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MCB 130L Part 2 Lecture 3 Transfection and Protein localization. Exploring protein function. 1) Where is it localized in the cell?. Approaches: a) Make antibodies - immunofluorescence. b) “Express” the protein in cells with a tag  Fuse to GFP. 2) What is it doing in the cell?.

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MCB 130L Part 2 Lecture 3

Transfection and

Protein localization


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Exploring protein function

1) Where is it localized in the cell?

Approaches:

a) Make antibodies - immunofluorescence

b) “Express” the protein in cells with a tag

 Fuse to GFP

2) What is it doing in the cell?

Approaches:

a) Reduce protein levels - RNA interference

b) Increase protein levels “over-express”

c) “Express” mutant versions


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Exploring protein function

1) Where is it localized in the cell?

Approaches:

a) Make antibodies - immunofluorescence

b) “Express” the protein in cells with a tag

 Fuse to GFP

2) What is it doing in the cell?

Approaches:

a) Reduce protein levels - RNA interference

b) Increase protein levels “over-express”

c) “Express” mutant versions

Transfection!!!!


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Transfection =

Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein

= “expressed”


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Direct introduction of the DNA

Electroporation - electric field temporarily disrupts plasma membrane

Biolistics (gene gun)- fire DNA coated particles into cell

Microinjection


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Infection:

Use recombinant viruses to deliver DNA

Retroviruses

Adenoviruses

Virally-mediated introduction of the DNA


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Carrier-mediated introduction of the DNA

Positively charged carrier molecules are mixed with the DNA and added to cell culture media:

Calcium Phosphate

DEAE Dextran

liposomes

micelles

Carrier-DNA complexes bind to plasma membrane and are taken up


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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 resistance required

“stably transfected” cell line


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DNA “expression” vector transfected:

Insert gene

in here

For expression in cells

Polyadenylation

site

GFP

CMV

Promoter

SV40

Promoter

To generate stable cell line

pCMV/GFP

Ampicillin

resistance

Neomycin

resistance

For amplification of the plasmid in bacteria

Polyadenylation

site

pUC

Bacterial origin of replication


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Three ways to make Green fluorescent protein “GFP” fusion constructs:

PROTEIN X

GFP

GFP

PROTEIN Y

PROTEIN

GFP

Z


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EXPERIMENT: fusion constructs:

Transfect unknown GFP fusion protein

Protein X, Y or Z

Visualize GFP protein fluorescence by fluorescence microscopy in living cells

Counter-stain with known marker to compare localization patterns inliving cells

= “vital stain”


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Some Cellular Organelles fusion constructs:


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Nuclei fusion constructs:

Mitochondria

Secretory Pathway:

Endoplasmic Reticulum

Golgi Complex

Endocytotic Pathway:

Endosomes

  • Compartments/organelles examined

  • Protein sequences sufficient for localization

  • Vital stains


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Nucleus fusion constructs:

Transport through nuclear pore

signal = basic amino acid stretches

example: P-P-K-K-K-R-K-V



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Nuclear Stain: fusion constructs:

Hoechst 33258 binds DNA


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Mitochondria fusion constructs:

Transmembrane transport signal

Example: 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



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Mitochondrial dye = MitoTracker Red fusion constructs:

Diffuses through membranes

Non-fluorescent until oxidized

Accumulates in mitochondria and oxidized

Mitotracker

DNA


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lysosome fusion constructs:

plasma

membrane

late

endosome

nuclear envelope

endoplasmic reticulum

early

endosome

CYTOSOL

cis

Golgi

network

Golgi

stack

trans

Golgi

network

Golgi apparatus

Cellular components of the secretory and endocytic pathways


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Endoplasmic Reticulum fusion constructs:

Entry into E.R.:

Transmembrane transport signal

= hydrophobic amino acid stretches

Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q

at amino terminus

Retention in E.R. lumen:

Signal = K-D-E-L-COOH

at carboxy terminus


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Endoplasmic Reticulum marker fusion constructs:

ER-Tracker Blue-White

Live bovine pulmonary artery endothelial cells


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Mitotracker Red fusion constructs: and ER-blue/white


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Golgi fusion constructs:

From the ER, secreted and membrane proteins move to the Golgi, a

series of membrane-bound compartments found near the nucleus

nucleus


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Golgi marker fusion constructs:

BODIPY-TR ceramide

Ceramide = lipid

When metabolized, concentrates in the Golgi

Red fluorophore


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Cultured Epithelial Cells fusion constructs:

DNA (Hoechst)

Golgi (ceramide)

Steve Rogers, U. Illinois


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MDCK Cells fusion constructs:

Madin-Darby Canine Kidney

Polarized Epithelial Cells

DNA (Hoechst)

Golgi (ceramide)

Lysosomes (LysoTracker)

Molecular Probes, Inc.


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Endocytosis can be divided into 3 categories: fusion constructs:

1. Phagocytosis - “eating”

2. Pinocytosis - “sipping”

3. Receptor-mediated endocytosis:

deliberate uptake of specific molecules


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cis fusion constructs:

Golgi

network

Golgi

stack

trans

Golgi

network

Cellular components of the endocytic pathway

lysosome

plasma

membrane

late

endosome

nuclear envelope

endoplasmic reticulum

early

endosome

CYTOSOL

Golgi apparatus


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Endosomes - pinch off from plasma membrane fusion constructs:

Clathrin -coated pits and vesicles


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RECEPTOR-MEDIATED ENDOCYTOSIS occurs through special fusion constructs:

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.


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Iron is carried in fusion constructs:

blood by the protein

TRANSFERRIN

and is taken up into

cells by endocytosis

mediated by the

TRANSFERRIN

RECEPTOR

Inside the endosome

Fe3+ is released.

Transferrin receptors

then return to the

cell surface, where

the transferrin

dissociates


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Rhodamine transferrin fusion constructs:

Does the fluorescent green protein co-localize?


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  • TODAY: fusion constructs:

  • Transfect Cells transiently with unknown protein X, Y or Z fused to GFP

  • In two days:

  • Vital stain with another dye to compare

  • Visualize both GFP and dyein the same living cells! by fluorescence microscopy

Where are the unknown proteins localized???


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