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LESSONS IN CELL BIOLOGY Baltimore Excellence in STEM Teaching Project Summer 2011 BEST Project 1.

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Presentation on theme: "LESSONS IN CELL BIOLOGY Baltimore Excellence in STEM Teaching Project Summer 2011 BEST Project 1."— Presentation transcript:

1 LESSONS IN CELL BIOLOGY Baltimore Excellence in STEM Teaching Project Summer 2011 BEST Project 1

2 Subcellular Localization and Preliminary Functional Analysis of RNF 10 in the mitochondria of HeLa cells. Nicole Veltre and Digital Harbor High School and Dr. Mariusz Karbowski Center for Biomedical Engineering and Technology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 2

3 INTERNSHIP GOALS develop research and lab skills in cell biology translate laboratory experiences, techniques and cell biology concepts to a curriculum for high school students in various science courses deepen personal content literacy in biology. 3

4 This summer I worked in the lab of Dr. Mariusz Karbowski at Center for Biomedical Engineering and Technology and Department of Biochemistry and Molecular Biology at the University of Maryland School of Medicine, Baltimore, MD 4 Dr. Karbowski is a pioneer. Six years ago, no E3 ligases that work on the mitochondria were known. Now, through his work and the work of others who study mitochondria, there are several that have been proven to work on the outer membrane of the mitochondria.

5 The approaches that I learned this summer were cell culture and transfection, immunoflouresence and microscope imaging. I have also performed Western Blot to test the efficiency of the RNAi constructs that we were using. While Dr. Karbowski was there for every step, the person who really deserves the credit is Shan, because she has the patience of a saint. She and I are pictured here towards the end of my lab experience. 5

6 I also want to devote at least one slide to the book that my AP Biology students read this past summer, along with my scientist Dr. Mariusz Karbowski, The Immortal Life of Henrietta Lacks by Rebecca Skloot. I read this book 2 summers ago and was so moved by it I had my 10 th grade biology class read it last year for extra credit. My AP Biology students read it this past summer and I even convinced Dr. Karbowski to read it to. More on that later…. 6

7 7 There is a paragraph well into the book where Debra Lacks, Henrietta’s daughter, is pouring through biology books trying to figure out what it means that her mothers cells are alive. When I read this part, it in particular, resonated with me as it is how I feel about biology even now, after teaching it for over 10 year in Baltimore City. I want to absorb everything I can. And like Debra Lacks, I did not start out with very much biology content knowledge. It resonated with me even more just this past summer as I poured through the internet resources in order to understand just what I was doing in the lab.

8 MITOCHONDRIA Mitochondria are the powerhouses of most eukaryotic cells. The membrane-contained structures (organelles)that produce energy for cells in the form of ATP. This is accomplished by oxidizing the major products of glycolysis, in which energy is extracted from sugars. Mitochondria contain their own DNA and resemble bacterial cells in their structure, and probably arose by means of early cells engulfing bacteria-like ancestors. 8

9 MITOCHONDRIA In addition to energy production, mitochondria are also involved in programmed cell death (apoptosis), neuronal injury, cellular proliferation, regulation of the oxidative- reductive (redox) state of the cell, in synthesis of heme, the iron group in blood, and in steroid synthesis. 9

10 Parkinson's disease (PD) is neurodegenerative disorder that causes slowed movements, tremor, rigidity, and a wide variety of other symptoms. "Neurodegenerative" refers to the degeneration, or death, of neurons, the type of cell in the brain that is the basis for all brain activity. 10

11 Just like with our students, science becomes more meaningful when there is a personal connection. This past spring, a close friend and colleague let me know that he had been diagnosed with Parkinson’s. 11

12 RESEARCH RATIONALE RNF10 and Parkin act on mitochondria. Parkin is a protein which in humans is encoded by the PARK2 gene. Mutations in this gene are known to cause a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinson disease. 12

13 RESEARCH RATIONAL RING finger protein 10 is a protein that in humans is encoded by the RNF10 gene. The protein encoded by this gene contains a ring finger motif, which is known to be involved in protein-protein interactions. The specific function of this protein has not yet been determined. EST data suggests the existence of multiple alternatively spliced transcript variants, however, their full length nature is not known. [1] [1] 13

14 RESEARCH RATIONAL Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. 14

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16 Known Mitochondrial UB SYSTEM COMPONENTS Karbowski M, Youle RJ: Regulating mitochondrial outer membrane proteins by ubiquitination and proteasomal degradation. Curr Opin Cell Biol 2011, 23:

17 . 17 During my stay in Dr. Karbowski’s lab I worked on analyses of a novel, little characterized E3 ubiquitin ligase RNF10. Although this protein does not have TMD, I found that it is enriched in the mitochondria. To achieve this I performed immunofluorescence (using two distinct anti-RNF10 antibodies) and found a significant overlap between RNF10 and markers of mitochondria. You will see this overlap later in the slide presentation.

18 RESEARCH RATIONALE investigate the down regulation of RNF 10 methods to quantify downregulation- microimaging and immunofluorescence 18

19 E1 Yeast 1 Human~10 E2 Yeast 11 Human ~100 E3 Yeast 54 Human several hundreds Substrate recognition mediate rate-limiting steps in ubiquitin signaling. Target ubiquitin conjugation machineries to specific cellular locations. Redundant Specific Ubiquitin Protein+Ubiquitin Ubiquitin conjugation system: protein quality control

20 Quality control system Ubiquitination of the target proteins Retrotranslocation of the Ub-tagged protein out of the membrane Degradation by the proteasome in the cytosol

21 Other Work: 1. RNA interference mediated downregulation of RNF10 2. Western blot validation of RNF10 downregulation 3. Preliminary mitochondrial phenotype analysis (in progress)

22 RESEARCH PROTOCOLS culture immortal human HeLa cells expose cells to E3 Proteins treat cells with antibodies assay to detect and quantify the protein response elicited by the cells. 22 The picture below was taken with my own camera, through the lens of one of the lab microscopes. I learned this trick from my own students.

23 Cell Culture and Transfection HeLa cells were cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM Glutamax, 1 mM sodium pyruvate, MEM nonessential amino acids (Invitrogen, Carlsbad, CA), 100 U/ml penicillin, and 100 mg/ml streptomycin in 5% CO 2 at 37°C. Cells were transfected with FuGeneHD transfection reagent (Roche, Indianapolis, IN ), according to the manufacturer’s instructions. The fine-tuned transfection conditions resulted in at least 80% of cells being transfected. 23

24 Immunofluorescence ………. is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualisation of the distribution of the target molecule through the sample. Immunofluorescence is a widely used example of immunostaining and is a specific example of immunohistochemistry that makes use of fluorophores to visualise the location of the antibodies. [1] [1] 24

25 Immunofluorescence and fluorescence microscopy analyses were performed as previously described ( Benard et al., 2010 ). The primary antibodies used for immunofluorescence studies were anti-Tom20 or cytochrom c polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and poly and mono clonal RNF10 (Protein Group and Sigma). Immunofl uorescence labeling was performed as previously described ( Benard et al., 2010 ), with one modification. Prior to blocking with bovine serum albumin (BSA), nonspecific sites were blocked with ImageiTFX signal enhancer (Invitrogen). Immunofluorescence, fluorescence microscopy, and image analysis 25

26 We found that using this reagent prior to BSA blocking dramatically increased the signal-to-noise ratio in fluorescence images. For live cell imaging experiments, images were acquired using a Zeiss AxioObserver Z1 fluorescence microscope, equipped with a 100/1.45 a-Plan- FLUAR objective lens (Zeiss MicroImaging, Thornwood, NJ), an ApoTome unit (enabling high-resolution structured illumination image acquisition), environment control units (for temperature and pH control), a Definitive Focus module, and a high-sensitivity CCD camera (QuantEM 512SC; Photometrics, Tucson, AZ ). Immunofluorescence, fluorescence microscopy, and image analysis 26

27 Using this system, image acquisition took approximately 50–100 ms per channel, with low illumination levels enabling the acquisition of several hundred images without considerable photobleaching or cytotoxicity. Colocalization analyses were performed using the colocalization module of AxioVision 4 software (Zeiss MicroImaging). Several of these images appear on the next slide. Immunofluorescence, fluorescence microscopy, and image analysis 27

28 Treatment of Cells with RNF10 After plating, the cells were incubated at 37ºC/5%CO2 for h until a confluent monolayer had formed. The cells were then transfected with RNAi 1, RNAi 2 and GFP 28

29 Transfection is the process whereby the nucleic acid sequences are introduced by either biochemical or physical processes. Biochemical methods: DEAE-dextran, calcium phosphate, and liposome-mediated transfection methods. Physical transfection: direct micro-injection of materials, biolistic particle delivery and electroporation. Transfection =“transformation” + “infection”

30 Analysis 30 Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. [1] It is more specific than the general term in that spectrophotometry deals with visible light, near- ultraviolet, and near-infrared, but does not cover time- resolved spectroscopic techniques. [1] Lucky for me Dr. Karbowski has a Nano Drop, so once I calibrated the machine, all I had to do was drop one drop on the well to make the measurement. So easy a caveman could do it!

31 NANODROP RESULTS Data collection, recording, and analysis were significant components of the internship. These graphs reflect the levels of protein in each well. You need to load the same amount of protein in each well, so you must determine the concentration. If you don’t have similar concentrations of the cells, you probably shouldn’t run a gel. 31

32 Nanodrop analysis contiued… 32 In addition we analyzed the effect of RNF10 downregulation (mediated by RNAi) on the phenotype of mitochondria, which then becomes a good starting point for the next experiment. We look at the mitochondria in the absence of the normal levels of RNF10 and to investigate whether RNF10 downregulation affected mitochondrial distribution and subcellular organization. The microscope imaging showed the change in the structure of the mitochondria.

33 RNF10 Mito (cyt. c) overlay RNF10 subcellular localization: ANTIBODY #2 (polyclonal)

34 Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples. 1) Proteins are separated by gel electrophoresis 2) The proteins are transferred to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. 34

35 3) The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time. 4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed. 35

36 Figure 4 from Giovanni Benard et al. The EMBO Journal online publication 18 March 2010 doi: /emboj This gel image shows the downregulation of IBRDC2 which induces an abnormal accumulation of Bax. I was able to get similar results in my own research with RNF10 and could see very clearly on the gel where there was more RNF10. © 2010 European Molecular Biology Organization. Alot of things can and did go wrong between this step and the final results.

37 Novel mitochondria-associated E3 Ub ligase RNF10 RNF10 No transmembrane domain= Transient association !!! Previously identified mitochondrial E3 ligases RNF10 subcellular localization: ANTIBODY #1 (monoclonal)

38 Figure 1 from Giovanni Benard et al. The EMBO Journal online publication 18 March 2010 doi: /emboj Identification of IBRDC2, a different novel mitochondria- associated RING-finger protein. This is not from my summer research, but from research from my cooperating scientist, Dr. Mariusz Karbowski. © 2010 European Molecular Biology Organization.

39 CURRICULUM DEVELOPMENT Includes basic lab concepts and Literacy components. 39 The curricular activities I have developed incorporate laboratory techniques, internet research, and science skills/processes which are correlated with recognized by national and state science standards.

40 40 How much do you know about Parkinson’s disease? Test your grasp of Parkinson’s by identifying these seven statements as true or false. 1.Parkinson’s disease can be diagnosed with a blood test? 2.Memory loss and trouble thinking are early warning signs of Parkinson’s. 3.Parkinson’s disease is untreatable. 4.Environment can have an impact on Parkinson’s disease onset. 5.Parkinson’s disease usually develops later in life. 6.Women are more likely than men to suffer from Parkinson’s. 7.By the year 2030, one billion people on the planet will be over the age of 65.

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47 47 The end!!! At least for now…….


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