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.

The People in Shirihai lab

Orian Shirihai Associate Professor	Linsey Stiles Ph.D Student	Brigham Hyde, Ph.D Post doctoral fellow President/Director of Licensing at Relay Technology Management Inc.
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

Publications

1. Molina AJ, Shirihai OS.
   Monitoring mitochondrial dynamics with photoactivatable green fluorescent protein.
   Methods Enzymol. 2009;457:289-304.
   PMID: 19426874 [PubMed - in process]

2. Mouli PK, Twig G, Shirihai OS.
   Frequency and selectivity of mitochondrial fusion are key to its quality maintenance function.
   Biophys J. 2009 May 6;96(9):3509-18.
   PMID: 19413957 [PubMed - in process]
   

3. Elorza A, Hyde B, Mikkola HK, Collins S, Shirihai OS.
   UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis.
   J Biol Chem. 2008 Nov 7;283(45):30461-70. Epub 2008 Aug 7.
   PMID: 18687678

4. Twig G, Hyde B, Shirihai OS.
   Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view.
   Biochim Biophys Acta. 2008 Sep;1777(9):1092-7. Epub 2008 May 14. Review.
   PMID: 18519024

5. Danial NN, Walensky LD, Zhang CY, Choi CS, Fisher JK, Molina AJ, Datta SR, Pitter KL, Bird GH, Wikstrom JD, Deeney JT, Robertson K, Morash J, Kulkarni A, Neschen S, Kim S, Greenberg ME, Corkey BE, Shirihai OS, Shulman GI, Lowell BB, Korsmeyer SJ.
   Dual role of proapoptotic BAD in insulin secretion and beta cell survival.
   Nat Med. 2008 Feb;14(2):144-53. Epub 2008 Jan 27.
   PMID: 18223655

6. Twig G, Elorza A, Molina AJ, Mohamed H, Wikstrom JD, Walzer G, Stiles L, Haigh SE, Katz S, Las G, Alroy J, Wu M, Py BF, Yuan J, Deeney JT, Corkey BE, Shirihai OS.
   Fission and selective fusion govern mitochondrial segregation and elimination by autophagy.
   EMBO J. 2008 Jan 23;27(2):433-46. Epub 2008 Jan 17.
   PMID: 18200046 [PubMed - indexed for MEDLINE]

7. Haigh SE, Twig G, Molina AA, Wikstrom JD, Deutsch M, Shirihai OS.
   PA-GFP: a window into the subcellular adventures of the individual mitochondrion.
   Novartis Found Symp. 2007;287:21-36; discussion 36-46. Review.
   PMID: 18074629 [PubMed - indexed for MEDLINE]

8. Wikstrom JD, Katzman SM, Mohamed H, Twig G, Graf SA, Heart E, Molina AJ, Corkey BE, de Vargas LM, Danial NN, Collins S, Shirihai OS.
   beta-Cell mitochondria exhibit membrane potential heterogeneity that can be altered by stimulatory or toxic fuel levels.
   Diabetes. 2007 Oct;56(10):2569-78. Epub 2007 Aug 8.
   PMID: 17686943 [PubMed - indexed for MEDLINE]

9. Sheftel AD, Zhang AS, Brown CM, Shirihai OS, Ponka P.
   Direct interorganellar transfer of iron from endosome to mitochondrion.
   Blood. 2007 Mar 21; [Epub ahead of print]
   PMID: 17376890 [PubMed - as supplied by publisher]

10. Twig G, Graf SA, Wikstrom JD, Mohamed H, Haigh SE, Elorza A, Deutsch M, Zurgil N, Reynolds N, Shirihai OS.
    Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP.
    Am J Physiol Cell Physiol. 2006 Jul;291(1):C176-84. Epub 2006 Feb 15.
    PMID: 16481372 [PubMed - indexed for MEDLINE]

11. Heart E, Corkey RF, Wikstrom JD, Shirihai OS, Corkey BE.
    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 Jan;290(1):E143-E148. Epub 2005 Sep 6.
    PMID: 16144817 [PubMed - indexed for MEDLINE]

12. Twig G, Graf SA, Messerli MA, Smith PJ, Yoo SH, Shirihai OS.
    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 Jan;288(1):C169-75. Epub 2004 Sep 1.
    PMID: 15342341 [PubMed - indexed for MEDLINE]

13. Katzman SM, Messerli MA, Barry DT, Grossman A, Harel T, Wikstrom JD, Corkey BE, Smith PJ, Shirihai OS.
    Mitochondrial metabolism reveals a functional architecture in intact islets of Langerhans from normal and diabetic Psammomys obesus.
    Am J Physiol Endocrinol Metab. 2004 Dec;287(6):E1090-9. Epub 2004 Aug 31.
    PMID: 15339741 [PubMed - indexed for MEDLINE]

14. Graf SA, Haigh SE, Corson ED, Shirihai OS.
    Targeting, import, and dimerization of a mammalian mitochondrial ATP binding cassette (ABC) transporter, ABCB10 (ABC-me).
    J Biol Chem. 2004 Oct 8;279(41):42954-63. Epub 2004 Jun 23.
    PMID: 15215243 [PubMed - indexed for MEDLINE]

15. Twig G, Jung SK, Messerli MA, Smith PJ, Shirihai OS.
    Real-time detection of reactive oxygen intermediates from single microglial cells.
    Biol Bull. 2001 Oct;201(2):261-2. No abstract available.
    PMID: 11687412 [PubMed - indexed for MEDLINE]

16. Shirihai OS, Gregory T, Yu C, Orkin SH, Weiss MJ.
    ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation.
    EMBO J. 2000 Jun 1;19(11):2492-502.
    PMID: 10835348 [PubMed - indexed for MEDLINE]

Designed by Gil Walzer. Click here for contact information.