401Lecture3Sp2013post

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Transcript 401Lecture3Sp2013post

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
Lower-order
Cognitive Skills
Higher-order
Cognitive Skills
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?
3. Which element(s) of
the bipartite NLS did
this figure show to be
responsible for full
nuclear localization?
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)?
4. Which piece of
evidence most strongly
supports the hypothesis
that actin and
imp(alpha)-imp(beta)
compete for interaction
with MRTF-A RPEL?
Outline
1. Wrap up Nuclear Transport
2. Nuclear Environment
1. Disease associated with inappropriate
nuclear structure
2. Approaches to studying nucleus:
1. Biochemical fractionation (Nuclear
lamins)
2. Microscopy
3. GFP-tagging: Why and How
Schematic of Nuclear Pore
Complex (NPC)
Into the nucleus
Randependent
Nuclear
Import
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.)
How Do We Know What Makes Up
the Nuclear Environment?
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
Nuclear Lamin Fractionation
1.What information does a SDS-poly acrylamide gel give you?
Size and amount
2.What information does the control (cells) lane give you?
The total amount of lamins present prior to the fractionation
The size of the lamins prior to the fractionation
3.What cellular components would you expect to find in the pellet
fraction? In the supernatant?
pellet: Organelles, cytoskeleton
supernatant: soluble proteins
4.What do these data suggest about the location of lamins during
mitosis?
Shifts from an insoluble to a soluble fraction, suggesting
dissociation from the nuclear envelope during mitosis
5. Which of the following hypotheses do these data support? b
a) Nuclear envelope breakdown is mediated by proteolytic degradation
of nuclear lamins
b) Nuclear envelope breakdown is mediated by disassembly of nuclear
lamins
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:
Live imaging within a cell or organism
Do not have to process/kill a cell to view a protein
• Disadvantages:
Altering a protein’s structure and potentially function
Low resolution
http://www.nichd.nih.gov/news/releases/green_brown_mice.cfm
Spatial Organization in Nucleus:
Why do we care?