Download Biochemistry 4: Cellular Biochemistry

Biochemistry 4:
Cellular Biochemistry
Ulrike Gaul
Thomas Becker
Julia von Blume
Ralph Böttcher
Veit Hornung
Carsten Grashoff
Markus Moser
Boris Pfander
Teaching assistance:
Sara Batelli
Topics of the lecture course
Biochemistry 1 – Introduction and biochemistry of cellular processes
Biochemistry 2 – Metabolic and catabolic processes in the cell
Biochemistry 3 - Macromolecules (Protein, DNA, Interactions)
Biochemistry 4 – Cellular Biochemistry
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Introduction: internal organization of cells
Visualization of cells: light and electron microscopy
Membrane biology
Protein sorting into compartments and organelles
Cell-cell communication
Cytoskeleton and intracellular transport
Cell cycle
Cell death
Cell adhesion, junctions, polarity and migration
Technical matters
Course hours
Monday
Tuesday
9:00-10:30
9:00-10:30
Lynen Auditorium, Building A
Slides
Description of course and Powerpoint presentations available on the Gene Center website:
http://www.genzentrum.lmu.de/lehrplan/Course/19
Login: lectures
Password: lect2004
Reading material / Textbooks
Alberts, Bray, Hopkin, Johnson, Lewis, Raff, Roberts, Walter
Essential Cell Biology
3rd edition, 2010, Garland Science
Alberts, Johnson, Lewis, Raff, Roberts, Walter
Molecular Biology of the Cell
5th edition, 2008, Garland Science
Pollard and Earnshaw
Cell Biology - Das Original mit Übersetzungshilfen
2nd edition, 2008, Spektrum Akademischer Verlag
Cells
Using microscopy, biologists
discovered 170 years ago that
all living things are made of
cells
Simplest forms are solitary
cells
Higher organisms (plants,
fungi, animals) consist of
many different cell types,
often organized into organs,
such as brain, muscle, bone
gut, liver, kidney, which are
designed to perform highly
specialized functions
Differences between cells
sizes and shapes
nerve cell in cerebellum
paramecium
section through a young
plant stem
small bacterium
human white blood cell (neutrophil) eating a
red blood cell
environmental requirements
oxygen yes/no
air – sunlight – water – minerals
complex mixture of foods made by other
organisms.
Commonalities between cells/organisms
discoveries of molecular biology
and biochemistry:
DNA  RNA  protein
DNA acts as store of genetic
information
Proteins serve as structural
support, chemical catalysts,
molecular motors
All cells require energy to maintain
themselves
Commonalities between cells/organisms
Commonalities at molecular level: gene families shared by all organisms
Three major domains/divisions of life
bacteria – archaea – eukaryotes
derived of a common ancestor cell (based on rRNA sequence relationships)
Bacteria and archaea are prokaryotes
Shapes and sizes of some bacteria
Prokaryotic cells live in enormous variety of ecological niches, very varied in
their biochemical abilities… 99% unexplored
Eukaryotes
In contrast to prokaryotes, eukaryotes have a nucleus (eu karyon)
which contains the cell’s DNA.
Shapes of some single cell eukaryotes (protists)
Protist on the move
Eukaryotic cell organization – overview
Eukaryotic cells possess a variety of organelles, subcellular structures, that
perform specialized functions.
Eukaryotic cell organization – nucleus
Chromosome condensation
during cell division
Eukaryotic cell organization – mitochondria
Mitochondria generate usable
energy from food to power the
cell
TEM reveals two membranes,
outer and inner, inner is folded.
Mitochondria perform endoxidation:
sugars are oxidized using oxygen,
ATP and CO2 are produced
Eukaryotic cell organization – mitochondria
Mitochondria have their own DNA
most likely evolved from engulfed bacteria
Eukaryotic cell organization – chloroplasts
Chloroplasts capture energy
from sunlight
Large green organelles, only
found in plants and algae, not in
animals or fungi.
Two surrounding membranes,
internal stacks of membranes
that contain chlorophyll.
Photosynthesis: chlorophyll
traps energy from sunlight and
use energy to make sugars,
oxygen is released as a
byproduct.
Eukaryotic cell organization – chloroplasts
Like mitochondria, chloroplasts have their own DNA
most likely evolved from photosynthetic bacteria that were engulfed by an
early eukaryotic cell
Eukaryotic cell organization
additional compartments within eukaryotic cells:
membrane-enclosed organelles involved in import and export of materials
Eukaryotic cell organization – endoplasmic reticulum (ER)
ER – irregular maze of interconnected spaces enclosed by a membrane
the site where most cell membrane components, as well as materials
destined for export are made
Eukaryotic cell organization – Golgi apparatus
Golgi apparatus – stack of flattened discs, receives and chemically modifies
the molecules made in the ER and then directs them to the exterior of the cell
or to various locations inside the cell
Eukaryotic cell organization
Lysosomes – small acidified (pH 5) organelles for intracellular digestion
release nutrients from food particles and break down unwanted molecules for
recycling or excretion
Peroxisomes – peroxide-containing organelles for oxidation
Endocytosis and exocytosis
continuous exchange of vesicles
between the different cell
compartments and the plasma
membrane
Eukaryotic cell organization – cytosol
Apart from all the membranous
compartments, there is the cytosol
the largest compartment of most cells
full of small and large molecules – water
based gel
catabolic and metabolic processes; protein
biosynthesis (ribosomes)
Eukaryotic cell organization – cytoskeleton
Cytoskeleton:
actin, microtubules, intermediate filaments
generate morphology
movement of the cell
movements within the cell such as
distributing chromosomes in dividing cells
Where did eukaryotes come from?
Animal – plant –
bacterial cells
Summary
 Cells are the fundamental units of life. All present-day cells are believed to have
evolved from an ancestral cell that existed more than 3 billion years ago.
 All cells grow, convert energy from one form to another, sense and respond to their
environment, and reproduce themselves.
 All cells are enclosed by a plasma membrane that separates the inside of the cell
from the environment.
 All cells contain DNA as a store of genetic information and use it to guide the
synthesis of RNA molecules and of proteins.
 The simplest of present-day living organisms are prokaryotes: although they contain
DNA, they lack a nucleus and other organelles and probably resemble most closely
the ancestral cell.
 Different species of prokaryotes are diverse in their chemical capabilities and inhabit
an amazingly wide range of habitats. Two fundamental evolutionary subdivisions are
found: bacteria and archaea.
 Eukaryotic cells possess a nucleus and other organelles not found in prokaryotes.
They probably evolved in a series of stages. An important step was the acquisition
of mitochondria, which are thought to have originated from bacteria engulfed by an
ancestral eukaryotic cell.
 The nucleus is the most prominent organelle in most plant and animal cells. It
contains the genetic information of the organism, stored in DNA molecules.
 The cytoplasm includes all of the cell’s content outside the nucleus. It harbors a
variety of membrane-enclosed organelles with specialized biochemical functions.
Mitochondria carry out oxidation of food molecules. In plant cells, chloroplasts
perform photosynthesis. The endoplasmic reticulum and Golgi apparatus permit
cells to synthesize complex molecules for export from the cell and for insertion in
cell membranes. Other vesicles (endosomes, lysosomes, peroxisomes) are required
for import and digestion of large molecules.
 The cytosol contains a concentrated mixture of large and small molecules that carry
out many essential biochemical processes.
 The cytoskeleton extends throughout the cytoplasm. This system of protein
filaments is responsible for cell shape and movement and for the transport of
organelles and molecules within the cell.
 Free-living single-cell eukaryotes include some of the most complex eukaryotic cells
known, and they are able to swim, mate, hunt, and feed.
 Animals, plants and fungi consist of diverse eukaryotic cell types all derived from a
single fertilized egg cell; the number of cells in a large multicellular organism runs
into the billions.
 Cells in a multicellular organism, though they all contain the same DNA, can be very
different. They turn on different sets of genes according to their developmental
history and cues they receive from their environment.
Biochemistry 4:
Cellular Biochemistry
Ulrike Gaul
Thomas Becker
Julia von Blume
Ralph Böttcher
Veit Hornung
Carsten Grashoff
Markus Moser
Boris Pfander
Teaching assistance:
Sara Batelli
Visualization of cells by light
and electron microscopy
Today’s topic
 Cell biology started with light microscopy … and is still very
important in the form of fluorescence microscopy (in various
forms), but its resolution is limited by the wavelength of visible
light.
 By using a beam of electrons instead, electron microscopy can
image the macromolecular complexes within cells at almost
atomic resolution.
 Today, we will discuss both types of microscopes and specimen
preparation, beginning with light microscopy.
Scale – from macroscopic structures to atoms
resolving power of
eye, light and electron
microscope
Light microscopy
Light waves travel by slightly different routes, so that
they interfere with one another and cause optical
diffraction effects.
At high magnification, an edge appears as a series of
lines, a point of light as a concentric pattern
Light microscopy – spatial resolution
Resolution limit is determined by features
of the objective lens (numerical aperture,
NA) and the wavelength of the light ()
numerical aperture (NA)
Working
distance
α
barely resolved
resolved
α
α
α = 7º
α = 20º
α = 60º
NA = n sin(α)
n: medium refractive index
n(air) < n(water) < n(oil)
α: ½ of the objective angular aperture
radius airy disk
(=lateral resolution)
rAiry  0.61

NA
Rayleigh criterion:
Two adjacent object points are defined
as being resolved when the central
diffraction spot (airy disk) of one point
coincides with the first diffraction
minimum of the other point in the image
plane
Example:
for NA = 1.3; λ = 546 nm
lateral resolution = 0.61λ/NA = 260 nm
Types of light microscopy
Bright field
Dark field
- only scattered light enters objective
Phase contrast
Differential interference contrast
(DIC, Nomarski)
- exploits interference effects
Sample preparation
Intact tissues are usually fixed, stained
and sectioned before microscopy
Fluorescence microscopy
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can be used to visualize specific
molecules within cells
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certain dyes and proteins (!) can
be excited by absorption of light;
they revert to the ground state by
emitting a photon of longer wave
length
First excited
Singlet state
in most cases, the absorption
and emission spectra are
relatively broad, but emission is
always shifted towards longer
wave lengths (Stokes shift)
Ground
Singlet state
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Stokes
shift
Higher
vibrational states
Triplet state
Jablonski diagram: 3 level system
Absorption spectra
Emission spectra
Fluorescence microscopy
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can be used to visualize specific
molecules within cells
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certain dyes and proteins (!) can
be excited by absorption of light;
they revert to the ground state by
emitting a photon of longer wave
length
First excited
Singlet state
in most cases, the absorption
and emission spectra are
relatively broad, but emission is
always shifted towards longer
wave lengths (Stokes shift)
Ground
Singlet state

Stokes
shift
Higher
vibrational states
Triplet state
Jablonski diagram: 3 level system
Fluorescence microscopy – Green fluorescent protein (GFP)
original source: jellyfish
Absorption spectra
Emission spectra
400
450
Which dyes/proteins are good for biological experiments?
 High extinction coefficient (> 30,000 – 40,000 M−1cm−1)
 High quantum yield of fluorescence
For example: Cy5: Φ = 29%
Rhodamine 6G: Φ = 95%
EGFP : Φ = 60%
 Little photodegradation (photobleaching)
Differential photobleaching in multiply-stained tissue over time
 Absorption in the far red of the visible spectrum.
(limits photodamage to living cells, lower autofluorescence)
Immunhistochemistry and multiple fluorescent probe microscopy
DNA
microtubules
centromeres
Fluorescence microscopy – wide-field vs. confocal
Fluorescence microscopy – wide-field vs. confocal
Wide-field
microscope
Confocal
microscope
laser
laser
˗ Low z-resolution
(~ 2-3mm)
+ High z-resolution
(~ 600nm)
+ High time resolution
(up to 5 ms per
frame)
˗ Low time resolution
(secs to mins per
image)
Confocal microscopy – following cells/proteins live
Sensory neurons
cytoplasmic GFP
time lapse of epithelial closure
myristylated GFP + moesin GFP
Fluorescence recovery after photobleaching (FRAP)
Use strong focused light
beam to extinguish GFP
fluorescence in specific
region of the cell
 Analyze how the
remaining fluorescent
proteins move into the
bleached area as a
function of time
 Learn kinetic
parameters, such as
diffusion coefficients,
active transport rates,
binding/dissociation
rates from other proteins
Fluorescence resonance energy transfer (FRET)
 Used to determine whether two proteins interact inside the cell
 Proteins attached to different color variants of GFP (fusion proteins)
e.g. proteinX-CFP, proteinY-GFP
 when X and Y interact, excitation of CFP leads to activation of GFP, resulting in
green emission
 fluorophores have to be close (1-10 nm) for FRET to occur.
Transmission Electron microscopy (TEM)
Light
microscope
Electron
microscope
The electron microscope can
resolve the fine structures of
the cell
As with light, resolution limit is
a function of the wavelength
Electrons accelerated at
100,000 V  =0.004 nm
Aberrations of electron lenses
hard to correct, NA very small
In practice:
resolution is 1nm = 10 A
Electron microscopy
specimen preparation
 Hard fixation
(glutaraldehyde,
osmium tetroxide)
 Embedding into
plastic resin
 Thin sections (50-100
nm) using diamond
knife
 Place sections on
copper grid
Electron microscopy
Serial EM sections
combined with 3D
reconstruction reveals
complex cell
interdigitations
Electron microscopy
Specific macromolecules can
be localized by immunogold
electron microscopy
Scanning electron microscopy (SEM)
SEM uses electrons that
are scattered or emitted
from specimen
specimen preparation
 Fix – dry - coat with
thin layer of heavy
metal
 Or: rapid freezing,
place on cooled stage
 Great depth of field
Scanning electron microscopy (SEM)
Summary
 Many light microscope techniques are available for observing cells.
 Living cells can be seen with phase contrast, Nomarksi optics, dark, or bright field
microscopy.
 Cells that have been fixed and stained can be studied by a conventional bright field
microscopy
 When labeled by immune histochemistry or when expressing genetically encoded
fluorescent proteins, they can be imaged by different forms of fluorescence
microscopy, in particular confocal microscopy.
 The resolution of microscopes is determined by their numerical aperture and the
wavelength of the light.
 Confocal microscopes improve resolution in the z-axis by eliminating out-of-focus
information. 3D information can be generated from these thin optical sections by 3D
reconstruction.
 Techniques are now available for detecting, measuring and following almost any
desired molecule in a living cell. Fluorescent indicator dyes can be introduced to
measure the concentrations of specific ions within cells or specific regions. Virtually
any protein of interest can be genetically engineered as a fluorescent fusion protein
and then imaged in living cells by fluorescence microscopy.
 The dynamic behavior and interactions can be followed by a wealth of microscopic
techniques, including FRAP, photo bleaching, and FRET.
Summary cont.
 Determining the detailed structure of membranes and organelles in cells requires
the higher resolution attainable in a transmission electron microscope. Specific
macromolecules can be localized with colloidal gold linked to antibodies.
 3D views of cell surfaces and tissues are obtained by scanning electron microscopy.