1.Advanced Cell Biology 2014 1nd Semester Department of Animal Science Chungbuk National University 2ndt Lecture

2. 1st week : Introduction 3rd week :Research Strategies For Cell Biology 5nd week : Nucleus, Transcription and Splicing 7nd week : Membrane and Channel 9nd week : Membrane Trafficking 11nd week : Cell Signaling 13nd week : Cytoskeleton 15nd week : Cell Cycle 3. Cell and complicated Machine : The ways to study Cells 4. Component List What kinds of component are in the machine (or cell?) But How about the cell? 5. Component in the Cell : Protein (RNA) Genome Sequence : Now we have most of gene list in the genome But it does not means that we know the exact component list in a specific cells http://www.ncbi.nlm.nih.gov http://genome.ucsc.edu/ 6. Differences cells have different component (Proteins, RNA), although they have common genome How we can figure out the whole component list? 7. Expression profiling using Microarray or RNA-Seq What Kinds of mRNA is there? How much specific RNA is there? RT-qPCR or Northern Blot mRNA levels does not necessarily correlated with Protein Levels. 8. Nature, 2012 9. Proteomics ~less than 1,000 abundant Proteins 10. - We want to know where the specific component is located inside in the cell - We want to know whether two protein is interact each other in cell Immunofluorescence / Flurorescence reporter fusion Colocalization / Immunoprecipation - We want to see what will happen if the specific protein / RNA was devoided in cell RNAi Knockout (CRISPR/Cas9) Ectopic Expression of Dominant Negative Mutant Now lets assume that we have a list of component inside the cell. Now what? - We want to check how the shape of the protein looks like X-ray Crystallography 11. Immunoprecipitation 12. Optical Microscope : Main workhorse of Cell Research 13. Phase-Contrast Microscope Contrast-enhancing techniques 14. Differential-Interference-Contrast Microscope 15. Fluorescences Fluorescence : some molecules can absorb one color and emits different colors 16. Fluorescences Microscope 17. Filters: the key to successful fluorescence microscopy 18. Staining of different components of the cell Q : We want to localize the location of a specific protein in cell. How we can do that? A : Use Antibody! By labeling antibody with fluorescence, you can locate the desired protein in 19. Alpha-Tubulin Actin Mitochondria Synaptic Vesicle 20. Immunofluorescence - Direct Immunofluoresence * Antibody (or chemical) which can bind a desired protein is labeled with fluorochrome * Pros - Convienient - More Sensitive * Cons - You should have a primary antibody labeled with fluorochrome - If you dont have it, you should do it by yourself or use Indirect Immunofluoresence 21. Alexa-Phalloidin Phalloidin : Actin binding Chemical 22. - Indirect Immunofluoresence * Unlabeled Antibody is applied on the fixed tissue * Antibody was detected by secondary antibody conjugated with fluorochrome Primary Antibody Recognize Antigen Secondary Antibody recognize Primary Antibody It is labeled by fluorescence Pros You dont need to label primary antibody Based on the selection of secondary antibody, you can change wavelength of signal Cons * More complicated (Two step process) 23. Fixation and Section We need to stop the cellular process and preserve the component inside in cell. Crosslinking Fixation Commonly used for luoresence microscopy Generate covalent cross-links between intracellular components Most commonly used agent : aldehyde Formation of bond between amine grouop Glutaraldehyde formaldehyde - Precipitating Fixatives : Disctrupt hydrophobic interaction Denature proteins Methanol, Ethanol, Acetic Acid 24. Colocalization Using two different fluorophore with different wavelength, we can test cellular locations of Two protein simultaneously. A B 25. Choice of fluorophore * Choice of two closely distributed spectrum may cause bleeding 26. Fluorescence Protein as Reporter GFP Gene of Interest - Drawbacks of immunoflorescences You need to have (specific and high-quality) antibody against your protein of interest You need to fix a cell (i.e. Dead Cells), so you cannot observe live event in live cell - You need a probe which will work in In the Living cell (and even organism) GFP(RFP) Your Gene of Interest Transfection 27. Time-Lapse Imaging of Live Cell 28. Confocal Microscopy The main problem in the florescence microscopy is that strong illumination background from other focal planes 29. Biochemical Characterization of Cell 30. Pertubation of Component Loss-of-function Study - Knockdown of functions for GOI (Gene of Interest) - Find a Phenotype caused by the Ablation of Gene Function - Find a function of Gene/Protein - Can be classified as - RNAi - Morpholino - Dominant Negative Mutant Gain-In-Function Study - Introduction / overexpression of GOI - Find a Phenotype caused by the (over)expression of Gene 31. RNAi (RNA interferences) Temporal knockdown of desired gene Loss-of function Study 32. Transfection 33. Genome Engineering and Knockout 34. Dominant Negative Mutant These Proteins are active only if they are exists as dimer.. If we express truncated form of mutant, they will be inactivated regardless of presence of wild type molecule Endogenous expression 35. Uses of dominant negative Rho family GTPases (Rac, Cdc42, Rho) 36. X-ray Crystallography 37. Protein Productions - You need to have enough (5-10mg) pure (at least 95% purity) protein - Overexpression (Bacteria or Insect Cell or Mammalian Cell) or Natural Source - Purification 38. Crystallization - Concentrate Proteins (at least 5mg/ml) - Crystallization happens in the boundary of soluble and precipitation 39. Strong X-ray generated from synchroton is essential 40. Raw Data : Diffraction Images of Protein Crystal (several hundreds) Computer Analysis 41. Final Structure and Interpretations 42. Do I need to know how to solve protein structure? - Not really (In most cases..) Do I need to know how to check my favorite protein? - Yes for sure. http://www.rcsb.org 43. OK. Lets check some random guys structure 44. In old days, you need very expensive workstation-level computer To visualize Protein Structure.. Not anymore. Cheap PC or even your smartphone can do that. 45. Pymol (http://www.pymol.org) Demo