Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 361Bi 1 Lecture 10 Monday, April 17, 2005Diffusion and Molecular Motion in Biology;MicroscopesCDt =2Little Alberts Panel 1-1 Optical microscopy with unstained cellsbright-fieldphase-contrastdifferential-interference-contrast50 mSome microscopes detect the photons that have interacted with biological molecules. Some have been absorbed. Others have changed phase or velocity and can therefore interfere with photons of unchanged characteristics.3 m 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 DiffusionMechanical PumpsIntracellularExtracellular 1 m 1 mm“blood-brain barrier”Lecture 2 capillary spacingHow molecules move in the body4 DtrDtMC4expπ8223CDt =C = concentration of moleculeM = initial moles of moleculeD = diffusion coefficientt = timer = distance or radiusDiffusion from a Point SourceMath 2a will treat probability and statistics.Here’s a note from a previous core math course:“Distributions that should be your good buddies:Bionomial (see Bi 1 lecture 9)Normal = Gaussian (see below)Poisson”5time to spread, tmolecularweight(MW)Diffusion coefficient D(m)2 /msx = 0.1 m(~ synaptic cleft) sx = 10 m(~ single cell) msx = 1 mm(~ brain region) sO2 or Na+32 or 23 15 s50 500Neurotransmitteror low-MW drug200 0.317 s170 1700protein 50,000 0.150 s500 5000Some diffusion constants and distancesDtxxrms22CDt =6All I really need to know about lifeI learned in Bi 1 3. Most processes follow an exponential time course 4. Most processes end with a Gaussian distribution1. If you want a job done right, get a protein2. Electrical circuits explain many processes7cytosolreceptorcytosolsynaptic clefttransmitter moleculesreceptorreceptorDiffusion across the synaptic cleft takes a negligible time at synapsespresynaptic terminalpostsynaptic dendritedirection of information flow50 nm= 500 Å= 0.05 mDiffusion time: a few s8These proteins have evolved in a natural—perhaps necessary--way to provide that•The resting potential arises via selective permeability to K+This selective permeability also leads to the Nernst potential. Transient breakdowns in membrane potential are used as nerve signals.•Neuronal and non-neuronal cells also signal via transient influxes of Na+ and Ca2+.3 classes of proteins that transport ions across membranes:Little Alberts 12-4© Garland Ion channels that flux many ions per eventIon-coupled transporters“Active” pumps that split ATPfrom Lecture 59Ca2+ has a diffusion coefficient ~ 100-fold less than that of other ions in the cytosol, because Ca2+ spends 99% of its time bound to proteinsCa2+10Calcium-sensitive fluorescent dyesfluo-3fluo-311Little Alberts Panel 1-1 exciting light onlyemitted light onlybeam-splitting(“dichroic”)mirrorGreek,2 colors12Fluorescence measurements of a Ca2+ transient in a cell“false color”131/s1 + 1/s2 = 1/fL1 / L2 = s1/s2s2s1L1 L2Thin lens equations:It’s time to learn about microscopes141/s1 + 1/s2 = 1/fL1 / L2 = s1/s2s2s1L1 L2151/s1 + 1/s2 = 1/fL2 / L1 = s2/s1s2s1L1 L2= n sinHow to read a microscope objective lens160/0.1716Tutorial on magnification using the lens equationhttp://www.micro.magnet.fsu.edu/primer/java/lenses/magnify/index.html17http://www.micro.magnet.fsu.edu/primer/java/nuaperture/index.htmlFluorescence efficiency is proportional to the 4th power of numerical aperture18 m 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 No energy required:diffusionDiffusionMechanical PumpsIntracellularExtracellular 1 m 1 mm“blood-brain barrier”capillary spacing(sometimeswith fewer dimensions)How molecules move in the body197-Helix Receptors Coupled to G proteins and Ion ChannelsTo be treated in Lecture 12, ThursdayGTP GDP + PiEffector: enzyme or channeloutsideNeurotransmitter or hormonebinds to receptoractivatesG proteininside20Membrane proteins encounter each other more frequently, because they are restricted to 2-dimensional diffusionhydrocarbon “tails” anchor the molecules in the membrane lipidsoutsideinside21RNA polymerase promoterRNA polymeraseDNAStep1:bind “nonspecifically” to DNAStep 2:bind “specifically” to promoterOne-dimensional diffusion: a protein bound to DNA22 m 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 No energy required:diffusionDiffusionMechanical PumpsIntracellularExtracellular 1 m 1 mmMolecular MotorsEnergy required:Molecular Motors“blood-brain barrier”capillary spacing(sometimeswith fewer dimensions)How molecules move in the body23electron micrographsATP-dependent motorscytosolcytoskeletonLittle Alberts 17-17© GarlandTwo molecular motors that travel along the cytoskeleton24NeurotransmitterandATPkinesincell bodypresynapticterminal~ 20 distinct proteinsvesicle transport;pumping protons;pumping neurotransmitter; docking;fusion;recycling.cytosol50 nmSynaptic vesicles are moved by molecular motorsfrom Lecture 925The chemist’s method for fluorescent labeling: attach a small fluorescent molecule to a proteinproteintetramethylrhodamine (red)fluorescein (green)26Often, we couple label fluorescent molecules to an antibody.This provides a specific label for the antigenpart of Little Alberts 4-32© Garlandtetramethylrhodamine27Some molecules discussed by Mary Kennedy, Lecture 9(Black background usually implies fluorescence microscopy)28Examples of antibody-labeled cytoskeletal proteins in single fixed, permeabilized * cells 50 mactin microtubules “intermediate filaments”*dead29Swiss-PDB viewer requiredhttp://www.its.caltech.edu/~lester/Bi-1/gfp-for-viewing.pdb30ExpressDNAThe biologist’s method for fluorescent labeling of living cells:attach a fluorescent proteinGene for your favorite proteinGene for GFPprotein31Single Green Fluorescent Protein (GFP)-tagged protein moleculesseconds(unlike this example,most fluorescent molecules bleach permanently after emitting ~ 106 photons)Data of Emil Kartalov ‘9632GFP-tagged proteins moving reversibly from the cytosol to the membrane in response to activation of a receptorbefore 90 sec after stimulus 300 sec