physicsfor biology: scheme i. introduction: a few
TRANSCRIPT
Physics for biology: scheme
I. Introduction: a few biological systems, and some physics tools
II. Electrostatics and thermodynamics of salty solutions
III. Diffusion in cells
IV. Macromolecules: statistical physics and micromanipulations
V. Molecular motors
Lab visits
References
Molecular Biology of the cellAlberts, Johnson, Lewis, Raff, Roberts, WalterGarland Science
Physical Biology of the Cell, R Philipps, J Kondev, J TheriotGarland Science
Biological Physics: Energy, Information, LifeP. Nelson, W. H. Freeman
http://www.ibiology.org/ibioseminars/lectures-by-name.html
Physique et Biologie : de la molécule au vivant, ed. JF Allemand et P. Desbiolles, EDP Sciences, Physics and biology: from molecules to life, World Scientific
Physics for biology
Sylvie HénonSteve Donaldson
Master 1 – I-CFPEcole Normale Supérieure
2016-2017
I. Introductiona few biological systems, and some physics tools
Scales of living systems
Light microscopy (late 1500s)
1. Physicists build tools used by biologists (and physicians)
A. Physics and biology
Robert Hooke 1665: « cells » in cork slices Anton van Leeuwenhoek 1675: micro-organisms and unicellular organisms
laser surgery… etc
NMR: image of brain (wikipedia)
X-ray diffraction: structure of proteins
echography
Use of fondamental concepts of physics: mechanics, thermodynamics, statistical mechanics, dynamical systems. Biological systems also obey physical laws.
Use of theoritical and methodological tools for analysis of a system and building of models: identify the important parameters, neglect less relevant phenomena, integrate different scales.
Different or new approaches to explore living beings
Innovative techniques for the study of biological systems (confocal microscopy, 2 photon microscopy, microfluidic, optical tweezers, nanotechnologies…)
2. Physics point of view in biology
Today: interdisciplinary research teams, with both biologists and physicists working together.
Complementarity between physics and biology
Living systems: very complex, tremendous diversity: thousands of different molecules interactingwith each other.
Biologists have nowadays extensive knowledge of systems composition and interaction diagrams
Physicists seek for universality in observed phenomena.
The two points of view are both necessary and complementary
a et b integrins
a actinin tallin
vinculin
An example: cell/matrix adhesion
The problem seen by biologists
A. Nicolas, B. Geiger and S. SafranPNAS 2004
The problem seen by physicists
B. Light microscopy
objective
eyepiece
condenser
light source
1. Principle
Microscopes today
computer
sensitive detectors
electronics
different light sources
diffraction :
D = d*2.44*l/Ø
2. Resolution
Ø<l D
d
Optical resolution : r = 1.22*l/(2*NA)
numerical aperture: NA= n sina
Better resolution: small l (blue better than red, Xrays or electrons even better)high NA: large a (small working distance, large lenses)
large n (immersion objectives)
a
a
a
(a) a = 7° NA = 0.12(b) a = 20° NA = 0.34(c) a = 60° NA = 0.87
n
n = 1
depth of field = distance between the nearest and farthest objects acceptably in focus in an imageincreases with NA
3. Phase contrast
Images of transparent objects like micro-organisms and cells
d = 2p(no-ne)e/l
e
no
ne
E(t,z) = E0 cos(wt-kz)
E(t,z) = E0 cos(wt-kz+d)same intensity
phase shifts are converted into amplitude differences
Eyes and detectors are sensitive to colour and intensity, not to phase
Application: observation of living cells in culture, without fixation and/or coloration
20µm
bright field phase contrast
4. Fluorescence microscopy
fluorescent molecule (fluorophore): absorbs light energy (excitation light or absorbed light) and rapidly restitutes it as fluorescent light (emitted light).
a. Principle
abso
rpti
on
10-1
5s
inte
rnal
Con
vers
ion
10-1
2s
pho
spho
rese
cnce
10-4
s -
102
s
non
radi
ativ
e pr
oces
s1
0-8s
proc
essu
s no
n r
adia
tifs
10-1
1s
-10
2s
fluo
resc
ence
10-8
s
S0
S2
S1
linear regime:
Ifluo Nfluorophores Iexc
DAPI 4',6-Diamidino-2-PhenylIndole
TRITCTetra methyl Rhodamine Iso Thio Cyanate
FITCFluoresceine Iso Thio Cyanate
400
wavelength (nm)
500 600 700
400 500 600300
Texas Red, Oregon Green, Acridine Orange, Lucifer Yellow, Alexa Fluor, YOYO …
b. Fluorophores
blue green
green red
blueUV
wavelength (nm)
wavelength (nm)
+ coupling to (chemical or antigen/antibody)
Organic molecules
DNAmicrotubulesactin filaments
10µm
directly binds to DNAanti-tubulin + fluorescent group bound to secondary antibodyfluorescent group bound to phalloidin
San Diego beach scene drawn with living bacteriaexpressing 8 different colors of fluorescent proteins
chimeric protein allows live-imaging
mutants: eGFP, YFP, CFP, RFP, mCherry, mBanana …
400wavelength (nm)
500 600
4 nm
GFP (Green Fluorescent Protein)
extracted from jellyfish Aequorea aequorea or Aequorea victoria
Osamu Shimomura, Martin Chalfie, Roger Y. Tsien, Nobel prize in Chemistry 2008
Quantum dots
nanocrystals of semi-conductors like CdSe : Ø = a few nms
broad absorption spectrum, narrow emission spectrum depending on size
CdSe core
ZnS shell
advantages : photostability, visible in electron microscopy
c. Resolution in fluorescence microscopy
distance between two distinguishable pointsr = 0.61*l/NA
objects smaller than r can be imagedbut objects closer than r cannot be resolved
5µm
microtubules
ø ~ 25 nm
chromosomes
5µm
DNA ø ~ 2nm
Recent ultra-high resolution techniques
stochastic optical reconstruction microscopy: STORM
photo activated localization microscopy: PALM
stimulated emission depletion: STED
5µm
microtubules in a Hela cell
r 25nm
Eric Betzig, Stefan W Hell, William E Moerner, Nobel prize in Chemistry 2008
5. Electron microscopy
r = 0.61*l/NA decreases with l use of electron beam, l down to sub-atomic
1µm
limitations thin samplesstaining with heavy atomsdamage of samples
Transmission Electron Microscope
C. Cells and their constituants
1. Overview
Eukaryote vs prokaryote
4 classes of macromolecules: phospholipids, DNA, proteins, glycoproteins
Animal cell vs plant cell
Golgi apparatus
Nuclear membrane
Cell membrane
2. MembranesEndoplasmic reticulum
3. Nucleus and DNA
Structure of DNA
cytosine
guanine
thymine
adenine
Specific interactions: AT et G-C DNA is oriented 2 antiparallel strands
A, C, T, G, sequence = genetic information
desoxy-ribo nucleic acid
4 nucleotides (bases)
(desoxy)ribose
DNA structure: Crick and Watson 1950’s Nobel Prize 1962
DNA condensation
histones
the coded genetic information
nucleotide codon gene
Transcription/translation
4. Proteins
DNA
mRNA
Protein
Translation
Secondary structure of proteins
a helix
Exemple: rhodopsine (transmembrane receptor)
b sheet Exemple: protein silk I (from silk worm)
Exemple: protein silk I (from silkworm)
5. Cytoskeleton: filaments and motors
- actin filaments or microfilaments ø ~ 6-8 nm
- microtubules ø ~ 25 nm
- intermediate filaments ø ~ 7-11 nm
actin microtubules intermediate filaments(keratin, vimentin, desmin, lamin)
+ end
- end
actine: 42 kDmost abondant protein in eukaryote cellsable to bind ATP
Actin filament (F-actin) = polymer of globular actin (G-actin)
polarized filament
Fuel of the cell: ATP (and GTP)
ATP = Adenosin TriPhosphate
Tri/di/monophosphateATP/ADP/AMP
Synthetised in mitochondria
ATP + H2O → ADP + Pi
ΔG˚ = −30.5 kJ/mol (−7.3 kcal/mol)ΔG˚ = − 12.2 kBT = 0.32 eV
Roles of actin in cells
contractile ring
microvilli contractile bundles
lamellipodiaand filopodia
endothelial cell of bovine aorta(J. Bishop-Steward et P. Millard Molecular probes)
Microtubules
Structure of a microtubule
Tubulin: polymer of a and b tubulin
55kD
polarized filament
14nm25nm
8nm
GTPGDP
+ end
13 protofilaments
Organisation of microtubules
aster of microtubules from centrosome (1-2µm ; 2 centrioles)
Role of microtubules
interphase mitosis
ciliated cell
cilium / flagellum
basal body
nervous cell
pole of the spindle
myosins
kinesins
dyneins
# of human proteins(approx, from genome)
50
75
10
representative functions
muscle contractionvesicle transport sensory cell function
axonal transportother vesicle transportmitosis
cilia/flagella beatingvesicle transportmitosis
Molecular motors
Directed movement (or rotation) thanks to conformationnal changesEnergie = ATP hydrolysisForce generation and transport along filaments
Acto myosin
Microtubules kinesin and dynein
vesicle trafficking in cellsflagellar motion (sliding of microtubules)
from « inner live of cell »