|SES #||TOPICS||LECTURE SUMMARIES||READINGS|
|1||General Introduction||We will introduce ourselves and discuss our backgrounds. The student will talk about their reasons for taking the course and what they hope to get out of it. The instructors will give an overview of the syllabus and the aims of the course. Then we will present a background to the field of fluorescence imaging: what is fluorescence, why use fluorescence detection, what sort of chemical structures are fluorescent, and how is fluorescence observed? We will discuss the format of a primary research paper and suggest how to critically analyze a paper. We will introduce the two papers for the following week.|
|2||Discovery of Green Fluorescent Protein|| |
Green Fluorescent Protein (GFP) triggered a revolution in our understanding of gene expression and signaling in live cells because the protein was fluorescent in the absence of any other cofactor. Such cofactors, which are needed for the fluorescence of proteins such as phycoerythrin or phytochrome, are only synthesized in certain organisms and are hard to deliver from outside the cell. We will examine the historical background of the course by reading about the first purification and analysis of GFP and then the first demonstration that GFP would produce fluorescence when cloned into prokaryotic or eukaryotic hosts. This discovery opened the way for the general use of GFP fusions as markers for protein localization.
Note: The manuscript for your written assignment will be handed out at the end of this class.
Morise, H., O. Shimomura, F. H. Johnson, and J. Winant. "Intermolecular Energy Transfer in the Bioluminescent System of Aequorea." Biochemistry 13, no. 12 (June 4, 1974): 2656-62.
Chalfie, M., Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher. "Green Fluorescent Protein as a Marker for Gene Expression." Science 263, no. 5148 (February 11, 1994): 802-5.
|3||Fluorescent Protein Engineering||The Green Fluorescent Protein initially recovered from jellyfish was far from an ideal tool. It was dim, it did not fold at 37°C, and its fluorescence was pH-sensitive and not photostable. Gradually, by a combination of screening and directed evolution these faults were corrected. These approaches, along with the isolation of fluorescent proteins from other organisms, also allowed selection of fluorescent proteins spanning the spectrum from blue to red. This allowed tracking of multiple proteins at once in the same cell. We will discuss the crystal structure of GFP, which illustrates the chemical reaction that makes the protein fluorescent and provides a basis for rational engineering. We will then talk about the Herculean effort to make a red fluorescent protein suitable for tracking of cellular proteins.|| |
Yang, F., L. G. Moss, and G. N. Phillips, Jr. "The Molecular Structure of Green Fluorescent Protein." Nat Biotechnol 14, no. 10 (October 1996): 1246-51.
Campbell, RE., O. Tour, AE. Palmer, P. A. Steinbach, G. S. Baird, D. A. Zacharias, and R. Y. Tsien. "A Monomeric Red Fluorescent Protein." Proc Natl Acad Sci U.S.A. 99, no. 12 (June 11, 2002): 7877-82.
|4||Photoconversion of Fluorescent Proteins Variants to Probe Cellular Dynamics||Many fluorescent proteins emit at a single color, but a few special ones can emit at two colors or can be non-fluorescent in the natural state but turned on upon photoactivation. Fluorescence emission depends on the chemical environment near the chromophore, such as the 3D arrangement of amino acids and/or protonation state of certain amino acids. In this class, we will examine two fluorescent protein variants. One can change from green to red over time, while fluorescence of the other can be induced by irradiation with violet light. We will discuss how these fluorescent protein variants have been used to study gene expression during embryonic development and protein trafficking between lysosomes.|| |
Terskikh, A., A. Fradkov, G. Ermakova, A. Zaraisky, P. Tan, A. V. Kajava, X. Zhao, S. Lukyanov, M. Matz, S. Kim, I. Weissman, and P. Siebert. "Fluorescent Timer: Protein that Changes Color with Time." Science 290, no. 5496 (November 24, 2000): 1585-8.
Patterson, G. H., and J. Lippincott-Schwartz. "A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells." Science 297, no. 5588 (September 13, 2002): 1873-7.
|5||Labeling Probes Other than Fluorescent Proteins|| |
All methods of labeling and imaging proteins have weaknesses. Fluorescent fusion proteins are not ideal for every application. Their large size means that they can disrupt the function of the protein to which they are fused. Also, fluorescent proteins do not provide assistance for the targeting of small molecules such as dyes. A number of ingenious methods have been developed to target small molecules to specific peptides or proteins in living cells. Since there are thousands of proteins made up of the same amino acids, this is a great chemical challenge. In this class we will discuss one method of targeting that depends upon small-molecule recognition of a specific peptide sequence and another method that depends on an engineered enzyme-substrate interaction. From our discussion of these papers, we will attempt to define the features of an ideal system for cellular labeling.
Note: You are required to hand in your written assignment at the start of this class.
Griffin, B. A., S. R. Adams, and R. Y. Tsien. "Specific Covalent Labeling of Recombinant Protein Molecules Inside live Cells." Science 281, no. 5374 (July 10, 1998): 269-72.
Juillerat, A., C. Heinis, I. Sielaff, J. Barnikow, H. Jaccard, B. Kunz, A. Terskikh, and K. Johnsson. "Engineering Substrate Specificity of O6-Alkylguanine-DNA Alkyltransferase for Specific Protein Labeling in Living Cells." Chembiochem 6, no. 7 (July 2005): 1263-9.
|6||Visit to a Fluorescence Microscopy Facility||We will visit a facility for fluorescence microscopy in the MIT Center for Cancer Research to see how some of the principles covered in this course are put into practice. In particular, we will see live-cell imaging of cells expressing fluorescent proteins of different colors and examples using photoactivatable GFP fused to RNA-binding proteins (class 3 and 4). We will also see inorganic fluorophores that provide an alternative to fluorescent proteins for protein tracking (class 5). Practical issues of the microscope and software set-up for live-cell imaging will be discussed. We will also continue our discussions based on your written assignments.|
|7||Visualizing the Central Dogma of Molecular Biology|| |
In 1958, Francis Crick coined the term the "Central Dogma" to characterize the all-important cellular processes whereby DNA is "transcribed" into RNA, and RNA is "translated" into protein. Since then, researchers have typically examined individual aspects of the Central Dogma in isolation, by developing separate systems for studying transcription or translation. In this class, we will first discuss a system in which DNA, RNA and protein are tracked together in living cells by the ingenious use of fluorescent proteins. This technique reveals how the genome organization is changed upon transcriptional activation in mammalian cells. Then we will discuss an alternative method, using fluorescently labeled RNA "beacons" to track the migration of a specific mRNA required for embryonic development.
Janicki, S. M., T. Tsukamoto, S. E. Salghetti, W. P. Tansey, R. Sachidanandam, K. V. Prasanth, T. Ried, Y. Shav-Tal, E. Bertrand, R. H. Singer, and D. L. Spector. "From Silencing to Gene Expression: Real-Time Analysis in Single Cells." Cell 116, no. 5 (March 5, 2004): 683-98.
Bratu, D. P., B. J. Cha, M. M. Mhlanga, F. R. Kramer, and S. Tyagi. "Visualizing the Distribution and Transport of mRNAs in Living Cells." U.S.A. Proc Natl Acad Sci 100, no. 23 (November 11, 2003): 13308-13.
|8||Fluorescent Sensors of Cell Signaling: FRET|| |
Cell signaling occurs on the timescale of milliseconds and with spatial compartmentalization over micrometers. This is clearly illustrated by neurons, which can have rapid firing of one synapse while a thousand other synapses on the same neuron are silent. Conventional approaches to analyze signaling cascades involved fixing or freezing thousands of cells at a defined time-point and grinding up the cells for subsequent assays on the signaling molecules involved. With the use of fluorescent reporters, cell signaling events could be observed in living cells and living organisms in real time. The first fluorescent reporters of cell signaling were based on dyes that had to be microinjected into cells or diffuse into cells from the cell medium. With the advent of fluorescent proteins, a new generation of fluorescent reporters became possible that could be genetically targeted to specific cellular compartments or cell-types. These reporters depended upon the phenomenon of fluorescence resonance energy transfer (FRET). We will explain the principle of FRET and, based on the first paper, we will discuss how the first genetically-encoded FRET reporter was constructed. We will then see how later generation FRET reporters compare, in the context of imaging calcium changes as fruitflies contract their muscles.
Note: You are required to have chosen your paper for the Oral Assignment and submitted it for approval to the instructors by the beginning of the class.
Miyawaki, A., J. Llopis, R. Heim, J. M. McCaffery, J. A. Adams, M. Ikura, and RY. Tsien. "Fluorescent Indicators for Ca2+ based on Green Fluorescent Proteins and Calmodulin." Nature 388, no. 6645 (August 28, 1997): 882-7.
Reiff, D. F., A. Ihring, G. Guerrero, E. Y. Isacoff, M. Joesch, J. Nakai, and A. Borst. "In Vivo Performance of Genetically Encoded Indicators of Neural Activity in Flies." J Neurosci 25, no. 19 (May 11, 2005): 4766-78.
|9||Quantitative and Ultra-Sensitive Fluorescent Imaging||Cell biologists have sometimes been accused of producing only pretty pictures and drawing only qualitative conclusions. However, the direct fusion of GFP to gene of interest in a 1:1 ratio, providing sufficient signal without the need of amplification, forms a good basis for quantitative analysis. We will discuss how fluorescent protein fusion was used to quantify the movement of proteins throughout the nucleus and between nuclear compartments. Biologists conventionally study hundreds to millions of molecules at a time. Improvements in lasers, cameras, microscope design and fluorescent proteins mean that biologists are starting to explore the behavior of molecules one at a time. Such ultra-sensitive detection unveils new features of cellular behavior, including stochastic phenomena, and allows resolution well below that limited by the diffraction of light. We will illustrate ultra-sensitive detection with a paper that looks at the diffusion of single neurotransmitter receptors over the surface of a neuron with 45 nm accuracy.|| |
Phair, R. D., and T. Misteli. "High Mobility of Proteins in the Mammalian Cell Nucleus." Nature 404, no. 6778 (April 6, 2000): 604-9.
Tardin, C., L. Cognet, C. Bats, B. Lounis, and D. Choquet. "Direct Imaging of Lateral Movements of AMPA Receptors inside Synapses." EMBO J 22, no. 18 (September 15, 2003): 4656-65.
|10||Student Presentations||Each member of the course will present one paper on fluorescent imaging. The paper should be selected to demonstrate the strength of fluorescent imaging over other methods to address a biological question, such as gene expression or cell signaling. The length of the presentation will be around 10-20 minutes, followed by 3-5 minutes of questions and discussions. It is suggested that PowerPoint be used as a visual aid. The goal is to develop your ability to communicate science to your peers. The first aim of the presentation is to introduce the background of the paper and then explain clearly what experiments were done. You should propose one future experiment in your last slide.|
|11||Power of Fluorescent Imaging for High-throughput Analysis||High-throughput techniques, such as mass spectrometry and microarrays, have become standard tools to study the expression of multiple gene/gene products in one experiment. However, despite their usefulness, these methods only give information that is an average from a number of cells and cannot provide significant temporal or spatial resolution. The possibility to distinguish multiple colors from the same volume of space makes fluorescence-based imaging an ideal tool to study many cellular processes in parallel. In this class, we will examine two fluorescence-based imaging assays that can extract multiple parameters, including gene expression profiles, subcellular organization and cell cycle states, from each single cell in a high-throughput fashion.|| |
Wheeler, D. B., S. N. Bailey, D. A. Guertin, A. E. Carpenter, C. O. Higgins, and D. M. Sabatini. "RNAi Living-Cell Microarrays for Loss-Of-Function Screens in Drosophila Melanogaster Cells." Nat Methods 1, no. 2 (November 2004): 127-32. Epub (October 21, 2004).
Neumann, B., M. Held, U. Liebel, H. Erfle, P. Rogers, R. Pepperkok, and J. Ellenberg. "High-Throughput RNAi Screening by Time-Lapse Imaging of Live Human Cells." Nat Methods 3, no. 5 (May 2006): 385-90.
|12||Fluorescent Imaging in Living Organisms||Fluorescent proteins have often been used in isolated cells, to study dynamics of proteins and mRNA or to understand cell signaling. However, fluorescent proteins are also powerful tools in whole organisms to track cells and cell signaling. We will discuss the extra difficulty of fluorescent imaging in large organisms and how methods such as two-photon microscopy have helped to overcome some of these difficulties. We will compare the use of fluorescence imaging to other imaging methods, such as Magnetic Resonance Imaging (MRI) and Positron Emission Tomography (PET), which are used in hospitals. The first paper looks at the trafficking of T cells in a living mouse, to try to understand how the immune response is initiated. The second paper describes how GFP has been used to understand the fate of malaria parasites injected inside the body by a mosquito bite.|| |
Miller, M. J., S. H. Wei, I. Parker, and MD. Cahalan. "Two-Photon Imaging of Lymphocyte Motility and Antigen Response in Intact Lymph Node." Science 296, no. 5574 (June 7, 2002): 1869-73.
Amino, R., S. Thiberge, B. Martin, S. Celli, S. Shorte, F. Frischknecht, and R. Menard. "Quantitative Imaging of Plasmodium Transmission from Mosquito to Mammal." Nat Med 12, no. 2 (February 2006): 220-4.