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