165a, MCDB Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
PART I: Multiple choice. Each question = 2 points. Each question has only one best answer.
1. Fixation and embedding are required for:
A) Creating tissue sections
B) Creating optical sections
C) Creating BOTH tissue and optical sections
D) Creating NEITHER tissue nor optical sections
2. Which statement is true about a fluorescent protein:
A) It emits light at a wavelength lower than the excitation wavelength.
B) It emits light at a wavelength higher than the excitation wavelength.
C) Its excitation and emission maxima are usually the same.
D) It absorbs lightwhat you see is a false colored negative image.
3. Confocal microscopy allows you to image a single section of a 3-D specimen because:
A) Excitation of fluorophores occurs only at a single focal point.
B) Fluorescent proteins are expressed only in a single plane.
C) A pinhole permits fluorophore emissions from only a single plane to reach the detector.
D) Tissues are sectioned before imaging and each section is imaged one by one.
4. The image of the cell to the right is:
A) A phase contrast image
B) A differential interference contrast image
C) A transmission electron micrograph
D) A scanning electron micrograph
5. Which of the following will affect the transition temperature of the cell membrane?
A) The relative amount of unsaturated phospholipids in the membrane
B) The degree of phosphorylation of membrane proteins
C) The sugar modifications of membrane proteins
D) All of the above.
6. Ras is farnesylated, making it a lipid-modified protein attached to the inner leaflet of the plasma
membrane. If you were to permeabilize a cell and expose it to trypsin, ras would likely be:
A) Completely impervious to trypsin digestion
B) Completely degraded by trypsin
C) Partially degraded by trypsin
D) Digested only when it was phosphorylated
7. Which statement about Fluorescence Recovery After Photobleaching (FRAP) is false:
A) It provides a measure of protein mobility.
B) It could be performed with FLAG, DAPI, or GFP labeling.
C) It could be performed using confocal or multiphoton microscopy.
D) It cannot be performed in fixed cells.
8. Which of the following is false about steroid (aka nuclear hormone) signaling pathways:
A) They are an example of juxtacrine signaling.
B) They typically do not involve a second messenger.
C) They involve translocation of a protein from the cytoplasm to the nucleus.
D) They do not require a membrane protein.
MCDB 165a, Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
9. Which statement about a standard co-immunoprecipitation experiment is false:
A) Antibodies to at least two different proteins are required to perform the experiment.
B) The antibodies used for precipitation and for blotting must have been created in different
animals.
C) Co-IP can detect direct interactions between two proteins or indirect interactions within a protein
complex.
D) A Western blot is typically the final step in a Co-IP experiment.
10. Which of the following is NOT a second messenger:
A) diacylglycerol
B) cyclic AMP
C) The Grb2 adaptor protein
D) calcium
11. Growth factors activate receptor tyrosine kinases by:
A) Phosphorylating them
B) Promoting their endocytosis
C) Causing them to dimerize
D) Cleaving PIP2
12. The gels to the right were blotted with either a phospho-specific antibody
that recognizes activated VEGFR2 or an antibody recognizing total VEGFR2
(actin blotting is a loading control). The quantification is at the bottom. Decursin
is a drug that may affect VEGF signaling. This data indicate that:
A) Decursin does not affect VEGFR2 activity.
B) Decursin causes VEGFR2 internalization.
C) Decursin potentiates VEGFR2 activity.
D) Decursin inhibits VEGFR2 activity
13. Which is a true statement about G-protein signaling:
A) The G alpha subunit is activated when it is bound to GDP
B) The beta/gamma subunits are active when alpha is bound to GTP
C) G proteins always activate a MAP kinase pathway
D) Active G alpha can translocate to the nucleus
14. Which of the following is false about basement membranes:
A) They contain laminin
B) They contain collagen IV
C) They are also known as connective tissues
D) They are required for proper organ development
15. In the image of the corneal stroma to the left, each black dot seen in
cross-section and each black line seen in longitudinal section represents:
A) A single trimeric molecule of collagen
B) A bundle of collagen trimers
C) A collagen IV/laminin/nidogen complex
D) A fibroblast cell laying down collagen fibers
MCDB 165a, Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
Part II. Short Answer. Each questions = 8-12 points. Be concise, clear, precise, and write
neatly. If an answer is unreadable or ambiguous it will be marked wrong.
1. You have discovered a complex of four membrane-associated proteins expressed by neurons and
wish to determine if they are integral membrane proteins or peripheral proteins. To study these
proteins you have created specific antibodies that recognize each one. You grow neurons in culture
and analyze three groups by western blot:
i) untreated neurons
ii) trypsin-treated neurons
iii) permeabilzed and trypsin-treated neurons
These are the results:
A) Which are the largest and smallest proteins? (2 points)
Largest: B (migrated most slowly through the gel)
Smallest: C (migrated fastest through the gel)
B) Which, if any, of the proteins are likely to be peripheral proteins (i.e., not in the membrane)?
Specify if they are on the inside or outside of the cells. (2 points)
Proteins A and C are peripheral proteins inside the cell, since cells must be permeabilized for trypsin
to destroy them.
C) Which, if any, of the proteins are integral membrane proteins? (2 points)
Proteins B and D are integral membrane proteins since they are not completely digested by trypsin
treatment in permeabalized or unpermeablized cells.
D) Which, if any, of the proteins are inserted into one leaflet of the plasma membrane? (2 points)
None of them. B and D are in the plasma membrane but must span the entire plasma membrane
since they are affected by both extracellular and intracellular trypsin.
E) Protein B exists in two forms, as evidenced by the doublet band on the Western Blot. You
hypothesize that these are either: i) two forms that are differentially glycosylated (i.e. one form has a
sugar modification and the other doesnt), or ii) two forms that are differentially phosphorylated (i.e.
one is phosphorylated and the other isnt). Based on the western blot, which of these hypotheses
is most likely? Briefly explain why. (2 points)
Protein B is morely likely phosphorylated on cytoplasmic residues. Glycosylations occur on only the
outside of cells and there are still two isoforms after treating the outside of cells with trypsin. On the
other hand, phosphorylation occurs inside the cell. Treating permeabilized cells with trypsin destroys
the distinction between the two isoforms (now there is only one), implying that the difference between
the two forms of Protein was intracellular.
MCDB 165a, Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
2. The mobility of proteins in the plasma membrane was demonstrated in 1970 by Frye and Edidin
using fused human and mouse cells. These hybrid cells (heterokaryons) were fixed at various
points after fusion, and the location of plasma membrane proteins was determined with
immunofluorescence.
A) Design a modern version of this experiment with LIVE hybrid mouse/human cells. Specify what
materials and what kind of microscopy will be used to accomplish this. (6 points)
There are multiple possible answers for this question, but this is the most straight-forward:
Tag a mouse integral membrane proteins with GFP
Tag a human integral membrane proteins with RFP
Express the two tagged proteins in mouse and human cells, respectively, and use these cells to form
a heterokaryon (fused cell). You can then image the fusion process with fluorescence microscopy.
An answer involving FRAP could also be acceptable here if properly designed.
B) Would it have been possible to perform the Frye and Edidinin experiment with two mouse cells of
the same type (rather than a human and mouse cell)? Why or why not? (3 points)
No, the antibodies would have recognized the same antigen on both cellsthere would be no way to
distinguish them.
C) Would it be possible to perform your proposed experiment from part A with two mouse cells of the
same type? Why or why not? (3 points)
Yes. You could tag mouse proteins with two different fluorescent proteins (e.g. RFP and GFP) in two
different cells. You could then monitor mixing of RFP and GFP with fluorescent microscopy.
3. You are studying a growth factor signal transduction pathway that activates a classic MAP kinase
pathway. One group of cultured cells was stimulated with growth factor and one was not. You image
several proteins in the pathway using immunofluorescence. The two columns on the right show
staining with DAPI and the MAPK scaffold protein. For the cells in the box, shade in the region
where you expect the MAPKKK and MAPK proteins to localize if stimulation was successful.
(8 points)
The MAPKKK will remain in the cytoplasm
in both conditions (like scaffold). The
MAPK will be in the cytoplasm before
stimulation (like scaffold) and in the
nucleus after stimulation (like DAPI). Active
MAPK translocates to the nucleus, but
upstream components of the MAPK
pathway (i.e. The MAPKK) remain in the
cytoplasm.
MCDB 165a, Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
4. Figure 9 from Nydegger et al.
(2006) is shown to the right. In
this experiment cells were
tranfected with HIV proteins Gag
and/or Env and the degree to
which these proteins colocalized with CD63 and CD9
was measured in
immunofluorescence images.
A) Do these experiments support or refute the hypothesis that Gag and Env require each other to
localize to CD63/CD9 microdomains? Briefly explain your reasoning. (5 points)
They support the hypothesis that they require each other. Colocalization of Env and Gag with CD63
and CD69 increase when both are present. This is most striking for Env, which substantially
increases its colocalization with the tetraspanins in the presence of Gag.
B) You hypothesize that individual tetraspanin molecules can move between microdomains. Briefly
describe an experiment to test this idea and state the expected results if the hypothesis is correct or
incorrect. (5 points)
Multiple answers are possible, but one clear way would be to use a FRAP experiment. For example,
you could tag a tetraspanin with a fluorescent protein, which would allow you to visualize
microdomains. You could then photobleach half of a cell and determine whether proteins repopulated
microdomains in this area (making sure that the number of microdomains increasedin other words,
its not just the existing microdomains that have moved as a unit). Doing this in the presence of a
protein synthesis inhibitor would be a good idea, since it would ensure that the reappearance of
microdomains was not due to new proteins added to the membrane.
5. Figure 1A from Lampugnani et al (2006) is shown to the right.
A) How were the blue and green fluorescence in this figure generated?
(2 point)
Blue: DAPI staining of nuclei
Green: Immunofluorescence using antibodies against VEGFR2.
Images were visualized with fluorescent microscopy.
B) The authors claim that the green fluorescence in this figure
represents internalized vesicles. How do they know that? (4 points)
After allowing internalization, the antibodies on the surface were removed with an acid wash, thus the
only antibodies remaining were in internal compartments.
C) According to this figure, what conditions promote internalization of the VEGF receptor? (4 points)
VEGF stimulation and sparse conditions both increase VEGFR2 internalization.
MCDB 165a, Midterm 1, 1-31-2012
Name:__________________________ UID: __________________________
6. The figure to the right from Lampugnani et al
(2006) investigates the consequences of knocking
down clathrin with an siRNA.
A) According to the figure, what is the consequence
of knocking down clathrin on VEGFR2 signaling?
(4 points)
Knocking down clathrin reduces VEGFR2 signaling,
as evidenced by less phosphorylated VEGFR2 and
less phosphorylated p44/42 MAPK.
B) Does knocking down clathrin affect VEC null or VEC positive cells more? Explain why this
difference occurs. (4 points)
VEGFR2 signaling is affected more in VEC null cells. In VEC positive cells, VEC is inhibiting
VEGFR2 internalization and signaling, so the VEGFR2 signaling is already very low. Knocking down
clathrin cannot reduce this substantially further. However, in VEC null cells, internalization and
signaling are high, so knocking down clathrin, which inhibits internalization and, as a consequence,
signaling, has a strong negative affect on VEGFR signaling.
C) According to this data, how do you expect the presence of clathrin to affect the localization of
p44/42 MAP kinase? (2 point)
Knocking down clathrin should reduce the amount of MAPK in the nucleus. Activated (i.e.
phosphorylated) MAPKs translocate to the nucleus. Since clathrin knockdown reduces signaling and
phosphorylation of MAPK, it should also prevent its translocation.
In other words:
Normal clathrin: more MAPK in the nucleus/less in the cytoplasm
Reduced clathrin: less MAPK in the nucleus/more in the cytoplasm