1 Bi 1 Lecture 10 Monday, April 17, 2005 Diffusion and Molecular Motion in Biology; Microscopes C Dt...
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Transcript of 1 Bi 1 Lecture 10 Monday, April 17, 2005 Diffusion and Molecular Motion in Biology; Microscopes C Dt...
1
Bi 1 Lecture 10
Monday, April 17, 2005
Diffusion and Molecular Motion in Biology;
Microscopes
C
Dt =
2Little 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.
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
4 Dtr
Dt
MC 4exp
π8
2
23
C
Dt =
C = concentration of moleculeM = initial moles of moleculeD = diffusion coefficientt = timer = 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”
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
O2 or Na+ 32 or 23 1 5 s 50 500
Neurotransmitter
or low-MW drug
200 0.3 17 s 170 1700
protein 50,000 0.1 50 s 500 5000
Some diffusion constants and distances
Dtxxrms 22 C
Dt =
6
All 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 distribution
1. If you want a job done right, get a protein
2. Electrical circuits explain many processes
7
cytosol
receptor
cytosolsynaptic cleft
transmitter molecules
receptor
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
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 Ca2+.
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
9
Ca2+ has a diffusion coefficient ~ 100-fold less than that of other ions in the cytosol,
because Ca2+ spends 99% of its time bound to proteins
Ca2+
10
Calcium-sensitive fluorescent dyes
fluo-3
fluo-3
11Little Alberts Panel 1-1
exciting light only
emitted light only
beam-splitting(“dichroic”)
mirrorGreek,2 colors
12
Fluorescence measurements of a Ca2+ transient in a cell
“false color”
13
1/s1 + 1/s2 = 1/f
L1 / L2 = s1/s2
s2
s1
L1
L2
Thin lens equations:
It’s time to learn about microscopes
14
1/s1 + 1/s2 = 1/f
L1 / L2 = s1/s2
s2
s1
L1
L2
15
1/s1 + 1/s2 = 1/f
L2 / L1 = s2/s1
s2
s1
L1
L2
= n sin
How to read a microscope objective lens
160/0.17
16
Tutorial on magnification using the lens equation
http://www.micro.magnet.fsu.edu/primer/java/lenses/magnify/index.html
17
http://www.micro.magnet.fsu.edu/primer/java/nuaperture/index.html
Fluorescence efficiency is proportional to the 4th power of numerical aperture
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
(sometimeswith fewer dimensions)
How molecules move in the body
19
7-Helix Receptors Coupled to G proteins and Ion ChannelsTo be treated in Lecture 12, Thursday
GTP GDP + Pi
Effector: enzyme or channel
outside
Neurotransmitter or hormonebinds to receptor
activatesG protein
inside
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
21
RNA polymerase promoter
RNA polymerase
DNA
Step1:bind “nonspecifically”
to DNAStep 2:
bind “specifically” to promoter
One-dimensional diffusion: a protein bound to DNA
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
(sometimeswith fewer dimensions)
How molecules move in the body
23
electron micrographs
ATP-dependent motors
cytosol
cytoskeleton
Little Alberts 17-17© Garland
Two molecular motors that travel along the cytoskeleton
24
Neurotransmitter
and
ATP
kinesin
cell body presynapticterminal
~ 20 distinct proteins
vesicle transport;pumping protons;pumping neurotransmitter; docking;fusion;recycling.
cytosol
50 nm
Synaptic vesicles are moved by molecular motorsfrom Lecture 9
25
The chemist’s method for fluorescent labeling: attach a small fluorescent molecule to a protein
protein
tetramethylrhodamine (red)fluorescein (green)
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
27
Some molecules discussed by Mary Kennedy, Lecture 9(Black background usually implies fluorescence microscopy)
28
Examples of antibody-labeled cytoskeletal proteins in single fixed, permeabilized * cells
50 m
actin microtubules “intermediate filaments”
*dead
29
Swiss-PDB viewer required
http://www.its.caltech.edu/~lester/Bi-1/gfp-for-viewing.pdb
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
31
Single Green Fluorescent Protein (GFP)-tagged protein molecules
seconds
(unlike this example,most fluorescent molecules bleach permanently after emitting ~ 106 photons)
Data of Emil Kartalov ‘96
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
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
(sometimeswith fewer dimensions)
34
Video 17.6 from “Little Alberts” CD
“Organelles moving on Microtubules”
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
36
End of Lecture 10
C
Dt =