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ROLE OF INTRACELLULAR SPHINGOSINE AND SPHINGOSINE-1PHOSPHATE
IN CALCIUM SIGNALING
This research project is supported by the R01-GM67892
grant from the National Institutes of Health (NIGMS)
Alterations in calcium (Ca) signaling are implicated in human pathologies
ranging from cancer to neurodegenerative diseases. The spatio-temporal
characteristics of Ca signals can regulate the activity of transcription
factors and directly affect gene expression. Ca is generally mobilized
from intracellular stores by inositol 1,4,5-trisphosphate (InsP3), which
activates specific receptors containing an intrinsic Ca channel (InsP3Rs)
and are localized to the endoplasmic reticulum. Notably, many cell-surface
receptors that increase InsP3 induce the simultaneous intracellular production
of sphingosine and sphingosine-1 phosphate (sphingosine-1P) as well.
A Ca-mobilizing role for these two sphingolipids has been proposed, but
their mechanism of action is unclear and the intracellular Ca channel(s)
they activate still unidentified. Furthermore, their functional interactions
with InsP3 have not been characterized. We aim to test the hypothesis
that both intracellular sphingosine and sphingosine-1P mobilize Ca through
InsP3Rs.
This project aims to:
- Characterize the Ca-mobilizing action of intracellular
sphingosine and sphingosine-1P - acting separately or in
combination with InsP3 - using caged-molecules that will
be photo-activated inside living cells and their effects
analyzed by single-cell microfluorimetry and digital imaging.
The reciprocal interactions between InsP3 and the two sphingolipids
during Ca signaling following the stimulation of several
plasma membrane receptors will also be investigated;
- Establish whether the expression of different InsP3R-subtypes
can determine the type of Ca signals evoked by intracellular
sphingosine and sphingosine-1P. Experiments using cells
genetically engineered to express specific InsP3R-subtypes
will be combined with studies performed with mammalian cell
lines transfected with distinct InsP3R-subtypes;
- Ascertain whether sphingosine and sphingosine-1P open
the InsP3R-channel and/or functionally modulate the action
of InsP3. To this end, single-channel recording from purified
and recombinant InsP3R subtypes reconstituted in planar
lipid bilayers will be employed.
Our long-term objective is to identify the mechanisms and
intracellular channels through which sphingosine and sphingosine-1P
mobilize intracellular Ca - acting alone or in combination
with InsP3 - and characterize their role in cellular signaling
following the stimulation of plasma membrane receptors.
Why use
caged compounds?
Caged compounds are molecules that
are rendered biologically inert by chemical modification
of their active functionality with a photo-removable
protecting group. Irradiation produces a concentration
jump from the caged compound to a biologically active
molecule. Photo-release of bioactive molecules (nucleotides,
peptides, neurotransmitters, second messengers, proteins)
has been used to study and dissect the myriad of specific
molecular interactions that control the function of
living cells in real time. This photochemical approach
has many advantages compared to other methods for changing
solute concentration, namely release can be (1) highly
localized, (2) ultra-fast, (3) timed, controlled and
repeated at any point during an experiment, (4) intracellular,
and (5) physically non-perturbing. |
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The
development of other technologies such as the patch
clamp technique and confocal microscopy has had a fundamental,
broad ranging impact on biological sciences, and has
thus revolutionized our understanding of many cellular
functions. Caged compounds complement these techniques
by providing both temporal and spatial control of cellular
chemistry. A particularly significant feature of the
uncaging technique for our studies is that its use
allows us to "by pass" the normal physiological
means of producing the second messenger (InsP3 or sphingolipids),
and so permits us to isolate the down-stream effects
of release from the up-stream production of each second
messenger. Thus, photorelease of a second messenger
is an ultimate reductionist approach to the dissection
of intracellular signaling pathways: if uncaging of "X" produces
effect "Y", then we have established definitely
that X alone is sufficient, and probably necessary,
for Y.
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Cells loaded with caged Nitric Oxide,
which is then photo-released by three consecutive flashes
of UV-light of increasing intensity and illuminating
the center of the field. The release of nitric oxide
within the cell is detected by the fluorescent probe
DAF-FM and is expressed as an increase in fluorescence.
Cells excluded by the UV-light illumination (red arrows)
do not release nitric oxide and display a stable fluorescent
signal.
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