Orian Shirihai Lab

(visit Orian Shirihai's personal webpage or send an email to Orian Shirihai)

Themes and structure:

Mitochondrial oxidative damage plays a key role in 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.

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.

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. 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.

Life cycle of the mitochondrion (See Twig et al EMBO 2008)

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 II 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.

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).

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 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:

Mitochondrial transporters of heme- producing cells 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. To explore their function in heme biosynthesis, we are using knockout mice as well as ABC-me 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. Experiments in the lab are focusing on the role of ABCme in heme production. To our surprise we found that UCP2, on the contrary, does not influence heme biosynthesis. Rather, it is a regulator of cell expansion during differentiation. 

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.

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.

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.

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.

BEND program.

Since 2004, Dr. Shirihai has been serving as the co-director of a program that exposes engineering students to the challenges of medical research. This program has led to a number of fruitful projects that have generated new tools for cell based research. These include image analysis programming, a perfusion apparatus for the LiveCell Array, and automation of individual cell picking from the Array.

RESPIROMETRY ASSAYS DEVELOPED AT THE SHIRIHAI LAB

(Download detailed respirometry protocols for islets here and for isolated mitochondria here)

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).x

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)

The People in Shirihai lab

Orian Shirihai Associate Professor	Linsey Stiles Ph.D Student	Fernanda M. Cerqueira PhD student
Anthony Molina Post Doctoral position	Jakob Wikstrom Post Doctoral position	Guy Las Post Doctoral position
Wei Qiu Ph.D Student	Marc Liesa Roig Post Doctoral position	Vered Levy Molecular Cytomics affiliate
	Prof. Daniel Dagan Consultant

Shirihai Lab ‘Alumni’

Gil Walzer Lab Manager	Hibo Mohamed Ph.D	Tal Drori Summer Projects
Solomon Graf	Shana Katzman	Gilad Twig MD/Ph.D
Sarah Haigh Ph.D	Erica Corson	Prof. Alvaro Andres Elorza Godoy, Ph.D
	Brigham Hyde, Ph.D President/Director of Licensing at Relay Technology Management Inc.

Publications

1. Las G, Shirihai O S. The role of autophagy in beta-cell lipotoxicity and type 2 diabetes. Diabetes Obes Metab 2010; (12 Suppl 2): 15-19.
   

2. Sahin E, Colla S, Liesa M, Moslehi J, Muller F L, Guo M, Cooper M, Kotton D, Fabian A J, Walkey C, Maser R S, Tonon G, Foerster F, Xiong R, Wang Y A, Shukla S A, Jaskelioff M, Martin E S, Heffernan T P, Protopopov A, Ivanova E, Mahoney J E, Kost-Alimova M, Perry S R, Bronson R, Liao R, Mulligan R, Shirihai O S, Chin L, Depinho R A. Telomere dysfunction induces metabolic and mitochondrial compromise. Nature 2011.
   

3. Twig G, Shirihai O S. The interplay between mitochondrial dynamics and mitophagy. Antioxid Redox Signal 2010.
   

4. Gurumurthy S, Xie S Z, Alagesan B, Kim J, Yusuf R Z, Saez B, Tzatsos A, Ozsolak F, Milos P, Ferrari F, Park P J, Shirihai O S, Scadden D T, Bardeesy N. The Lkb1 metabolic sensor maintains haematopoietic stem cell survival. Nature 2010; (468): 659-663.
   

5. Twig G, Liu X, Liesa M, Wikstrom J D, Molina A J, Las G, Yaniv G, Hajnoczky G, Shirihai O S. Biophysical properties of mitochondrial fusion events in pancreatic beta-cells and cardiac cells unravel potential control mechanisms of its selectivity. Am J Physiol Cell Physiol 2010; (299): C477-C487.
   

6. Zhong L, D'Urso A, Toiber D, Sebastian C, Henry R E, Vadysirisack D D, Guimaraes A, Marinelli B, Wikstrom J D, Nir T, Clish C B, Vaitheesvaran B, Iliopoulos O, Kurland I, Dor Y, Weissleder R, Shirihai O S, Ellisen L W, Espinosa J M, Mostoslavsky R. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 2010; (140): 280-293.
   

7. Hyde B B, Twig G, Shirihai O S. Organellar vs cellular control of mitochondrial dynamics. Semin Cell Dev Biol 2010; (21): 575-581.
   

8. Wang T, Si Y, Shirihai O S, Si H, Schultz V, Corkey R F, Hu L, Deeney J T, Guo W, Corkey B E. Respiration in adipocytes is inhibited by reactive oxygen species. Obesity (Silver Spring) 2010; (18): 1493-1502.
   

9. Liu S, Okada T, Assmann A, Soto J, Liew C W, Bugger H, Shirihai O S, Abel E D, Kulkarni R N. Insulin signaling regulates mitochondrial function in pancreatic beta-cells. PLoS One 2009; (4): e7983.
   

10. Tagen M, Elorza A, Kempuraj D, Boucher W, Kepley C L, Shirihai O S, Theoharides T C. Mitochondrial uncoupling protein 2 inhibits mast cell activation and reduces histamine content. J Immunol 2009; (183): 6313-6319.
    

11. Chen W, Paradkar P N, Li L, Pierce E L, Langer N B, Takahashi-Makise N, Hyde B B, Shirihai O S, Ward D M, Kaplan J, Paw B H. Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria. Proc Natl Acad Sci U S A 2009; (106): 16263-16268.
    

12. Liu X, Weaver D, Shirihai O, Hajnoczky G. Mitochondrial 'kiss-and-run': interplay between mitochondrial motility and fusion-fission dynamics. EMBO J 2009; (28): 3074-3089.
    

13. Getty-Kaushik L, Richard A M, Deeney J T, Krawczyk S, Shirihai O, Corkey B E. The CB1 antagonist rimonabant decreases insulin hypersecretion in rat pancreatic islets. Obesity (Silver Spring) 2009; (17): 1856-1860.
    

14. Molina A J, Wikstrom J D, Stiles L, Las G, Mohamed H, Elorza A, Walzer G, Twig G, Katz S, Corkey B E, Shirihai O S. Mitochondrial networking protects beta-cells from nutrient-induced apoptosis. Diabetes 2009; (58): 2303-2315.
    

15. Wikstrom J D, Twig G, Shirihai O S. What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy? Int J Biochem Cell Biol 2009; (41): 1914-1927.
    

16. Molina A J, Shirihai O S. Monitoring mitochondrial dynamics with photoactivatable green fluorescent protein. Methods Enzymol 2009; (457): 289-304.
    

17. Mouli P K, Twig G, Shirihai O S. Frequency and selectivity of mitochondrial fusion are key to its quality maintenance function. Biophys J 2009; (96): 3509-3518.
    

18. Elorza A, Hyde B, Mikkola H K, Collins S, Shirihai O S. UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis. J Biol Chem 2008; (283): 30461-30470.
    

19. Twig G, Hyde B, Shirihai O S. Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim Biophys Acta 2008; (1777): 1092-1097.
    

20. Danial N N, Walensky L D, Zhang C Y, Choi C S, Fisher J K, Molina A J, Datta S R, Pitter K L, Bird G H, Wikstrom J D, Deeney J T, Robertson K, Morash J, Kulkarni A, Neschen S, Kim S, Greenberg M E, Corkey B E, Shirihai O S, Shulman G I, Lowell B B, Korsmeyer S J. Dual role of proapoptotic BAD in insulin secretion and beta cell survival. Nat Med 2008; (14): 144-153.
    

21. Twig G, Elorza A, Molina A J, Mohamed H, Wikstrom J D, Walzer G, Stiles L, Haigh S E, Katz S, Las G, Alroy J, Wu M, Py B F, Yuan J, Deeney J T, Corkey B E, Shirihai O S. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J 2008; (27): 433-446.
    

22. Haigh S E, Twig G, Molina A A, Wikstrom J D, Deutsch M, Shirihai O S. PA-GFP: a window into the subcellular adventures of the individual mitochondrion. Novartis Found Symp 2007; (287): 21-36.
    

23. Wikstrom J D, Katzman S M, Mohamed H, Twig G, Graf S A, Heart E, Molina A J, Corkey B E, de Vargas L M, Danial N N, Collins S, Shirihai O S. beta-Cell mitochondria exhibit membrane potential heterogeneity that can be altered by stimulatory or toxic fuel levels. Diabetes 2007; (56): 2569-2578.
    

24. Sheftel A D, Zhang A S, Brown C, Shirihai O S, Ponka P. Direct interorganellar transfer of iron from endosome to mitochondrion. Blood 2007; (110): 125-132.
    

25. Heart E, Yaney G C, Corkey R F, Schultz V, Luc E, Liu L, Deeney J T, Shirihai O, Tornheim K, Smith P J, Corkey B E. Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose. Biochem J 2007; (403): 197-205.
    

26. Deutsch M, Deutsch A, Shirihai O, Hurevich I, Afrimzon E, Shafran Y, Zurgil N. A novel miniature cell retainer for correlative high-content analysis of individual untethered non-adherent cells. Lab Chip 2006; (6): 995-1000.
    

27. Twig G, Graf S A, Wikstrom J D, Mohamed H, Haigh S E, Elorza A, Deutsch M, Zurgil N, Reynolds N, Shirihai O S. Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP. Am J Physiol Cell Physiol 2006; (291): C176-C184.
    

28. Heart E, Corkey R F, Wikstrom J D, Shirihai O S, Corkey B E. Glucose-dependent increase in mitochondrial membrane potential, but not cytoplasmic calcium, correlates with insulin secretion in single islet cells. Am J Physiol Endocrinol Metab 2006; (290): E143-E148.
    

29. Twig G, Graf S A, Messerli M A, Smith P J, Yoo S H, Shirihai O S. Synergistic amplification of beta-amyloid- and interferon-gamma-induced microglial neurotoxic response by the senile plaque component chromogranin A. Am J Physiol Cell Physiol 2005; (288): C169-C175.
    

30. Katzman S M, Messerli M A, Barry D T, Grossman A, Harel T, Wikstrom J D, Corkey B E, Smith P J, Shirihai O S. Mitochondrial metabolism reveals a functional architecture in intact islets of Langerhans from normal and diabetic Psammomys obesus. Am J Physiol Endocrinol Metab 2004; (287): E1090-E1099.
    

31. Graf S A, Haigh S E, Corson E D, Shirihai O S. Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me). J Biol Chem 2004; (279): 42954-42963.
    

32. Best J L, Ganiatsas S, Agarwal S, Changou A, Salomoni P, Shirihai O, Meluh P B, Pandolfi P P, Zon L I. SUMO-1 protease-1 regulates gene transcription through PML. Mol Cell 2002; (10): 843-855.
    

33. Twig G, Jung S K, Messerli M A, Smith P J, Shirihai O S. Real-time detection of reactive oxygen intermediates from single microglial cells. Biol Bull 2001; (201): 261-262.
    

34. Shirihai O S, Gregory T, Yu C, Orkin S H, Weiss M J. ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation. EMBO J 2000; (19): 2492-2502.

Designed by Gil Walzer. Click here for contact information.