Lab 5A and 5B Overview Investigating protein sorting signals using cloning, transfection, GFP-fusion proteins, and vital stains for cellular compartments 2. Protein sorting and membrane trafficking - or - How cells deliver things to the right place 1. Fluorescent proteins and cool things we can do with them Today 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 Next Week

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 ENDOCYTOSIS (Endo = within Cyto = cell) EXOCYTOSIS (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 nuclear envelope endoplasmic reticulum lysosome early endosome late endosome Golgi apparatus cis Golgi network trans Golgi network Golgi stack CYTOSOL plasma membrane secretory vesicle

Protein sorting involves a “bucket brigade” through a series of membrane-bound compartments and vesicles

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.

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.

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.

Transport of secreted proteins from the ER to the Golgi and from the Golgi to the cell surface involves vesicle movement along microtubules. 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) Mitochondrial import 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 Endoplasmic Reticulum import H 2 N-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 P-P-K-K-K-R-K-V K-R-P-A-A-T-K-K-A-G-Q-A-K-K-K-K Endoplasmic Reticulum retention K-D-E-L-COOH

Protein XGFP Protein X-GFP translational fusion GFP-Protein X translational fusion Signal Sequence (Nuclear Localization Signal) No localization signal Will be sorted to the NUCLEUS Fusion proteins will be targeted according to the signal sequences they encode.

**** Permits dynamic and in vivo analysis**** of biological processes 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. neurons zebrafish pigs!!!?

Tyrosine 66 Glycine 65 Serine 67 Both chemical and biochemical fluorophores contain extended networks of conjugated double bonds GFP Fluorescein Rhodamine The “β-barrel” structure of GFP provides a special environment inside the living cell that enables the fluorophore to work

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. Variants of Green Fluorescent Protein and DsRed have been engineered to have different excitation and emission spectra, and other useful properties

Tracking gastrulation in a living Drosophila embryo using moesin-GFP GFP has been harnessed to study an enormous variety of biological processes

Imaging of whole organisms expressing GFP GFP has been harnessed to study an enormous variety of biological processes

Protein XGFP Protein X-GFP translational fusion GFP-Protein X translational fusion Signal Sequence (Nuclear Localization Signal) No localization signal Will be sorted to the NUCLEUS Fusion proteins will be targeted according to the signal sequences they encode.

18 Escherichia coli Bateria are Essential tools in DNA Cloning 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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Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria) GFPProtein XProtein YGFPin ZProteGFP Fusion proteins are usually introduced into cells as DNA constructs pCMV: Strong, constitutive promoter Neomycin: Selectable marker for mammalian cells BGH pA: Polyadenylation sequence Ampicillin: Selectable marker for bacterial cells pUC: Origin of replication for bacterial cells Proteins can be tagged with GFP at either end or internally

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. GFPProtein XProtein YGFPin ZProteGFP Fusion proteins are usually introduced into cells as DNA constructs

22 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

23 Step 1) digest DNA inserts with restriction enzyme(s).

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25 Step 5) select plasmid-containing (transformed) bacteria on agar plates with antibiotics. Negative control Experiment

26 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

27 Step 8) screen for plasmids containing DNA insert