Introduction to Biomedical Engineering – December 2008 How to visualize and study the kinetics of gene expression (transcription and mRNA processing) inside living cells with the highest spatial and temporal resolution? Biology Microscopy Eukaryotic Cell Transcription Translation SPLICING Splicing Splicing is a modification of a RNA molecule, in which non-coding sequences of pre-RNA, the sequences that have no expression, are removed. These sequences are called introns and the sequences that codify proteins are called exons. By Splicing, the pre-RNA becomes mRNA (the mature form), in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins. Gene Expression 1.Transcription 2.Translation Transcription occurs inside cell nucleus. The transcription is the synthesis of a RNA molecule from a DNA molecule,. First, a pre-RNA molecule is formed, complementary to the DNA molecule. This pre-RNA must be transformed to mRNA, by removing the parts that do not code for a protein – a process called splicing. Translation is the process by which the final transformation (mRNA to protein) takes place. It occurs in the cytoplasm, in an organelle of the cell – ribosome – that has the function to read the mRNA sequence. While reading takes place, the aminoacids which are being transported by tRNA connect to mRNA. mRNA is composed by nucleotides, and each sequence of three nucleotides (codon) codifies an aminoacid. At the end of Translation, the protein is formed. Problem Epifluorescence microscope Fluorescence GFP Structure Differences between Widefield and Confocal Confocal microscopy 3D Reconstruction Confocal microscopes are able to image optical slices in the sample, using a pinhole aperture that rejects most out-of-focus light. Laser scanning confocal microscopes are among the state of the art microscopy technologies available today. Confocal microscopes can be divided in single point scanning systems, line scanning systems and spinning disc. GFP-labeled rotavirus-derived particles STED (Stimulated Emission Depletion) FRAP FRAP (Fluorescence Recovery Afther Photobleaching) Confocal microscope Uses a pinhole placed in front of the detector, rejecting light outside plane of focus Scans the sample point by point and in different z-sections, constructs a 3D image adding all this sequential information Increases the resolution Decreases the excessive background New techniques Reduces the minimum resolvable details Increases the resolution Break the diffraction limit STORM (Stochastic Optical Reconstruction Microscopy) Image molecules with a high resolution Imaging single molecules Storm microscope – imaging single molecules Makes use of Photobleaching Measure the recovery of fluorescence as a function of time Alternative Splicing Imaging Splicing In IMM the main objective is to visualize gene expression in living cells (splicing). In order to visualize single gene expression sites within the nucleus of living cells, were generated human cells that contained 30 copies of a single gene integrated in their genome. These single genes are expressed when an antibiotic is present in the cell medium. To image the cells using fluorescence, each gene of interest originates mRNA which has a particular sequence specific for binding of a protein termed MS2. This protein is not generated endogenous by the cell. To visualize cells, MS2 is fused to a red fluorescent protein (mCherry – derived from GFP). mRNA is observed as a red dot in the nucleus of cell. In order to visualize the transcription itself, cells are also transfected with Polymerase II (Pol II) fused to GFP. Cells are also treated with an inhibitor of Endogenous Polymerase II. This treatment assures that Pol II – GFP is present in transcription and we are able to see a green dot in the nucleus of cell too. This green dot colonizes with the red dot inside the nucleus creating a yellow dot. Since Leeuwenhoek, microscopes are certainly one of the most useful and fundamental tools in what concerns biological research and clinical pathology. In fact, the examination of microscopic specimen opened way to many of the major discoveries ever made. Since Leeuwenhoek, microscopes are certainly one of the most useful and fundamental tools in what concerns biological research and clinical pathology. In fact, the examination of microscopic specimens opened the way to many of the major discoveries ever made. Fluorescence brought major improvements in optical microscopy, since living cells become nearly invisible in the brighfield mode and most stains are toxic. Two major applications are: Immunofluorescence GFP GFP can be fused to other proteins and co-expressed to intrinsically label proteins in cells. An Optical Microscope can be characterized by 3 main properties: Magnification: The ability to generate a magnified image of the specimen; Resolution: The ability to separate details in the image; Contrast: The capacity to makedetails visible to the eye, camera, or other image device. The Eukaryotic cell is the basic unit of life in complex organisms. One of the processes that occurs inside it is Gene Expression. Gene Expression consists in the translation of information encoded in a gene (in DNA) into a protein. This process occurs in two phases: Transcription and Translation. Alternative Splicing Resolution Contrast Fluorescence Difference in light intensity between the specimen image and the adjacent background relative to the overall background intensity. Shortest distance between two points on a specimen that can still be distinguished by the observer or camera system as separate entities. Optical phenomena, in which the molecular absorption of a photon triggers the emission of a photon with a longer (less energetic) wavelength. Fluorescence mechanism Jellyfish Aequoria victoria GFP (Green fluorescent protein) is derived from one species of jellyfish, Aequorea victoria. Several variants of GFP with different colors and properties have been generated from the wild type protein and are available today. Photobleaching is one of the biggest limitations in fluorescent microscopy. This effect is characterized by the permanent lost of fluorescence, after prolonged periods of fluorophore exciation by light. Sometimes takes place an Alternative Splicing event. In this case exons can be skipped, or extended, or introns can be retained and different mRNA is formed. These alterations in the structure of mRNA create a lot of diversity in animal’s proteins. In some cases however, alternative splicing causes some diseases like: cancer, autoimmune diseases, muscular atrophy and others. Relation with NA (Numerical Aperture) of the objective. Numerical aperture index of refraction of the medium half-angle of the maximum cone of light Conclusions: STED is very slow, not being compatible with the time of splicing  No better than Confocal STORM isn’t possible in living cells, so it can’t be applied to our problem, because it’s being studying the process while his occurrence. Confocal (FRAP) is the method of choice Researchers from all over the world are developing newer and better techniques to solve our problem In the future new developments will offer microscopy a better balance between contrast, resolution and magnification, because we are in fact only one step ahead from new exciting scientific discoveries Nucleus of Eukaryotic Cell marked with GFP and mCherry