1. An approach to compare internalization of clustered protein in cells expressing the wild-
type and mutant forms of VSV-G protein
1Andalus Ayaz, 2Dr. Deborah Brown
Stony Brook University1, Department of Biochemistry and Cell Biology, State University of New York At Stony Brook2
INTRODUCTION
It is known that if proteins on the plasma membrane are
clustered, they can be internalized into the cells. Not much is
known about this process.
In our lab we use antibodies to cluster proteins. Our hypothesis
is that when the antibodies bind to the proteins, the proteins form
clusters and consequently, recruit a lipid molecule in the plasma
membrane called PIP2. We believe this is possible because the
cytoplasmic tail of the protein contains basic (positive) amino
acids which can electrostatically attract the negatively charged
head groups PIP2.
plasma membrane
PIP2 molecules in orange
MUTAGENESIS:
We used PCR to mutate the basic amino acids lysine and arginine in the cytoplasmic region of
VSV-G into neutral amino acids, alanine, to see if internalization was affected in the mutant form of
the protein.
Cytoplasmic tail of wild-type VSV-G (blue indicates basic amino acids; red indicates acidic amino
acids):
L R V G I H L C I K L K H T K K R Q I Y T D I E M N R L G K *
Cytoplasmic tail of mutant VSV-G:
L R V G I H L C I A L A H T A A A Q I Y T D I E M N R L G K *
This research was supported by a grant from SUNY RF for
Explorations in STEM Research program
INTERNALIZATION OF WILD-TYPE VSV-G:
We bound mouse anti-VSVG antibodies to the wild-type VSV-G
protein on the plasma membrane for clustering. Next, we bound goat
anti-mouse green fluorescent antibodies to the mouse anti-VSVG
antibodies at 37oC for specific times to allow internalization to take
place in cells. Later we bound red fluorescent cow anti-goat antibodies
to the remaining VSV-G on the plasma membrane on ice. We noticed
the rate of internalization was affected by the amount of protein being
expressed by the cell. We thought the higher the amount of protein
present in a cell, the more internalization there would be. However, we
saw that cells which expressed lower amounts of VSV-G showed faster
internalization.
The yellow color shows co-localized green and red fluorescence ,
identifying the VSV-G protein on the plasma membrane. The green
dots show the internalized VSV-G because they are not on the plasma
membrane.
COMPARISON OF CELLULAR DISTRIBUTION AND EXPRESSION
BETWEEN WILD-TYPE AND MUTANT FORMS OF VSV-G USING
CONFOCAL MICROSCOPY
We bound mouse anti-VSVG antibodies to wild-type and mutant VSVG on the plasma
membrane, followed by fluorescent goat anti-mouse antibodies. We quantitated the
expression levels of the wild-type and the mutant forms of VSV-G through mean
fluorescence intensity using confocal microscopy. It seemed both protein types were
being expressed at the same level on the plasma membrane. In another experiment, we
used mean fluorescence intensity to quantitate total cellular expression for both wild-type
and mutant VSVG. We fixed and permeablized cells so that mouse anti-VSVG
antibodies, followed by fluorescent goat anti-mouse antibodies, would have access to
wild-type and mutant VSVG inside the cells. We also used rabbit anti-VSVG antibodies
followed by fluorescent goat antibodies in this experiment for a separate set of cells. Our
results showed that even though cells stained with rabbit anti-VSVG antibodies were
brighter, both protein types were being expressed in cells at the same level.
FURTHER STUDIES:
• Since we know that lower expressing cells with wild-type
VSV-G internalize faster, we can try to use confocal
microscopy to see internalization in cells expressing low
amounts of mutant VSV-G protein by quantitating how much
protein has been taken in. Even though the mutant protein is
not present as much as the wild-type protein in the cells, we
are interested in the amount of protein present at the plasma
membrane and our results seem to indicate that they amount
of protein at the plasma membrane is the same.
• Also, to investigate why mutant VSV-G is not expressed as
much as the wild-type VSVG in the cell as a whole, we can
carry out experiments to see if there are any problems with the
transport of mutant VSV-G protein or if it’s being broken
down while travelling to the plasma membrane.
APPROACHES:
Our current assay for quantitating internalization rates
works by quantitating internalized fluorescent
antibodies after binding to VSV G on the plasma
membrane in the cold, warming to allow
internalization and then stripping remaining surface-
bound antibodies with acid. To compare
internalization of different forms of a protein with this
assay, they must be expressed on the plasma
membrane at the same level. However, this may not
be true of wild type and mutant VSV G. Hence, we
have started developing a new assay in which total
surface bound or internalized antibodies are analyzed
by quantitative Western blotting using the Odyssey
machine. We first bind mouse anti-VSVG antibodies
to cells in the cold and then probe them with green
fluorescent donkey anti-mouse antibodies. So far,
we’ve used this assay for wild-type VSVG. Once we
figure it out, we will use it for mutant VSVG as well.
Cytoplasm of cell
However, when we compared the cellular expression
levels of the wild-type and mutant VSV-G using
western blots, we saw that the mutant VSV-G was
expressed significantly lesser than the wild-type
VSV-G.
Left to right: 2 duplicate samples
of 0.4ug of total bound antibody.
Last lane was successfully
stripped off antibodies with acid.
Total cellular expression for wild-
type VSV-G
Total cellular expression for mutant
VSV-G
Low expression of wild-type VSVGHigh expression of wild-type VSV-G
Left to right: (1) cellular expression
of wild-type VSVG; (2) cellular
expression of mutant VSVG
Antibodies (in blue)
bound to proteins (in
green)
0
100
200
300
400
500
600
Meanfluorescentintensity
wild-type
mutant
Left to right: (1) wild-type and
mutant VSVG bound with mouse
anti-VSG antibodies; (2) wild-type
and mutant VSVG bound with
rabbit anti-VSVG antibodies.