Themes and structure
Mitochondrial oxidative damage plays a key role in cellular degeneration, aging and metabolic diseases. Our goal is to determine how damage is prevented or contained, how dysfunctional mitochondria are recognized and removed, and how mitochondrial networks participate in these processes.
We study two disease models in which oxidative damage to mitochondria play a key role in the development of pathology. In diabetes, nutrient-induced oxidative damage has been shown to be a major mediator of endocrine dysfunction and beta cell loss. In bone marrow, oxidative damage induced by iron and heme-intermediates, leads to the development of sideroblastic anemia and myelodysplastic syndrome.
Cellular imaging is central to our research and much effort is dedicated to developing of novel techniques for monitoring living cells under the microscope. The lab has enjoyed long term collaborations in both academia and industry. Funding is divided such that 25% is received from industry and the rest from NIH.
We are a very social group. Favorite activities include lab retreats such as kayaking, picnics, dinners or just hanging out together after work (see pictures). Over the years we have gathered a diverse group of people who worked in the lab and are still staying in touch with the people and the active research topics of our lab.
We study two disease models in which oxidative damage to mitochondria play a key role in the development of pathology. In diabetes, nutrient-induced oxidative damage has been shown to be a major mediator of endocrine dysfunction and beta cell loss. In bone marrow, oxidative damage induced by iron and heme-intermediates, leads to the development of sideroblastic anemia and myelodysplastic syndrome.
Cellular imaging is central to our research and much effort is dedicated to developing of novel techniques for monitoring living cells under the microscope. The lab has enjoyed long term collaborations in both academia and industry. Funding is divided such that 25% is received from industry and the rest from NIH.
We are a very social group. Favorite activities include lab retreats such as kayaking, picnics, dinners or just hanging out together after work (see pictures). Over the years we have gathered a diverse group of people who worked in the lab and are still staying in touch with the people and the active research topics of our lab.
A clonal beta cell stained with JC1.
This image by Jakob Wikstrom won a photography competition at the MBL, Woods Hole. JC1 tends to aggregate in the periphery of the cell, where its concentration builds up faster.
Diabetes
Induction of mitochondrial fusion can protect from nutrient-induced beta cell death
Mitochondria in β-cells play a key role as integrators of nutrient signals and insulin secretion. One significant manifestation of diabetes is the gradual reduction in mitochondrial capacity to produce signals in response to fuels. The cause of this gradual deterioration is not yet understood. Our goal is to understand the mechanisms that underlie deterioration of mitochondrial function during the development of β-cell dysfunction and diabetes.
We have shown that β-cells respond to the chronic exposure to high levels of glucose and fatty acids with a drastic reduction in mitochondrial networking through fusion and fission. This phenomenon precedes a gradual deterioration of mitochondrial function that is characterized by the generation of a subpopulation of mitochondria with reduced membrane potential. Remarkably, under these conditions, induction of mitochondrial fusion in the β-cell prevents the appearance of mitochondria with reduced membrane potential and protects from the detrimental effects of chronic exposure to a nutrient rich environment (see publications list: Molina et al. 2009)
We have shown that β-cells respond to the chronic exposure to high levels of glucose and fatty acids with a drastic reduction in mitochondrial networking through fusion and fission. This phenomenon precedes a gradual deterioration of mitochondrial function that is characterized by the generation of a subpopulation of mitochondria with reduced membrane potential. Remarkably, under these conditions, induction of mitochondrial fusion in the β-cell prevents the appearance of mitochondria with reduced membrane potential and protects from the detrimental effects of chronic exposure to a nutrient rich environment (see publications list: Molina et al. 2009)
Mitochondrial fusion, fission and autophagy:
A novel axis for quality control and an explanation for the long term, cumulative effect of diet on beta cell aging.
By tagging and tracking individual mitochondria in intact β-cells we discovered the existence of a quality control mechanism that relies on both fusion and fission. Following mitochondrial fission some daughter units depolarize. These units display a lower likelihood for subsequent fusion and are apparent targets of autophagy (see Twig et al. 2008). Moreover, this model predicts that the inhibition of mitochondrial dynamics (MtDy) by Gluco-lipo-toxicity (GLT) may have a cumulative effect and result in an increased portion of dysfunctional units over time. Such enrichment of dysfunctional mitochondria could explain the long lasting effect of GLT, a phenomenon that has been shown to impact animals’ prognosis many months after a high fat diet has been discontinued (see Liesa and Shirihai, 2013).
Life cycle of the mitochondrion
See Twig et al EMBO 2008; Liesa Shirihai Cell Metabolism 2013
Mitochondrial metabolic oscillations in normal and diabetic animals.
Oscillatory insulin secretion is a hallmark of a healthy response to nutrient intake. Early in the development of the disease, Type 2 diabetics have irregular insulin secretion oscillations. This observation puts the mechanism of β-cell oscillation and synchronization in the focus of diabetes research today. Recent studies provide increasing evidence that mitochondria function as the oscillator of the beta cell.
We hypothesized that oscillations in mitochondrial oxidative phosphorylation (OXPHOS) are synchronized across the pancreatic islet of Langerhans and that the regularity and orchestration of these oscillations are disrupted in diabetes.
We hypothesized that oscillations in mitochondrial oxidative phosphorylation (OXPHOS) are synchronized across the pancreatic islet of Langerhans and that the regularity and orchestration of these oscillations are disrupted in diabetes.
Islets stained with membrane potential sensitive dye Rhodamine 123
See Katzman et al 2004 AJP
To study mitochondrial metabolic oscillations and synchronization within the intact islet we utilize voltage sensitive fluorescent dyes and time lapse confocal / 2-photon microscopy. By monitoring the mitochondrial activity of multiple cells within an intact islet, we have shown that metabolic oscillations are coordinated across the islet. We found that the islet is composed of a metabolically heterogeneous population of cells, which respond to different glucose concentrations and oscillate in distinctive frequencies. Using a gerbil model for type II diabetes, we showed that the coordination and the regularity of the oscillations in mitochondrial activity are altered in diabetes.
To analyze a large population of cells we are currently developing a model system comprised of a dispersed islet on a cell chip, where the activity of several thousand cells can be monitored continuously. These studies are done in collaboration with Molecular Cytomics (see Technology section bellow).
To analyze a large population of cells we are currently developing a model system comprised of a dispersed islet on a cell chip, where the activity of several thousand cells can be monitored continuously. These studies are done in collaboration with Molecular Cytomics (see Technology section bellow).
Mitochondrial oxidative damage and the heme biosynthetic pathway.
While essential for OXPHOS, heme biosynthesis introduces three of the most effective generators of reactive oxygen species to the mitochondria, ALA, heme, and iron. Remarkably, OXPHOS and heme synthesis are co-dependent and thus mutations in mitochondrial DNA or increased mitochondrial oxidative damage result in defects in heme synthesis and the accumulation of iron. Differentiating red blood cells produce 85% of the body’s heme. As such, erythroid cells from bone marrow make a robust model in which the relationship between heme synthesis and oxidative damage can be investigated.
Our study focuses on two disease models: Myelodysplastic syndrome and anemia of chronic disease in the elderly. These are divided into three main projects as follows:
Our study focuses on two disease models: Myelodysplastic syndrome and anemia of chronic disease in the elderly. These are divided into three main projects as follows:
Mitochondrial transporters function to prevent oxidative damage.
We have identified two inner membrane transporters that are induced by GATA1 during erythroid differentiation: ABC-me (ABCB10) and UCP2. Animals deficient in each of these transporters show increased oxidative damage in heme producing cells (Hyde et al., 2012; Elorza et al., 2008). To explore their function in heme biosynthesis and protection form oxidative damage, we are using knockout mice as well as ABCB10 and UCP2 deficient cell culture models. We have established that differentiating bone marrow progenitor cells that are deficient in ABC-me have severe reduction in their ability to form mature red blood cells (Hyde et al., 2012). Furthemore, we have shown that ABCB10 is required to protect from oxidative stress associated with ischemia-reperfusion in the heart (Liesa et al., 2011). Experiments in the lab are focusing on the role of ABCB10 in heme production and protection from oxidative stress in erythroid and non-erythroid cells (reviewed in Liesa et al., 2013).
Technology and Collaborations
The study of heterogeneous populations of cells using the LiveCell Array technology.
To this end, and in collaboration with Molecular Cytomics, we have participated in the development of a revolutionary tool in cell biology, the LiveCell Array (see figure bellow). When used in combination with an imaging apparatus and analysis programs, the Array facilitates real-time image acquisition at the resolution of a single cell from thousands of cells per experiment.
In the case of beta cells each array is imaged before and after stimulation with glucose. After imaging, the array with the cells inside is returned to the incubator so that the same cells can be analyzed again after 24 hours incubation with a drug. Our goal is to identify and classify specific functional subpopulations of β-cells, and to define the molecular and pharmacological characteristics of each group.
Once specific cells have been identified, they may be extracted from the Array for downstream processing (cell expansion or single cell PCR). We use a micromanipulator to recover cells.
In the case of beta cells each array is imaged before and after stimulation with glucose. After imaging, the array with the cells inside is returned to the incubator so that the same cells can be analyzed again after 24 hours incubation with a drug. Our goal is to identify and classify specific functional subpopulations of β-cells, and to define the molecular and pharmacological characteristics of each group.
Once specific cells have been identified, they may be extracted from the Array for downstream processing (cell expansion or single cell PCR). We use a micromanipulator to recover cells.
Molecular Cytomics Cell Array
The cell array is transparent and can load 10,000 cells, including non-adherent cells. After loading, cells can be perfused with solutions for the purpose of staining or treatment, without being displaced. A dedicated imaging software stores the array coordinates so that each cell can be identified based on its location even after the array returns from a treatment or overnight incubation at the tissue culture incubator.
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Image analysis
In collaboration with Molecular Devices and with Molecular Cytomics, we have developed a number of image processing and analysis algorithms for the cell array and for images obtained by confocal microscopy. These applications allow for the analysis of large movies, where each frame undergoes numerous steps of processing and analysis.
One popular application that has been developed in the lab allows for the tracking and monitoring of mitochondrial membrane potential in individual mitochondria in intact cell over time. The application can process a movie of hundreds of images into a membrane potential time chart within a few seconds.
One popular application that has been developed in the lab allows for the tracking and monitoring of mitochondrial membrane potential in individual mitochondria in intact cell over time. The application can process a movie of hundreds of images into a membrane potential time chart within a few seconds.
Tagging and tracking mitochondrial networks in the living cell by photo-conversion of mitochondrial PA-GFP.
We have developed a technique that allows for the photolabeling of an individual mitochondrion within a living cell. The technique can determine the boundaries and size of the labeled mitochondria. It also enables the tracking of movement, interactions with other mitochondria through fusion, as well as membrane potential over time. Using this approach, we have characterized the life cycle of mitochondria and the criteria for selection of fusion mates.
Using a 2-Photon laser, specific mitochondria are photo-labeled and tracked. Red: Beta cell mitochondria, Green: Photo-converted GFP.
Respirometry Assays Developed at the Shirihai Lab
(Download detailed respirometry protocols for islets and isolated mitochondria at Tools - > Respirometry Protocols)
Our lab has developed different protocols to study mitochondrial function, using confocal microscopy, image analysis and respirometry. During 2009, Jakob Wikstrom M.D., Ph.D., developed the Islet Capture Microplate for the XF24 Analyzer, together with Seahorse Biosciences. This plate allows studying mitochondrial function in intact pancreatic islets and its real-time response to different metabolic fuels (see Figure 1). For further questions, please contact Sam Sereda (samsereda@gmail.com).
Our lab has developed different protocols to study mitochondrial function, using confocal microscopy, image analysis and respirometry. During 2009, Jakob Wikstrom M.D., Ph.D., developed the Islet Capture Microplate for the XF24 Analyzer, together with Seahorse Biosciences. This plate allows studying mitochondrial function in intact pancreatic islets and its real-time response to different metabolic fuels (see Figure 1). For further questions, please contact Sam Sereda (samsereda@gmail.com).
Figure 1. Respirometry of human islets. Trace showing % changes in OCR (oxygen consumption rates), three replicates ± SEM, related to basal oxygen consumption (5 mM glucose). The injection in the different ports is shown as a vertical line. A- 15 mM Glucose. B- 5 µM Oligomycin. C- 1 µM FCCP. D- 5 µM Rotenone/ Antimycin A
In addition, respirometry of isolated mitochondria is a very powerful methodology to determine mitochondrial function independently of changes in cellular mitochondrial mass and in extra-mitochondrial metabolism/transport of the fuels oxidized by mitochondria. Furthermore, it allows the study of mitochondrial oxidative phosphorylation at a high resolution level (i.e. the contribution of complex I, II to oxygen consumption and the capacity of complex IV). We have set up a protocol to measure oxygen consumption in isolated mitochondria using the XF24 (see Figure 2). This protocol has been developed by Marc Liesa Ph.D, together with a former member of our lab, Prof. Alvaro E. Elorza Ph.D, Seahorse Biosciences (George W. Rogers Ph.D., David Ferrick Ph,D), David Nicholls Ph.D., Martin Brand Ph.D. and Anne N. Murphy Ph.D. For further questions about this protocol please contact Marc Liesa Ph.D. (liesa@bu.edu) and/or Seahorse Biosciences (George W. Rogers Ph.D., www.seahorsebio.com).
Figure 2. Respirometry of isolated mitochondria from murine heart (20 µg of protein loaded per well). OCR (oxygen consumption rates, pmoles O2/ min). Average values of four replicates ± SEM. Basal: state II measurements in the presence of Pyruvate + Malate (P+M) 5mM. A- ADP 0.25 mM (state 3). B- Oligomycin 2 uM (state 4). C- DNP 100 µM (uncoupled). D- Antimycin 4 µM (non- OXPHOS respiration)
