Microscopy and internal organization of a cell'

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Microscopy and internal organization of a
cell. Lecture 10, Fall 2003
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Looking at cells.
A sense of scale - what can be seen with
microscopes.
Limit of resolution for a light microscope is set
by wavelengths of visible light - limit is 1/2
the wavelength, which is 400 to 700 nm.
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• To observe a specimen with light microscopy
• it must absorb particular wavelengths of light
  (which results in color)
• or it must slow the light so that different paths
  of light are out of phase.

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Bright-field microscopy
Bright-field microscopy is poor for viewing
colorless samples such as most animal cells. It
cannot detect phase shifts.
Phase-contrast microscopy
The optics of a phase-contrast microscope brings
parallel paths of light together so that
constructive and destructive interference occurs.
Note how the cell is outlined by a white halo
that enhances the distinction between the cell
and its surroundings.
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Fluorescence microscope is very useful because it
allows visualization of specific molecules in
cells.
Emitted light
Incident light
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Stain for DNA.
A variety of fluorescent molecules are used to
visualize cells. The fluorescent molecules
absorb light at specific wavelengths and emit
light of longer wavelengths. Through judicious
choice of tags, one can visualize more than one
protein in a sample. Fluorescence microscopes
are designed so one can easily change the
excitation wavelength and the emission wavelenght
that can be seen.
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Often, a two antibody method is used to detect
the location of an antigen with fluorescence.
The method is referred to as indirect
immunofluorescence or indirect immunocytochemistry
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• Primary antibodies are produced by the
  experimentor.
• Secondary antibodies are available from
  companies. These antibodies are produced by
  injecting one species of animal with antibodies
  from another species of animals.

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Another fluorescence approach is to express a
protein as a fusion with green fluorescent
protein (GFP).
The chromaphore is generated by a self catalyzed
reaction of 3 amino acid side chains in the
protein
GFP can be fused either to the N-terminus or
C-terminus.
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2 views of a protein in the ER.

• Indirect immunofluorescence
• Dead cells.
• Fixed and permeabilized.
• Primary antibody binds antigen.
• Secondary antibody contains fluorescent tag.

• GFP-tagged protein
• View live cells.
• GFP fusion needs to behave properly.

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Confocal fluorescence microscopy is a
sophisticated variation of fluorescence
microscopy that allows one to focus on a precise
point. By moving the incident beam of light and
collecting the pattern of emitted light in a
computer, one can generate precise optical
sections or reconstruct 3D images.
Optical sections
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Transmission electron microscopy - TEM
Specimen scatters electrons
Image consists of the electron beam minus the
scattered electrons. The image is analogous to
looking at a shadow.
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Electron microscopy requires the use of heavy
metal atoms.
The small atoms of life (C,H,O,N,P,S) do not
scatter electrons very well so they are poorly
detected by the EM. Hence, samples are almost
always stained with heavy metals such as uranium,
lead, platinum, osmium or gold since these do
scatter electrons. The heavy metal stains are
applied so that they associate with different
components in the sample. For example, osmium
tetroxide reacts with membranes so it serves to
outline organelles. One can attach gold
particles to antibodies and determine the
location of a particular protein by incubating
the sample with gold-tagged antibodies.
For TEM, the samples must be extremely thin,
100nm.
This is less than the diameter of a cell so cells
must be fixed, thinly sliced and then stained in
order to be viewed.
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Internal organization of cells.
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Internal organization of bacteria

• No internal compartments.
• Genetic processes occur in cytosol.
• Electron transport chains and ATP synthase reside
  in plasma membrane.
• Glycolysis and citric acid cycle in the cytosol.

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The outer surface of a gram negative bacteria
like E.coli.

• Outer membrane - lipopolysaccharides,
  phospholipids, and protein.
• Porin provides a channel for diffusion of
  metabolites.
• Periplasmic space - peptidoglycan layerand
  soluble proteins.
• lysozyme cleaves the peptidoglycan layer causing
  cells to burst.
• Ampicillin inhibits synthesis.
• Inner membrane - phospholipids and proteins
• Electron transport chain, ATP synthase,
  transporters.

Gram positive bacteria like Bacillus lack the
outer lipid layer.
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An example of the subcellular organization of a
eukaryotic cell.
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Typical eucaryote contains multiple internal
membrane compartments.
Cytosol - most protein synthesis, glycolysis,
metabolic pathways synthesizing amino acids and
nucleotides.
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Plasma membrane - channels and carrier proteins
to regulate transport of molecules in and out of
the cell, receptors for signaling molecules.
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Nucleus - contains main genome, DNA and RNA
synthesis.

• Nuclear envelope consists of two phospholipid
  bilayers.
• Transport in and out of the nucleus is through
  the nuclear pores.

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• Mitochondria (plant and animal cells) - 2
  membranes
• ATP synthase and the respiratory chain reside in
  the inner membrane.
• Citric acid cycle occurs in the matrix.
• Genetic processes related to the organelles
  genome occur in the matrix.

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Chloroplasts (plants) - 3 membranes Thylakoid
membrane - light harvesting centers, electron
transport chains, ATP synthase. Stroma - genetic
processes, carbon fixation.
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Peroxisomes - single membrane Contains enzymes
that are involved in oxidation of a variety of
small molecules and in detoxification.
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ER, Golgi, endosome, lysosome and secretory
vesicles are organelles surrounded by single
membranes and interact with each other via small
transport vesicles.
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Endoplasmic reticulum - synthesis of membrane
proteins and secreted proteins, initiation of
N-linked glycosylation, protein folding and
disulfide bond formation, lipid synthesis,
detoxification of lipophillic compounds.
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Golgi - protein and lipid glycosylation, sorting
of proteins destined for the secretory vesicles,
plasma membrane or endosome.
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Endosomes - intermediate compartment where
proteins from the golgi and many proteins
endocytosed from the plasma membrane meet before
being transported to the lysosome.
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Lysosome - degradation of macromolecules that are
typically endocytosed from the plasma membrane
and organelles that are old. Lumen is about pH 5.
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Secretory vesicles - store molecules that are
secreted when cells receive appropriate signals
such as hormones.
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The cytoskeleton gives the cell shape and
organizes the internal components of the cell.
It consists of 3 types polymers assembled from
distinct proteins.
Lodish 19-50
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Microtubules are dynamic polymers of tubulin.
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Lodish 19-11
Force can be generated by assembly and
disassembly of microtubules and by ATP powered
motor proteins called kinesin and dynein
Lodish 19-26
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Kinesin and dynein use ATP hydrolysis to walk
along microtubules.
Alberts 16-59
Type of movement depends of the organization of
the motors.
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The ER and Golgi appear to be supported by
microtubules since drugs that depolymerize
microtubules (colchicine, nocadozole) cause the
ER to collapse and the Golgi to fragment.
untreated
Red tubulin Green golgi
nocadozole
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Microfilaments are dynamic polymers of Actin.
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Most microfilaments are located near the cell
membrane and function in cell shape and motility.
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Myosin uses ATP hydrolysis to move along
microfilaments with a ratcheting motion.
In muscles, myosin is organized so opposing actin
filaments are pulled towards each other.
Lodish 18-29
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Intermediate filaments are relatively static
polymers of long rod-like proteins.
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Intermediate filaments give individual cells
mechanical strength and strengthen epithelial
tissue by forming networks connected at cell-cell
junctions.
The nuclear lamina is composed of a type of
intermediate filament protein called lamins.