yet unknown factors are likely involved in regulating mitochondrial dynamics. We propose that mitochondria-associated F-box proteins Mfb1 and Mdm30 are key components of a complex machinery that regulates mitochondrial dynamics throughout yeast's entire life cycle.
Project description:Mitochondrial morphology and function are coupled in healthy cells, during pathological conditions and (adaptation to) endogenous and exogenous stress. In this sense mitochondrial shape can range from small globular compartments to complex filamentous networks, even within the same cell. Understanding how mitochondrial morphological changes (i.e. "mitochondrial dynamics") are linked to cellular (patho) physiology is currently the subject of intense study and requires detailed quantitative information. During the last decade, various computational approaches have been developed for automated 2-dimensional (2D) analysis of mitochondrial morphology and number in microscopy images. Although these strategies are well suited for analysis of adhering cells with a flat morphology they are not applicable for thicker cells, which require a three-dimensional (3D) image acquisition and analysis procedure. Here we developed and validated an automated image analysis algorithm allowing simultaneous 3D quantification of mitochondrial morphology and network properties in human endothelial cells (HUVECs). Cells expressing a mitochondria-targeted green fluorescence protein (mitoGFP) were visualized by 3D confocal microscopy and mitochondrial morphology was quantified using both the established 2D method and the new 3D strategy. We demonstrate that both analyses can be used to characterize and discriminate between various mitochondrial morphologies and network properties. However, the results from 2D and 3D analysis were not equivalent when filamentous mitochondria in normal HUVECs were compared with circular/spherical mitochondria in metabolically stressed HUVECs treated with rotenone (ROT). 2D quantification suggested that metabolic stress induced mitochondrial fragmentation and loss of biomass. In contrast, 3D analysis revealed that the mitochondrial network structure was dissolved without affecting the amount and size of the organelles. Thus, our results demonstrate that 3D imaging and quantification are crucial for proper understanding of mitochondrial shape and topology in non-flat cells. In summary, we here present an integrative method for unbiased 3D quantification of mitochondrial shape and network properties in mammalian cells.
Project description:Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.
Project description:Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.
Project description:The two striatal projection neuron subtypes (medium spiny neurons- MSNs), those enriched in dopamine receptor 1 versus 2 (D1-MSNs and D2-MSNs), display dichotomous properties at the level of the transcriptome, projections, morphology, and electrophysiology. Recent work illustrates dichotomous mitochondrial length in NAc MSN subtype dendrites after cocaine self-administration, with a shift toward smaller mitochondria, due to enhanced fission, occurring in D1-MSN dendrites and a shift toward larger mitochondria in D2-MSN dendrites. However, to date there has been no comparison of mitochondrial morphological properties between MSN subtypes. In this study, we examine mitochondrial morphology in NAc D1-MSNs versus D2-MSNs. We observe an increase in the frequency of smaller length mitochondria in D2-MSN dendrites relative to D1-MSN dendrites, while D1-MSN dendrites display an increase in larger length mitochondria. The differences in mitochondrial length occur in both NAc core and shell, although to a greater extent in NAc core. Finally, we demonstrate that the mitochondrial fusion molecule, Opa1, is differentially expressed in NAc MSN subtypes, with D1-MSNs displaying higher expression of Opa1 ribosome-associated mRNA. The difference in Opa1 levels may account for the bias toward enhanced smaller mitochondria in D2-MSNs and enhanced larger mitochondria in D1-MSNs. Collectively, our study demonstrates differential mitochondrial size and a potential molecular mediator of these mitochondrial differences in NAc MSN subtypes.
Project description:Mitochondria form intricate networks through fission and fusion events. Here, we identify mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively) anchored in the mitochondrial outer membrane. MiD49/51 form foci and rings around mitochondria similar to the fission mediator dynamin-related protein 1 (Drp1). MiD49/51 directly recruit Drp1 to the mitochondrial surface, whereas their knockdown reduces Drp1 association, leading to unopposed fusion. Overexpression of MiD49/51 seems to sequester Drp1 from functioning at mitochondria and cause fused tubules to associate with actin. Thus, MiD49/51 are new mediators of mitochondrial division affecting Drp1 action at mitochondria.
Project description:<h4>Aims/hypothesis</h4>Mitochondria frequently change their shapes by fusion and fission and these morphological dynamics play important roles in mitochondrial function and development as well as programmed cell death. The goal of this study is to investigate whether: (1) mitochondria in mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit increased fragmentation; and (2) chronic treatment with a superoxide anion (O(2)(-)) scavenger has a beneficial effect on mitochondrial fragmentation in MCECs.<h4>Methods</h4>MCECs were freshly isolated and lysed for protein measurement, or cultured to determine mitochondrial morphology and O(2)(-) production. For the ex vivo hyperglycaemia experiments, human coronary endothelial cells were used.<h4>Results</h4>Elongated mitochondrial tubules were observed in MCECs isolated from control mice, whereas mitochondria in MCECs from diabetic mice exhibited augmented fragmentation. The level of optic atrophy 1 (OPA1) protein, which leads to mitochondrial fusion, was significantly decreased, while dynamin-related protein 1 (DRP1), which leads to mitochondrial fission, was significantly increased in MCECs from diabetic mice. Diabetic MCECs exhibited significantly higher O(2)(-) concentrations in cytosol and mitochondria than control MCECs. Administration of the O(2)(-) scavenger TEMPOL to diabetic mice for 4 weeks led to a significant decrease in mitochondrial fragmentation without altering the levels of OPA1 and DRP1 proteins in MCECs. High-glucose treatment for 24 h significantly induced mitochondrial fragmentation, which was restored by TEMPOL treatment. In addition, excess O(2)(-) production, either in cytosol or in mitochondria, significantly increased mitochondrial fragmentation.<h4>Conclusions/interpretation</h4>These data suggest that lowering the O(2)(-) concentration can restore the morphological change in mitochondria and may help improve mitochondrial function in diabetic MCECs.
Project description:Although it is clear that mitochondrial morphogenesis is a complex process involving multiple proteins in eukaryotic cells, little is known about regulatory molecules that modulate mitochondrial network formation. Here, we report the identification of a new yeast mitochondrial morphology gene called MFB1 (YDR219C). MFB1 encodes an F-box protein family member, many of which function in Skp1-Cdc53/Cullin-F-box protein (SCF) ubiquitin ligase complexes. F-box proteins also act in non-SCF complexes whose functions are not well understood. Although cells lacking Mfb1p contain abnormally short mitochondrial tubules, Mfb1p is not essential for known pathways that determine mitochondrial morphology and dynamics. Mfb1p is peripherally associated with the mitochondrial surface. Coimmunoprecipitation assays reveal that Mfb1p interacts with Skp1p in an F-box-dependent manner. However, Mfb1p does not coimmunoprecipitate with Cdc53p. The F-box motif is not essential for Mfb1p-mediated mitochondrial network formation. These observations suggest that Mfb1p acts in a complex lacking Cdc53p required for mitochondrial morphogenesis. During budding, Mfb1p asymmetrically localizes to mother cell mitochondria. By contrast, Skp1p accumulates in the daughter cell cytoplasm. Mfb1p mother cell-specific asymmetry depends on the F-box motif, suggesting that Skp1p down-regulates Mfb1p mitochondrial association in buds. We propose that Mfb1p operates in a novel pathway regulating mitochondrial tubular connectivity.
Project description:Axonopathy is critical in the early pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Axonal swellings such as globules and spheroids are a distinct feature of axonopathy and our recent study showed that transgenic (tg) mice expressing DLB-linked P123H ?-synuclein (P123H ?S) were characterized by P123H ?S-immunoreactive axonal swellings (P123H ?S-globules). Therefore, the objectives of this study were to evaluate ?-synuclein (?S)-immunoreactive axonal swellings (?S-globules) in the brains of tg mice expressing human wild-type ?S and to compare them with the globules in P123H ?S tg mice.In ?S tg mice, ?S-globules were formed in an age-dependent manner in various brain regions, including the thalamus and basal ganglia. These globules were composed of autophagosome-like membranous structures and were reminiscent of P123H ?S-globules in P123H ?S tg mice. In the ?S-globules, frequent clustering and deformation of mitochondria were observed. These changes were associated with oxidative stress, based on staining of nitrated ?S and 4-hydroxy-2-nonenal (4-HNE). In accord with the absence of mitochondria in the P123H ?S-globules, staining of nitrated ?S and 4-HNE in these globules was weaker than that for ?S-globules. Leucine-rich repeat kinase 2 (LRRK2), the PARK8 of familial PD, was detected exclusively in ?S-globules, suggesting a specific role of this molecule in these globules.Lysosomal pathology was similarly observed for both ?S- and P123H ?S-globules, while oxidative stress was associated with the ?S-globules, and to a lesser extent with the P123H ?S-globules. Other pathologies, such as mitochondrial alteration and LRRK2 accumulation, were exclusively detected for ?S-globules. Collectively, both ?S- and P123H ?S-globules were formed through similar but distinct pathogenic mechanisms. Our findings suggest that synuclein family members might contribute to diverse axonal pathologies.
Project description:Mitochondria are dynamic organelles that divide and fuse to modulate their number and shape. We have previously reported that the loss of dynamin-related protein 1 (Drp1), which mediates mitochondrial division, leads to the degeneration of cerebellar Purkinje cells in mice. Because Drp1 has been shown to be important for apoptosis and necroptosis, it is puzzling how Purkinje neurons die in the absence of Drp1. In this study, we tested whether neurodegeneration involves necrotic cell death by generating Purkinje cell-specific Drp1-knockout (KO) mice that lack the receptor-interacting protein kinase 3 (Rip3), which regulates necroptosis. We found that the loss of Rip3 significantly delays the degeneration of Drp1-KO Purkinje neurons. In addition, before neurodegeneration, mitochondrial tubules elongate because of unopposed fusion and subsequently become large spheres as a result of oxidative damage. Surprisingly, Rip3 loss also helps Drp1-KO Purkinje cells maintain the elongated morphology of the mitochondrial tubules. These data suggest that Rip3 plays a role in neurodegeneration and mitochondrial morphology in the absence of mitochondrial division.