Lecture 1 – 1/8How do we figure out what an organelle does?- 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 assays- Combine immunology with microscopy : visualize where enzymes are in a cello Stain cells :a. cells need to be fixed – formaldehyde or glutaraldehyde are used as embalming fluid for cellsb. cells need to be permeabilized – treat with detergent o 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 only on DEAD/FIXED cells- Stain tissue for EM: 1. use a metal stain, such as Osmium tetroxide or Uranyl acetate2. need very thin sections - proteins can be localized to structures using immunogold labelingo gold particle linked primary antibody against antigen Ao 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 gold is e- dense and shows up dark on EMo problems : difficult to label to proteins at once, difficult to generate 3D view of structure, often need to count number of particles associated with a structure to demonstrate localization- Jellyfish - fluorescent protein that is green when excited with blue light (GFP)o Many colors—for co-localization need pairs whose spectra don’t overlap- 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 α - Screening for temperature sensitive lethal mutationso Add mutagen to yeast in liquid culture and distribute smaller aliquotso Incubate at 23C for 5hr, plate out individual aliquots, 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- MB 1. Fluid – accommodate changes in cell growth, shape, motility2. Selective barrier – maintains environment but imports energy, etc.o 50% lipids + 50% proteins- 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- 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 bleaching 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 - Regulation of Fluidity: homeoviscous Adaptation o Van der waals bond ~ 0.1 kcal Distance dependento Regular saturated bonds or longer side chains = higher area interacting = increased VDW forces = increased TTo Unsaturated side chains or short tails = can’t fully line up and distancebetween tails is larger = decreased VDW forces = decreased TT- Temp where MB goes from liquid to solid = transition temperature (TT)o At lower temperatures, the TT decreases and the MB remains fluid longer by adding double bonds or shorter tailso At higher temperatures, the TT increases and the MB remains gel longer by adding saturation or longer tails- Cholesterol o As much as 50% of MBo OH polar head group, rigid cyclic structure, nonpolar tailo H-bonding between cholesterol and lipid C=Oo Decreases permeability and moderates fluidity  Low temp : increases fluidity (acts as kink that blocks tight packing) High temp : decreases fluidity (due to rigid structure)Lecture 3 – 1/15o Lipids made on cytoplasmic face of ERo Flip-flop 10^5 X faster than predicted (must be able to flip flop so that there isn’t a build up of lipids on one side of MB) translocation proteins/flippases can be specific for head groupo flip PC (phosphatidylcholine – uncharged & large) but not PS (phosphatidylserine – neg. charged) Generates asymmetrieso PE: phosphatidylethanolamine – uncharged- Living cell: PC on outside, PE and PS insideo PS flippase uses ATP to flip PS to inside (to keep the asymmetry) ON in living cellso Scramblase lets things come to an equilibrium OFF in living cell- Dead cell: o ATP levels drop due to death, so PS flippase can’t worko Scramblase turns ON so that some of the PS inside can come outside and reaches equilibrium = cell death signalo Macrophages recognize these PS on the outside and eats these cells- 3 types of transport 1. Diffusion a. Small hydrophobic or nonpolar moleculesb. Even for these molecules, sometimes it’s too slow2. Passive : transport down [ ] gradienta. Uncharged molecules – [ ] gradient onlyb. Charged molecules – [ ] and charge affect transporto Electrochemical gradient – nucleic acids play key role in maintaining net negative charge inside cell o Donnan Effect : if equal amounts of Na on outside and inside & if there is neg. charged DNA on inside and Cl- on outside (net MB