lab 5a and 5b overview investigating protein sorting signals using cloning, transfection, gfp-fusion...
TRANSCRIPT
Lab 5A and 5B Overview Investigating protein sorting signals using cloning, transfection,GFP-fusion proteins, and vital stains for cellular compartments
1. Protein sorting and membrane trafficking- or -
How cells deliver things to the right place
2. Fluorescent proteins are critical tools in Cell biologyLast Week
3. Transfection and transgene expression - or -
How we get DNA into cellsto express “designer” genes
4. Fluorescent markers for differentcompartments of the secretory
and endocytic pathways
This
Week
Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria)
GFPProtein X
Protein YGFP
in ZProte GFP
Fusion proteins are usually introduced into cells as DNA constructs
pCMV: Strong, constitutive
promoter
Neomycin:Selectable marker formammalian cells
BGH pA:Polyadenylationsequence
Ampicillin:Selectable marker for bacterial cells
pUC:Origin of replication
for bacterial cells
Effectene®
The negatively-charged phosphate backbone of DNAmust be neutralized by positively charged counterions
to allow transport across the plasma membrane.
TRANSFECTION - from trans, meaning “across”
DNA is a large, charged molecule that normally doesn’t cross cell membranes… so we have to use tricks to get it into cells
Principle of transfectionwith Effectene® reagent
+ Enhancer
Outline of transfection protocol (Qiagen)
There are many points along the way where transfection efficiency can be compromised
Proton sponge hypothesis: sequestration of cations by DNA leads to the osmotic
swelling and rupture of endosomes, releasing DNA vector into the cytoplasm
Only a fraction of treated cells will be successfully transfected, and thus the expression of the transgenewill be quite VARIABLE - here GFP is used as a marker
of transfection
Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria)
GFPProtein X
Protein YGFP
in ZProte GFP
Fusion proteins are usually introduced into cells as DNA constructs
pCMV: Strong, constitutive
promoter
Neomycin ResistanceGene: Selectable markerFor mammalian cells
BGH pA:Polyadenylationsequence
Ampicillin:Selectable marker for bacterial cells
pUC:Origin of replication
for bacterial cells
Alternative strategies to ectopically express genes/siRNAs
Retroviral Vectors
-High Efficiency-Stable DNA integration-Replication Incompetent-Level 2 Bio-Safety
Transfect Retrovirus
Electroporation
-High Efficiency-Many Cells/even intact tissues-Cell Fusion-Loss of intracellular components
Gene Gun
-Applicable to many tissues-Penetrates Mitochondria/Chloroplasts-Shallow penetration of particles-Cell damage
Microinjection
-No selection process-DNA delivery accurately controlled-Minimal perturbation of cells-Technically difficult/few cells
Alternative strategies to ectopically express genes/siRNAs
4 transfectedconstructs
Z
Y
X
U
5 vital dye counterstains
Golgi
Endosome
ER
Mitochondria
Nucleus
Ceramide is a LIPID that gets trapped after modification
in the Golgi apparatus
Label Ex Em
BODIPY-TR
Green Red
Fluorophore
BODIPY®-TR Ceramide (Molecular Probes)
Steve Rogers, U. Illinois
Golgi (ceramide)
DNA (Hoechst)
Cultured Epithelial Cells
Iron is carried inblood by the proteinTRANSFERRINand is taken up intocells by endocytosismediated by the TRANSFERRIN
RECEPTOR.
EX EM
RedGreen
RhodamineRhodamine-labeled-labeledTRANSFERRIN proteincan be used to trackreceptor-mediatedendocytosis
MitoTracker Red CM-H2XRos
“…the reduced versions of these probes do not fluoresce until they enter an actively respiring cell, where they are oxidized to the fluorescent mitochondrion-selective probe and then
sequestered in the mitochondria.” MOLECULAR PROBES handbook
EX EM
RedGreen
Image from Nikon
Cultured Lung Epithelial Cells
Mitochondria (MitoTracker)DNA (DAPI)
Actin (Phalloidin)
“ER-Tracker Blue-White DPX is a highly selective and photostable stain for the ER in live cells…
Staining at low concentrations does not appear to be toxic to cells.”
(MOLECULAR PROBES Handbook)
ER-Tracker Blue-White DPX
EX EM
BlueUV
Image from Invitrogen
ER (ER-Tracker)
Cultured Endothelial Cells
4 transfectedconstructs
Z
Y
X
U
5 vital dye counterstains
Golgi
Endosome
ER
Mitochondria
Nucleus
20 DataSets
Potential Challenges Encountered in this Week’s Lab
Photobleaching
Autofluorescence - increases as cells die
Bleed-through between different filter sets
Nonspecific labeling of organelles
Data Management
Fluorescence MicroscopyStokes’ shift
intensity excitation
and emissionfilters
wavelength
Fluorophore (or “Fluorochrome”)
Excitation maximum
Emissionmaximum
Fluorescein 490 520
Rhodamine 550 580
DAPI or Hoechst 345 455
Fluorescence wavelength filters must be designed to match the
excitation/emission spectra of the fluorophores you plan to use.
Bodipy-TR ceramide
Red Channel
Autofluorescenceof dying cells
GFP
GFP Channel
Simultaneous localization of cellular components
To be useful, a “counterstain” should fluoresce at a wavelength
different from GFP - e.g. RED or BLUE
Mitochondria (MitoTracker)Lysosomes (Lyso-Tracker)
N
“Colocalization” can help to establish
that twomolecules are in the same place at the
same time.If the location of
one is known, it can reveal the
location of a less well-characterized
component.
Mitochondria(MitoTracker)
ColocalizationGFP Fusion Protein
-Beech et al. Science (2000)
-Invitrogen