Signal Transduction across the Plasma Membrane - Single Molecule Dynamics in Life Science - page 109

To gain insights into mechanisms of stochastic signal processing and transduc-
tion, it is important to elucidate experimentally the stochastic nature of signaling
molecules. For this purpose, we have developed single-molecule imaging techniques
in living cells, and have applied these techniques to chemotactic signaling systems in
Dictyostelium cells, which have successfully revealed the stochastic nature of the
signaling molecules responsible for chemotaxis [22 - 24]. We have also developed a
theoretical framework to analyze the impact of noise associated with the signal
transduction processes [25]. We begin with a brief introduction to chemotactic
signaling systems in Dictyostelium cells.

5.7
Chemotactic Signaling System of Eukaryotic Cells

Molecular mechanisms of chemotactic response are highly conserved among many
eukaryotic cells including human leukocytes and Dictyostelium cells. In Dictyostelium
cells, extracellular adenosine 3 0 ,5 0 -monophosphate (cAMP) molecules trigger che-
motactic signaling by binding to G protein-coupled cAMP receptor (cARs), which is
mediated through G protein-liked signaling pathways including heterotrimeric G
protein, Ras, PI3K, PTEN, PH-domain-containing proteins, guanylyl cyclase, PLC g

and PLA 2 [26, 27]. One of the key reactions in this signaling system is a distinctive
localization of phosphatidylinositol 3,4,5-trisphosphates (PI(3,4,5)P 3 ) on the mem-
brane exposed to a higher concentration of cAMP, which can be monitored by the
binding of PH-domain-containing proteins to PI(3,4,5)P 3 on the membrane. Similar
localizations of PH-domain-containing proteins have been observed in mammalian
leukocytes and fibroblasts. The localization of PI(3,4,5)P 3 depends on dynamic
signaling processes by PI3K and PTEN because they control the production and
degradation of PI(3,4,5)P 3 , respectively, inwhich reciprocal distributions of PI3K and
PTEN leads to the accumulation of PI(3,4,5)P 3 at the leading edge of chemotaxing
cells. PI(3,4,5)P 3 has been suggested to produce pseudopod extensions by recruiting
PH-domain-containing proteins to the side of membrane exposed to a higher
concentration of chemoattractants. Thus, chemotactic signaling systems can convert
small differences in extracellular signals into localized signals for promoting the
preferential formation of pseudopods. The localization of PI(3,4,5)P 3 takes place in
an all-or-nonemanner, suggesting that noisy input signals are somehow processed to
generate a clear signal re ecting the direction of the gradient of extracellular cAMP
within cells.

5.8
Stochastic Nature of Chemotactic Signaling Molecules

To monitor input signals for chemotactic response, we prepared a fluorescent analog
of cAMP (Cy3-cAMP) and used a total internal re ection fluorescence microscope
(TIRFM) with which Cy3 - cAMP binding to the receptors can be observed on the