2. 1 Bi 1 Lecture 10 Monday, April 17, 2005 Diffusion and Molecular Motion in Biology; Microscopes C Dt =

3. 2 Little Alberts Panel 1-1 Optical microscopy with unstained cells bright-field phase-contrast differential-interference-contrast 50  m Some 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.

4. 3 m 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 1 Diffusion Mechanical Pumps Intracellular Extracellular 1  m 1 mm “blood-brain barrier” Lecture 2 capillary spacing How molecules move in the body

5. 4 C Dt = C = concentration of molecule M = initial moles of molecule D = diffusion coefficient t = time r = distance or radius Diffusion from a Point Source Math 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”

6. 5 time to spread, t molecular weight (MW) Diffusion coefficient D (  m) 2 /ms x = 0.1  m (~ synaptic cleft)  s x = 10  m (~ single cell) ms x = 1 mm (~ brain region) s O 2 or Na + 32 or 231 5  s 50500 Neurotransmitter or low-MW drug 2000.3 17  s 1701700 protein50,0000.1 50  s 5005000 Some diffusion constants and distances C Dt =

7. 6 All I really need to know about life I learned in Bi 1 3. Most processes follow an exponential time course 4. Most processes end with a Gaussian distribution 1. If you want a job done right, get a protein 2. Electrical circuits explain many processes

8. 7 cytosol receptor cytosol synaptic cleft transmitter molecules receptor Diffusion across the synaptic cleft takes a negligible time at synapses presynaptic terminal postsynaptic dendrite direction of information flow 50 nm = 500 Å = 0.05  m Diffusion time: a few  s

9. 8 These 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 Ca 2+. 3 classes of proteins that transport ions across membranes: Little Alberts 12-4 © Garland Ion channels that flux many ions per event Ion-coupled transporters “Active” pumps that split ATP from Lecture 5

10. 9 Ca 2+ has a diffusion coefficient ~ 100-fold less than that of other ions in the cytosol, because Ca 2+ spends 99% of its time bound to proteins Ca 2+

11. 10 Calcium-sensitive fluorescent dyes fluo-3

12. 11 Little Alberts Panel 1-1 exciting light only emitted light only beam-splitting (“dichroic”) mirror Greek, 2 colors

13. 12 Fluorescence measurements of a Ca 2+ transient in a cell “false color”

14. 13 1/s 1 + 1/s 2 = 1/f L 1 / L 2 = s 1 /s 2 s2s2 s1s1 L1L1 L 2 Thin lens equations: It’s time to learn about microscopes

15. 14 1/s 1 + 1/s 2 = 1/f L 1 / L 2 = s 1 /s 2 s2s2 s1s1 L1L1 L 2

16. 15 1/s 1 + 1/s 2 = 1/f L 2 / L 1 = s 2 /s 1 s2s2 s1s1 L1L1 L 2 = n sin   How to read a microscope objective lens 160/0.17

17. 16 Tutorial on magnification using the lens equation http://www.micro.magnet.fsu.edu/primer/java/lenses/magnify/index.html

18. 17 http://www.micro.magnet.fsu.edu/primer/java/nuaperture/index.html Fluorescence efficiency is proportional to the 4th power of numerical aperture

19. 18 m 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 1 No energy required: diffusion Diffusion Mechanical Pumps Intracellular Extracellular 1  m 1 mm “blood-brain barrier” capillary spacing (sometimes with fewer dimensions) How molecules move in the body

20. 19 To be treated in Lecture 12, Thursday GTPGDP + P i Effector: enzyme or channel outside Neurotransmitter or hormone binds to receptor  activates G protein      inside

21. 20 Membrane proteins encounter each other more frequently, because they are restricted to 2-dimensional diffusion hydrocarbon “tails” anchor the molecules in the membrane lipids outside       inside

22. 21 RNA polymerase promoter RNA polymerase DNA Step1: bind “nonspecifically” to DNA Step 2: bind “specifically” to promoter One-dimensional diffusion: a protein bound to DNA

23. 22 m 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 1 No energy required: diffusion Diffusion Mechanical Pumps Intracellular Extracellular 1  m 1 mm Molecular Motors Energy required: Molecular Motors “blood-brain barrier” capillary spacing (sometimes with fewer dimensions) How molecules move in the body

24. 23 electron micrographs ATP-dependent motors cytosol cytoskeleton Little Alberts 17-17 © Garland Two molecular motors that travel along the cytoskeleton

25. 24 Neurotransmitter and ATP kinesin cell body presynaptic terminal ~ 20 distinct proteins vesicle transport; pumping protons; pumping neurotransmitter; docking; fusion; recycling. cytosol 50 nm Synaptic vesicles are moved by molecular motors from Lecture 9

26. 25 The chemist’s method for fluorescent labeling: attach a small fluorescent molecule to a protein protein tetramethylrhodamine (red) fluorescein (green)

27. 26 Often, we couple label fluorescent molecules to an antibody. This provides a specific label for the antigen part of Little Alberts 4-32 © Garland tetramethylrhodamine

28. 27 Some molecules discussed by Mary Kennedy, Lecture 9 (Black background usually implies fluorescence microscopy)

29. 28 Examples of antibody-labeled cytoskeletal proteins in single fixed, permeabilized * cells 50  m actin microtubules “intermediate filaments” *dead

30. 29 Swiss-PDB viewer required http://www.its.caltech.edu/~lester/Bi-1/gfp-for-viewing.pdb

31. 30 Express DNA The biologist’s method for fluorescent labeling of living cells: attach a fluorescent protein Gene for your favorite proteinGene for GFP protein

32. 31 Single Green Fluorescent Protein (GFP)-tagged protein molecules seconds (unlike this example, most fluorescent molecules bleach permanently after emitting ~ 10 6 photons) Data of Emil Kartalov ‘96

33. 32 GFP-tagged proteins moving reversibly from the cytosol to the membrane in response to activation of a receptor before 90 sec after stimulus 300 sec later Translocation of the GFP-tagged PKC-gamma C1A domain. Timepoints before (left), 90s after (middle) and 300s after (right) activation of the IgE receptor

34. 33 How molecules move in the body m 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 10 -1 1 No energy required: diffusion Molecular Motors Diffusion Mechanical Pumps Intracellular Extracellular 1  m 1 mm Energy required: Molecular Motors “blood-brain barrier” capillary spacing (sometimes with fewer dimensions)

35. 34 Video 17.6 from “Little Alberts” CD “Organelles moving on Microtubules”

36. 35 Optical microscopy with unstained cells 50  m Some 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. bright-field phase-contrast differential-interference-contrast

37. 36 End of Lecture 10 C Dt =