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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”

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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 =

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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

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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

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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

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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+

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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

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Fluorescence measurements of a Ca2+ transient in a cell

“false color”

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1/s1 + 1/s2 = 1/f

L1 / L2 = s1/s2

s2

s1

L1

L2

Thin lens equations:

It’s time to learn about microscopes

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1/s1 + 1/s2 = 1/f

L1 / L2 = s1/s2

s2

s1

L1

L2

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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

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Tutorial on magnification using the lens equation

http://www.micro.magnet.fsu.edu/primer/java/lenses/magnify/index.html

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http://www.micro.magnet.fsu.edu/primer/java/nuaperture/index.html

Fluorescence efficiency is proportional to the 4th power of numerical aperture

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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

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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

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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

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RNA polymerase promoter

RNA polymerase

DNA

Step1:bind “nonspecifically”

to DNAStep 2:

bind “specifically” to promoter

One-dimensional diffusion: a protein bound to DNA

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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

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electron micrographs

ATP-dependent motors

cytosol

cytoskeleton

Little Alberts 17-17© Garland

Two molecular motors that travel along the cytoskeleton

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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

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The chemist’s method for fluorescent labeling: attach a small fluorescent molecule to a protein

protein

tetramethylrhodamine (red)fluorescein (green)

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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

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

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Examples of antibody-labeled cytoskeletal proteins in single fixed, permeabilized * cells

50 m

actin microtubules “intermediate filaments”

*dead

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Swiss-PDB viewer required

http://www.its.caltech.edu/~lester/Bi-1/gfp-for-viewing.pdb

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Express

DNA

The biologist’s method for fluorescent labeling of living cells:attach a fluorescent protein

Gene for your favorite proteinGene for GFP

protein

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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

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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

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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)

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Video 17.6 from “Little Alberts” CD

“Organelles moving on Microtubules”

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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

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End of Lecture 10

C

Dt =