[email protected]: Thursday Pacific Hall 2130 5-6pmMidterm (Feb 12 in class)Final (March 19 3pm-6pm location TBA)40% Midterm + 60% Final OR 100% FinalProblems sets every week discussed in section—enroll for section onlineGoogle RedditLecture 1 – 1/8How do we figure out what an organelle does?- Case study: peroxisome- Gel filtration chromatography: purify an enzyme o layer sample on column, add buffer to wash, collect fractionso Separate based on physical properties (size, charge, etc.)- Differential centrifugation: purify an organelleo Largest things pellet firsto If it’s too big, then you can use equilibrium density centrifugation Buoyant force opposes gravitational force which allows it to pellet- Fractionate cells and look for proteins/enzyme activities that co-migrate with organelleo Then measure enzyme activity via assayso Can determine where organelle is by EM if you don’t have protein marker- Combine immunology with microscopy : visualize where enzymes are in a cello Stain cells :1. cells need to be fixed – treat with a chemical denaturant so cell doesn’t fall apart during treatmenta. formaldehyde or glutaraldehyde are used as embalming fluid for cells2. cells need to be permeabilized – treat with detergent (pokes holes in MB) or if working with tissue, the tissue must be sectionedo Immunofluorescence :  rabbit makes primary antibody against the enzyme (antigen A) goat then makes secondary antibody against rabbit antibodies- so you can detect where the rabbit antibodies are located- simple and get signal amplification fluorescence : molecular absorption of photon causes emission of longer wavelength photon- allow you to localize many antibodies at the same time- 3 seemingly different genetic diseases: o Zellweger Syndrome, Neotonal Adrenoleukodystrophy, Infantil Refsum Diseaseo Common to all of those diseases is they all appear to have a defect in peroxisome – seen by immunofluorescenceo Can use EM to see if the organelle is not made, or if the catalase is not able to be transported into the organelle- Stain tissue for EM: 1. use a metal stain, such as Osmium tetroxide or Uranyl acetate—these are electron dense and will block the beam and appear dark2. need very thin sections since a piece of paper can completely block an electron beam- proteins can be localized to structures using immunogold labelingo gold particle linked primary antibody against antigen Ao gold particles are electron dense and show up as dark areas on the EMo beads cluster where the protein is found—can see if the protein is inside or outside an organelle (fluorescence just shows you that the enzyme is there)o problems : difficult to label two proteins at once, difficult to generate 3D view of structure, often need to count number of particles associated with a structure to demonstrate localization- ideally, we want to fluorescently label proteins in live cells o avoid fixation artifactso understand how molecules/structures move in real timeo KEY: jellyfish- Jellyfish make fluorescent protein that is green when excited with blue light (GFP)o Many colors—for co-localization, need pairs whose spectra do not overlapo Found eight filament forming proteins Ability to form filaments is not caused by fusion to GFP and the GFP fusions have no detectable phenotypes- Yeast genetics: o Wild type (a) and mutant type (alpha)o A x alpha  A/alpha diploido Sporulation of A/alpha  2 wildtype haploid A + 2 mutant haploid alphao 2 haploidsdiploid sporulation and haploids form again- Screening for temperature sensitive lethal mutations o Add mutagen to yeast in liquid culture and distribute into smaller aliquotso Incubate at 23C for 5hro Plate out individual aliquotso Incubate at 23Co Replica-plate (make copy of the plate) and incubateo growth at 23C and no growth when temp. is shifted to 36C shows where the temp. sensitive mutants are- Epistasis :o A mutant gives repressed reporter expressiono B mutant gives constitutive reporter expressiono To see which acts first, make a double A/B mutant If it leads to repressed expression, then A activates expression and B blocks A If it leads to constitutive expression, then B blocks expression and A blocks B- To find where an enzyme is in a cell: EM, immunogold labeling, or GFP Lecture 2 - 1/10- Important organelles have separate functions o Mitochondria (ATP), nucleus (DNA), ER/golgi (transport), lysosome (degradation)o Free radicals made from ATP synthesis are compartmentalized away from other molecules (such as DNA) which could be damaged by theseradicalso Proteases would breakdown cell if not contained in lysosome- MB 1. Fluid – accommodate changes in cell growth, shape, motility2. Selective barrier – maintains environment but imports energy, etc.o ~ 5 nm thicko 50% lipids + 50% proteinso lipids are amphipathic- Hydrogen bonds o Energy of ~ 1 kcalo Acetone: O of H2O can bond to C2 or H of H2O bonds to Oxygen H bonds are easily exchangedo Every time a nonpolar molecule can’t H-bond, it loses E because the H bonds between water are broken for no reason (forms 2 layers)o Water becomes highly ordered (cage-like) around hydrophobic molecules because you cant easily exchange the H bonds- Amphipathic o Single tail  conical shape Forms micelle (how detergent works)o 2 tails  cylindrical forms capsulated bilayer- MB determined to be fluid via FRAP (fluorescence recovery after photobleaching)o Lipids labeled with GFPbleach so there is a bleached arealook at recovery into that areao Graph shows slow recovery over time after bleachingo Shows rubid lateral and rotational movement Takes ~ 1 sec to get to other side of bacterial cell Takes ~ 20 sec to get to other side of Euk. Cell- Flip-Flopping via ESR (electron spin resonance)o Attach spin label (dye with unpaired electrons) to lipidso Measure spin label  quench with Vitamin C (reduces label on outside only)  measure spin over timeo Graph : when quenched the label reduces by half, then there is very slow quenching afterwards (linear downward towards zero)o Flip-flop once a month – lipid bilayer is 2D fluid- Regulation of Fluidity: homeoviscous Adaptation o Want constant fluidity, but affected by temperatureo Alter lipids to maintain fluidityo Van der waals bond ~ 0.1 kcal Distance dependento Regular saturated bonds or longer side chains = higher area interacting = increased VDW