daw lecture 3

8/14/2019 Daw Lecture 3

1/34

MCB 130L Part 2 Lecture 3

Transfection andProtein localization


2/34

Exploring protein function1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

b) Express the protein in cells with a tag Fuse to GFP

Approaches:a) Reduce protein levels - RNA interference

b) Increase protein levels over - express

c) Express mutant versions


3/34

Exploring protein function1) Where is it localized in the cell?

2) What is it doing in the cell?

Approaches:a) Make antibodies - immunofluorescence

Approaches:a) Reduce protein levels - RNA interference

Transfection!!!!

b) Express the protein in cells with a tag Fuse to GFP

b) Increase protein levels over - express

c) Express mutant versions


4/34

Transfection =

Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein

= expressed


5/34

Direct introduction of the DNA

Electroporation - electric

field temporarily disruptsplasma membrane

Biolistics (gene gun)- fireDNA coated particles intocell

Microinjection


6/34

Infection:Use recombinant viruses to deliver DNA

RetrovirusesAdenoviruses

Virally-mediated introduction of the DNA


7/34

Positively charged carrier moleculesare mixed with the DNA and added tocell culture media:

Calcium Phosphate

DEAE Dextranliposomesmicelles

Carrier-DNA complexes bind to plasmamembrane and are taken up

Carrier-mediated introduction of the DNA


8/34

Types of Transfection

Transient: Expression assayed 24-48

hours post transfection

Stable: Integration of the transfected DNA

into the cell genome - selectable

marker like neomycin resistancerequired

stably transfected cell line


9/34

DNA expression vector transfected:

pCMV/GFP

Insert gene

in herePolyadenylation

site

PolyadenylationsitepUC

Bacterial origin of replication

Forexpressionin cells

Togeneratestable cellline

Foramplificationof theplasmid inbacteria


10/34

PROTEIN X

PROTEIN Y

GFP

GFP

GFP ZPROTEIN

Three ways to make Green fluorescentprotein GFP fusion constructs:


11/34

EXPERIMENT:

Transfect unknown GFP fusion proteinProtein X, Y or Z

Visualize GFP protein fluorescence by fluorescencemicroscopy in living cells

Counter-stain with known marker to compare

localization patterns in living cells = vital stain


12/34

Some Cellular Organelles


13/34

Compartments/organelles examined Protein sequences sufficient for localization Vital stains

Secretory Pathway: Endoplasmic Reticulum

Golgi Complex

Endocytotic Pathway: Endosomes

Mitochondria

Nuclei


14/34

Transport through nuclearporesignal = basic amino acid

stretchesexample: P-P- K-K-K-R-K -V

Nucleus


15/34

Import of proteins into nucleus through nuclear pore


16/34

Nuclear Stain:

Hoechst 33258binds DNA


17/34

Transmembrane transport signalExample: H 2 N-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

Mitochondria


18/34

Protein being transported across mitochondrial membranes


19/34

Mitochondrial dye =MitoTracker Red

Non-fluorescent untiloxidized

Accumulates inmitochondria and oxidized

Diffuses throughmembranes

Mitotracker

DNA


20/34


21/34


22/34

ER-Tracker Blue-White

Live bovine pulmonary arteryendothelial cells

Endoplasmic Reticulum marker


23/34


24/34


25/34


26/34

Steve Rogers, U. Illinois

Golgi (ceramide)DNA (Hoechst)

Cultured Epithelial Cells


27/34

Golgi (ceramide)

Lysosomes (LysoTracker)

DNA (Hoechst)

MDCK CellsMadin-Darby Canine KidneyPolarized Epithelial Cells

Molecular Probes, Inc.


28/34

Endocytosis can be divided into 3 categories:

1. Phagocytosis - eating

2. Pinocytosis - sipping

3. Receptor-mediated endocytosis:deliberate uptake of specific molecules


29/34

nuclear envelope

endoplasmic reticulum

lysosome

earlyendosome

lateendosome

Golgi apparatus

cis Golgi

network

trans Golgi

network

Golgistack

CYTOSOL

plasmamembrane

Cellular components of the endocytic pathway


30/34


31/34

RECEPTOR-MEDIATED ENDOCYTOSIS occurs through specialmembrane 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 coatedvesicles.


32/34

Iron is carried inblood by the proteinTRANSFERRIN and is taken up intocells by endocytosismediated by theTRANSFERRIN

RECEPTOR

Inside the endosome

Fe3+

is released.Transferrin receptorsthen return to thecell surface, wherethe transferrindissociates


33/34

Rhodamine transferrin

Does the fluorescent green protein co-localize?


34/34

TODAY: Transfect Cells transiently withunknown protein X, Y or Z fused to GFP

In two days: Vital stain with another dye to compare Visualize both GFP and dye in the sameliving cells! by fluorescence microscopy

Where are the unknown proteinslocalized???

daw lecture 3

Documents