II.  An investigational drug was synthesized by a graduate student in
your laboratory, and it appears SERTRAN increases the release of
serotonin (a neurotransmitter) in mice.   The drug itself is a small,
lipid soluble compound resembling a guanine nucleotide, but with a
long, hydrophobic side chain that could interact with the side chains
of amino acids on the surface of internal cellular proteins if brought
into close proximity.  Preliminary work consisting of in vivo labeling
of known serotonin producing cells (by treating mice with a FITC
fluorescent labeled drug) indicates this compound is associated with
neuronal axonal transport pathways.  You remember from your Cell
Biology II Course that small G-proteins, like Rab, are involved in
this pathway.

(A)  Describe a simple co-localization experiment that will show you
by light microscopy whether or not this drug is binding to a Rab
related protein.

(B)  Assuming you actually see a positive result in Part A, describe
an experiment that would show if this drug enhances the binding of Rab
to Syntaxin, a protein found on the inner face of the pre-synaptic
neuronal membrane.(i need all answer in deep, so pl discribe in brief)

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Request for Question Clarification by rbnn-ga on 23 Oct 2002 14:46 PDT

What does the phrase 

"(i need all answer in deep, so pl discribe in brief)"

mean?

Thank you for the question.

Because of the relatively high price for the question, I felt that it
was important to get the best possible answer from a microbiology
specialist as quickly as possible.

To do this, I contacted a professional microbiologist, who recommends
the following experimental protocols:

For part A:

1. Treat the known serotonin-producing cells (from paragraph one) with
a non-hydrolysable analogue of GTP such that all the vesicles fuse
with the target membranes

2. Treating simultaneously with the FITC-labelled drug in from the
first part, fix the  known serotonin-producing cells and stain the
preparation with a fluorescently-labelled antibody against Rab (use a
different fluorescence to FITC).

3. Take pictures using the fluorescent 'scope.

4. Use the camera's imaging software to superimpose the two images
(i.e. the green and red staining from the FITC-labelled drug and  the
labelled antibody)

5. Look for the third colour (i.e. yellow, in the areas of
co-localization).

----------------Part B-----------

For part B:

1. Using the serotonin-secreting cells from paragraph one, again treat
with the non-hydrolysable analogue of GTP to encourage all the
vesicles to fuse  with the pre-synaptic membrane, which should
encourage the Rab and syntaxin  to make contact.

2. Make cell lysates at this point and do a co-immunoprecipitation
experiment with the anti-Rab antibodies (from my answer in part A).

3. If Rab and syntaxin are associated, then syntaxin will be brought
down in the co-IP.

4. The co-IP'ed protein can then be resolved by SDS-PAGE and Western
blotted with anti-syntaxin antibodies.

Note: You will do appropriate controls and show that there is more
syntaxin brought down with Rab when the drug is used versus no drug
control; you have to control the IP too with appropriate negative
controls.

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Clarification of Answer by rbnn-ga on 23 Oct 2002 19:51 PDT

Regarding part A, since a fluorescent scope may not be permitted as a
light scope is requested, for a light scope, try the following:

Treat the serotonin-secreting cells with:

1. The drug, (that is supposed to increase vesicle fusion and
serotonin release).

2. No drug, (control; so you won't see vesicle release from the
pre-synaptic membranes).

3. A drug to inhibit the guanine nucleotide exchange factor which is
required such that Rab can exchange GDP for GTP and allow vesicle
fusion (so no vesicle fusion should be observed in this case).

These different conditions could be viewed under the light microscope;
you should be able to visualize the vescicle fusion and serotonin
release.