Download 1) digest DNA inserts with restriction enzyme(s).

Lab 5A and 5B Overview
Next Week
Today
Investigating protein sorting signals using cloning, transfection,
GFP-fusion proteins, and vital stains for cellular compartments
1. Fluorescent proteins and cool things
we can do with them
2. Protein sorting and membrane trafficking
- or How cells deliver things to the right place
3. Transfection and transgene expression
- or How we get DNA into cells
to express “designer” genes
4. Fluorescent markers for different
compartments of the secretory
and endocytic pathways
Cells need to take in molecules from their environment…
…and they need to target other proteins to their surfaces
and to specific compartments (organelles) within the cell
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ENDOCYTOSIS
EXOCYTOSIS
(Endo = within
Cyto = cell)
(Exo = out
Cyto = cell)
a.k.a. SECRETION
These processes involve membrane fusion to form new compartments
Cellular components of the secretory and endocytic pathways
lysosome
plasma
membrane
late
endosome
nuclear envelope
endoplasmic reticulum
early
endosome
CYTOSOL
cis
Golgi
network
Golgi
stack
trans
Golgi
network
Golgi apparatus
secretory
vesicle
Protein sorting involves a “bucket brigade” through a series
of membrane-bound compartments and vesicles
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RECEPTOR-MEDIATED ENDOCYTOSIS
is the way that cells deliberately pull in
specific molecules from outside.
To do this, cells express specific receptors on
their surfaces that bind to external molecules
and concentrate them in special coated vesicles.
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RECEPTOR-MEDIATED ENDOCYTOSIS occurs through special
membrane sites coated with the protein CLATHRIN.
Receptors interact with clathrin indirectly, through
ADAPTIN proteins.
Coated membrane buds that contain clathrin, adaptins, and receptors
bound to their ligands pinch off to form coated vesicles.
RECEPTOR-MEDIATED ENDOCYTOSIS occurs through special
membrane sites coated with the protein CLATHRIN.
Clathrin has a “triskelion” structure and forms polymers that
help to drive budding.
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Secreted proteins move from the Endoplasmic Reticulum (ER)
to the Golgi. There, they are tagged in a variety of ways so that
the cell can target them to the proper ultimate destination.
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Transport of secreted proteins from the ER to the Golgi
and from the Golgi to the cell surface
involves vesicle movement along microtubules.
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How would you expect these processes to be
affected by treatment with nocodazole?
Amino acid signal sequences or patches
direct protein sorting into some organelles
- they act like a subcellular ZIP code.
Nuclear Localization Signals (NLS)
P-P-K-K-K-R-K-V
K-R-P-A-A-T-K-K-A-G-Q-A-K-K-K-K
Mitochondrial import
H2N-M-L-S-L-R-Q-S-I-R-F-F-K-P-A-A-T-R-T-L-C-S-S-R-Y-L-L
Endoplasmic Reticulum import
H2N-M-M-S-F-V-S-L-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q
Endoplasmic Reticulum retention
K-D-E-L-COOH
Fusion proteins will be targeted according to the
signal sequences they encode.
Protein X
Signal Sequence
(Nuclear Localization Signal)
GFP
No localization signal
Protein X-GFP translational fusion
Will be sorted to the NUCLEUS
GFP-Protein X translational fusion
Will be sorted to the NUCLEUS
Green Fluorescent Protein (GFP)
Comes from a jellyfish, Aequorea victoria
Gene has been cloned and
transferred into a wide variety of
“heterologous” expression systems
… including Drosophila, mammalian cells,
C. elegans, yeast, zebrafish etc. etc.
**** Permits dynamic and in vivo analysis****
of biological processes
neurons
zebrafish
pigs!!!?
Both chemical and biochemical fluorophores contain extended
networks of conjugated double bonds
Fluorescein
GFP
Tyrosine 66
Glycine 65
Serine 67
Rhodamine
The “β-barrel” structure of GFP provides a
special environment inside the living cell
that enables the fluorophore to work
Variants of Green Fluorescent Protein and DsRed have been engineered
to have different excitation and emission spectra, and other useful properties
If two different FPs can be
separated by fluorescent filters, they are
useful for double-labeling experiments.
CFP (Cyan Fluorescent Protein) and
YFP (Yellow Fluorescent Protein)
provide a very useful combination.
GFP has been harnessed to study
an enormous variety of biological processes
Tracking gastrulation in a living Drosophila
embryo using moesin-GFP
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GFP has been harnessed to study
an enormous variety of biological processes
Imaging of whole organisms expressing GFP
Fusion proteins will be targeted according to the
signal sequences they encode.
Protein X
Signal Sequence
(Nuclear Localization Signal)
GFP
No localization signal
Protein X-GFP translational fusion
Will be sorted to the NUCLEUS
GFP-Protein X translational fusion
Will be sorted to the NUCLEUS
Escherichia coli Bateria are Essential
tools in DNA Cloning
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Generation of an enhanced bacterial cloning system:
1) Mutation of bacterial restriction modification systems (hsdR-).
2). Mutation of bacterial DNA recombination proteins (i.e. recA gene).
3). Mutation of endonuclease activity (i.e. endA gene) results in increased plasmid yields.
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Fusion proteins are usually introduced into cells as DNA constructs
Protein X
GFP
Prote
pCMV:
Strong, constitutive
promoter
Ampicillin:
Selectable marker
for bacterial cells
GFP
Proteins can be
tagged with GFP
at either end or
internally
Protein Y
GFP
in Z
BGH pA:
Polyadenylation
sequence
Neomycin:
Selectable marker for
mammalian cells
pUC:
Origin of replication
for bacterial cells
Cloning vector for expressing GFP fusion proteins
in mammalian cells (constructed in bacteria)
Fusion proteins are usually introduced into cells as DNA constructs
Protein X
GFP
Prote
GFP
Protein Y
GFP
in Z
YOUR MISSION: decipher the protein sorting information
in proteins U, X, Y, and Z by determining the localization
of the fusion proteins in mammalian cells.
Eight steps of DNA cloning:
1) digest DNA inserts with restriction enzyme(s).
2) digest DNA plasmid vector with restriction enzyme.
3) ligate digested DNA inserts and plasmid vector.
4) transform E. coli with the ligation reaction.
5) select plasmid-containing (transformed) bacteria on
agar plates with antibiotics.
6) amplify bacterial clones
7) extract and purify plasmid DNA
8) screen for plasmids containing DNA insert
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Step 1) digest DNA inserts with restriction enzyme(s).
Step 2) digest DNA plasmid vector with restriction enzyme.
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Step 5) select plasmid-containing (transformed) bacteria on agar plates with antibiotics.
Negative control
Experiment
Negative control
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Step 7) extract and purify plasmid DNA
*preparation and clearing of a bacterial lysate
*adsorption of DNA onto the QIAprep membrane
*washing and elution of plasmid DNA
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Step 8) screen for plasmids containing DNA insert
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