Announcements

• Writing Assignment #1 returned in section• This week in section:

– Writing assignment #2 due (questions on your fig.) Group presentations of figures from nuclear pore paper

– MCQ for your presentation

Bloom et al., 1956

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Example MCQs:Which require higher-order cognitive

skills?

1. What do you predict would happen if you repeated the transport experiment in the absence of G-actin?

2. In part (C) of figure 5, what can be concluded from the 3 pulldowns on the right (mut1 - major binding pocket, mut2 - minor binding pocket, mut1+2)?

3. Which element(s) of the bipartite NLS did this figure show to be responsible for full nuclear localization?  

4. Which piece of evidence most strongly supports the hypothesis that actin and imp(alpha)-imp(beta) compete for interaction with MRTF-A RPEL?

Outline1. Wrap up Nuclear Transport2. Nuclear Environment

1. Disease associated with inappropriate nuclear structure

2. Approaches to studying nucleus:1. Biochemical fractionation (Nuclear

lamins)2. Microscopy3. GFP-tagging: Why and How

Schematic of Nuclear Pore Complex (NPC)

Into the nucleus

Ran-dependent NuclearImport

What are the steps?

Fig. 13-36

What does “ring” staining represent?

Ran-dependent Nuclear Export:Similarities and Differences with Import?

Nuclear Transport Wrap Up• Small proteins (< 30 kDa can move through nuclear

pores by passive diffusion). Not considered “nuclear import” since occurs through diffusion.

• Larger proteins require ACTIVE transport (GTP, Ran & Nuclear import receptors Importin α/β)

• Practice thinking about what would happen if proteins involved in nuclear transport were altered (always active, non-functional, placed in a different compartment in cell)

• Compare and contrast Ran-dependent mechanisms of nuclear import and export

Remaining Questions About Nuclear Transport

Take 2-3 minutes and think about what you have learned about nuclear transport over the last 2 weeks

On index cards:

Write down one question you have about this topic (something that is unclear to you, something that you want to know more about etc.)

Biochemical approaches

• 35S-Methionine added to cells

• Lyse cells and centrifuge (fractionation, p. 94)

– Pellet = all things that sediment to bottom of tube

– Supernatant = all things that stay in solution

• Immunoprecipitate w/ antibody to Lamins

• Separate on a SDS polyacrylamide gel

– SDS coats proteins with a uniform negative charge

– Electrophorese proteins through a denaturing gel toward a positive electrode

How Do We Know What Makes Up the Nuclear Environment?

Nuclear Lamin Fractionation

1.What information does a SDS-poly acrylamide gel give you? 2.What information does the control (cells) lane give you?

3.What cellular components would you expect to find in the pellet fraction? In the supernatant?

4.What do these data suggest about the location of lamins during mitosis?

5. Which of the following hypotheses do these data support?a) Nuclear envelope breakdown is mediated by proteolytic degradation of nuclear laminsb) Nuclear envelope breakdown is mediated by disassembly of nuclear lamins

Size and amount

The total amount of lamins present prior to the fractionationThe size of the lamins prior to the fractionation

pellet: Organelles, cytoskeletonsupernatant: soluble proteins

Shifts from an insoluble to a soluble fraction, suggesting dissociation from the nuclear envelope during mitosis

b

What Initiates N.E. Breakdown?

• Phosphorylation of nuclear lamins by the cyclin-dependent kinase p34cdc2 initiates depolymerization of nuclear lamins resulting in nuclear envelope breakdown.

• What happens to lamins after mitosis?

Nuclear Envelope Re-assembly After Mitosis

Another role for Ran-GTP:• Ran GEF associates with Histone H2A/2B• Increased concentration of Ran GTP around chromatin promotes fusion of ER to form mini nuclei• Mini nuclei fuse together to form nucleus

Progeria: rare autosomal dominant disease due to mutation in Lamin A

http://ghr.nlm.nih.gov/condition/hutchinson-gilford-progeria-syndrome

Microscopy Tools• Different types of microscopy

– light microscopy– fluorescence & immunofluorescence microscopy– Fluorescence recovery after photobleaching (FRAP)

• What you should know:– What information can you learn using each general

type of microscopy?– What biological questions can you answer/not

answer with each type of microscopy?

Labeling Proteins with GFP

• GFP = Green florescent protein• Naturally fluorescent 30 Kda

protein• Used to “tag” individual proteins

in living cells and organisms• Create a fusion protein• Why would you want to tag a

protein with GFP?

FGFR-3 GFP

Imagine you want to determine the subcellular location of a fibroblast growth factor

receptor. How could you do this?

• What part(s) of the receptor gene will you include?

Hint: Where would you expect this protein to be located in the cell?

• Where will GFP be attached?• How could you visualize the location of your fusion

protein?• What additional scientific questions can you answer

with your new tool?• What assumptions are you making when you interpret

data from an experiment which uses a fusion protein?

Homework:

Imagine you want to determine at what developmental stage and in what tissues the -actin gene is expressed in a hydra

How could you design a GFP experiment to answer these questions?– What key part(s) of

the -actin gene would you need to include in your DNA construct?

GFP-fusion analysis

• Advantages:

• Disadvantages:

Live imaging within a cell or organism

Do not have to process/kill a cell to view a protein

Altering a protein’s structure and potentially functionLow resolution

http://www.nichd.nih.gov/news/releases/green_brown_mice.cfm

Spatial Organization in Nucleus:Why do we care?