FLUORESCENCE MICROSCOPY PROTEIN LABELING AND LOCALIZATION DNases, RNases, Proteinases siRNA

1. Greetings! Here are the lecture notes from today. Related information can be found in your book on the following pages. However, the information is not always as comprehensive so please look at the following slides for other details or examples. Pg 555-557 flourescence microscopy and immunoflourescence Pg 574-578 radioisotopes Pg 452 Figure 7-107 RNA interference Also, I will put a note on every slide to tell you what you should focus on when studying. Therefore you will have an idea of how you should study. I am interested in the major concepts that I present to you. STUDY GUIDE FOR TEST (the stuff I talked about in class): Review intro biology on central dogma, mitosis, meiosis, and cell cycle. Know main categories of life and viral genomes. Pay attention to viral intermediates. Vertical vs horizontal transmission Types of Gene duplications and how they are caused Orthologous and paralogous genes (Primate evolution tree example) Basic gene structure (promoters, coding regions) Microarray analysis

2. TECHNIQUES IN CELL AND MOLECULAR BIOLOGY The techniques presented here are used in all kinds of ways in research; thus it is important that you have a basic understanding of how they work. We will visit their use in context throughout the rest of the year. • FLUORESCENCE MICROSCOPY • PROTEIN LABELING AND LOCALIZATION • DNases, RNases, Proteinases • siRNA • RADIOLABELING OF NUCLEIC ACIDS • FLOW CYTOMETRY

3. Flourescence Microscopy All you need to know is that you can excite molecules at a wavelength and they emit at another wavelength. The filter used on the microscope allows you to look at one flourescent emission at a time. nobelprize.org

4. Immunofluorescence utilizes antibodies that have a specific target, i.e., proteins. www.serotec.com/ NOTE: Either primary or secondary antibodies can be tagged You can see where a protein is in a cell by using a flourescently labeled antibody that recognizes either a) your protein directly, or b) the antibody that binds to your protein (shown above).

5. HeLa Cells (HPV transformed) Primary Anti-tubulin mouse monoclonal Secondary goat anti-mouse conjugated to Alexa Fluor 568 TO-PRO-3 targets nucleus (DNA) This is an example of a primary antibody against tubulin and a secondary antibody with a fluorescent tag against the primary antibody. The nucleus lights up yellow because the cells have been stained with a flourescent tag that specifically binds DNA. http://www.microscopyu.com

6. Fluorescence microscopy utilizes a variety of natural and synthetic compounds: NATURAL: Phallotoxins are isolated from the mushroom, Amanita phalloides. They bind to and stabilize actin filaments by inhibiting depolymerization. Phalloidins and phallacidins are similar peptides, used more or less interchangeably to label filamentous actin. Wheat germ agglutinin (WGA), a lectin that binds sialic acid - an important constituent of glycoproteins and glycolipids. Green Flourescent Protein (GFP) is a spontaneously fluorescent protein isolated from the Pacific jellyfish, Aequoria victoria. SYNTHETIC: 4'-6-Diamidino-2-phenylindole (DAPI) is known to form fluorescent complexes with natural double-stranded DNA, showing a fluorescence specificity for AT, AU and IC clusters. Important information is underlined. The molecules here are used in all kinds of ways depending on what you want to look at. For example, if you want to know where a protein is in the cytoplasm, then perhaps using phalloidin conjugated with a flourescent molecule in ADDITION to your tagged protein (use a different flourescent molecule like GFP) is a way to PROVE it is in the cytoplasm (they co-localize).

7. GFP is a naturally occurring molecule found in jellyfish. It was originally identified at Friday Harbor Laboratories.

8. Rhodamine Phalloidin (actin) DAPI (DNA and RNA) Dr. Jalal Vakili VLST Corp. This is just an example of the use of Phalloidin conjugated to the flourescent molecule Rhadamine (you do not need to know the name); DAPI binds DNA to show you where the nucleus is at.

9. Plasma membrane localization of Beta-Catenin. Rhodamine immuno-fluorescence staining of methanol-fixed A-431 cells with a DAPI counterstain www.alzforum.org Here is an example of how one would use immunoflourescence to show where a protein is located in the cell (the plasma membrane in this case).

10. Nuclei were visualized with mouse anti-histones (core) primary antibodies, while the Golgi complex was stained with rabbit anti-giantin antibodies. Secondary antibodies were goat anti-mouse and anti-rabbit conjugated to Texas Red and Oregon Green 488, respectively to produce red nuclei and green Golgi cisternae. The filamentous actin network was counterstained with Alexa Fluor 350 conjugated to phalloidin. This is an example of how one would localize a protein that is thought to be in the Golgi. Note, that we know where these proteins localize, but in an experiment, that would be a hypothesis, particularly if you are working with a protein that you THINK localizes in the Golgi.

11. This is just showing you how merged images can be shown as one. www.gurdon.cam.ac.uk

12. This is another example of a merged image and what it would look like. DIC = differential interference contrast microscopy www.gurdon.cam.ac.uk

13. Figure 1, a triple-labeled Drosophila embryo at the cellular blastoderm stage. The specimen was immunofluorescently labeled with antibodies to three different proteins. After three corresponding images were collected in the red, green, and blue channels of the confocal system, the images could be rearranged by copying them to different channels. Illustrates the power of using flourescent molecules to look at gene expression. The proteins are transcription factors expressed in different areas of a developing embryo. http://www.microscopyu.com

14. Small intestine cross-section. Texas Red-X conjugated to wheat germ agglutinin (WGA), a lectin that binds to N-acetylglucosamine and N-acetylneuraminic (sialic acid) residues. Alexa Fluor 488 conjugated to phalloidin and DAPI. http://www.microscopyu.com/galleries One can use flourescence microscopy to look at tissues, such as a cross section of the small intestine.

15. Protein tagging Recombinantly created Synthetic epitopes/Antibody tags Flag CTag Chemical binders His tag Fluorescent tags Green fluorescent protein (GFP) Chemical modification Biotinyation • There are two main ways to tag proteins: • Create a tag by adding a sequence in frame with your protein, so when it is expressed it gives you a tagged protein (see next page). • Chemically modify it (just to let you know but do not memorize)

16. Protein Tagging Recombinant method Chemical modification Biotin molecule DNA encoding Protein X Fuse to synthetic sequence Linkage Chemistry Express protein These are just visual examples.

17. Synthetic epitopes • Recombinant epitope targeted by antibody • Flag tag Antibody can be conjugated to fluorescent tag or bound to a solid particle SO. . . You can track it with fluorescence or purify it! Some tags allow you to bind them to beads in order to purify them.

18. Synthetic epitopesTandem Affinity Purification (Tap) Tags A Tap tag is one such tag that allows you to purify the protein with beads.

19. Chemical Binding epitopes • PolyHistidine tag • Protein binds to resin conjugated to nickel Ni Ni Ni Ni Ni Multiple histidines at the end of the protein let you bind the protein to a Nickel column so you could purify it away from other proteins.

20. Fluorescent tags Green fluorescent protein (GFP) But there are other modified kinds too! UV photon Green photon Proteins can be tagged with the naturally occurring protein GFP. Thus you can visualize your protein in a cell. A flow cytometer can detect it as well.

21. DNASES Exonuclease I (ExoI) from E. coli degrades ssDNA in a 3'=>5' direction S1 nuclease is an endonuclease that is active against ssDNA and ssRNA DNase I, is an endonuclease acts on ssDNA and dsDNA, chromatin and RNA:DNA hybrids Applications: Degradation of DNA template in transcription reactions Removal of contaminating genomic DNA from RNA samples DNase I footprinting Nick Translation There are DNases that degrade DNA and they function on either single stranded or double stranded molecules.

22. RNASES RNase A is sequence specific for ssRNAs RNase I degrades all ssRNAs RNase H cleaves the RNA in a DNA/RNAduplex to produce ssDNA RNase P is unique from other RNases in that it is a ribozyme and functions to cleave off extra sequence of RNA on tRNA molecules RNase VI is non-sequence specific for dsRNAs RNase III is specific for dsRNA There are RNases that degrade RNA. Some recognize single stranded RNA, some recognize double stranded RNA, and others DNA/RNA duplexes.

23. 2006 Nobel Prize Winners for their discovery of RNA interference – gene silencing by double-stranded RNA Craig Mello Andrew Fire This is one example of why RNase is important. . .

24. This is one example of why RNase is important. . . RNA interference! It is activated by dsRNA and targets complementary mRNA. Thus, you can use it to “knockout” a gene within cells. Dicer is an RNase III Argonaute is part of RISC and has endonuclease activity, particularly against the complementary mRNA!

25. WHY DID THAT EVOLVE? • Innate immunity to incoming viruses that have a dsRNA intermediate • Family: Orthomyxoviridae (Genus Influenza) • Family: Picornaviruses (Rhinoviruses and Poliovirus) • Family: Reoviruses ( ) • NOTE: the genome is a perfect dsRNA molecule • B) Protection against double-stranded RNA from mobile genetic elements The life cycles of viruses often include dsRNA, and so it played a role in the evolution of DICER and RISC in RNA interference.

26. Proteases Details forthcoming! Basically, they degrade proteins. Serine proteases Threonine proteases Cysteine proteases Metalloproteases Aspartic acid proteases Glutamic acid proteases Just know that you can add a proteinase and degrade protein in your samples. This is important if you are wanting to study just the nucleic acids or something like that.

27. Radioisotopes and Autoradiography 32P-ATP, GTP, TTP, CTP, UTP is available for incorporation into nucleic acids (sometimes referred to as “labeling”) 35S available for incorporation into proteins Nucleic acids and proteins can be labeled with radioisotopes. We can use this procedure to a) track molecules in a cell, b) look at the molecules on a autoradiograph (film), or c) detect their presence in a sample. Alpha phosphate should be used in order to incorporate it into a strand of DNA or RNA.

28. Flow Cytometry • This technique allows us to measure: • Flourescent cells in a sample (example – GFP expressing cells or cells stained with DAPI that binds DNA) • The size and shape of the cell • One can even sort cells depending on if they are flourescent or not. • I drew a graph on the board of how this is used to measure a cell cycle profile. The y axis is cell number (total number of cells put through the machine) and the x- is DNA content. The stain is DAPI.