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MOLECULAR ENGINEERING APPLIED TO INTRACELLULAR SIGNAL TRANSDUCTION 
Roger Y. Tsien, Ph.D., Investigator 
Dr. Tsien's laboratory investigates intracellular 
signal transduction, with an emphasis on probing 
intact cells with carefully designed molecules that 
give optical readouts or controlled perturbations of 
internal biochemistry. 
Signal Transduction to the Nucleus 
Previous work from this laboratory showed that 
the catalytic subunit (C) of cAMP-dependent pro- 
tein kinase moves reversibly into and out of the nu- 
cleus upon elevation and depression of cAMP levels. 
Because such translocation may be a key step in 
carrying cAMP signals into the nucleus to affect gene 
expression, Dr. Alec Harootunian investigated its 
mechanism to decide whether nuclear import and 
export of C are active processes, perhaps controlled 
by phosphorylation, or merely passive diffusion. 
Covalent inactivation of the phosphorylating ac- 
tivity of C or co-injection of a competitive inhibitor 
of active nuclear transport did not prevent entry of 
REF-52 fibroblasts into the nucleus. Even when 
cAMP is high, exit of C from the nucleus could be 
demonstrated by increasing the laser power on the 
confocal microscope to photobleach the fluores- 
cein label on the C that remains in the cytoplasm. 
Afterward, the unbleached C in the nucleus reequili- 
brated out into the cytoplasm to the same final con- 
centration ratio and with the same kinetics as it had 
initially entered the nucleus. Therefore nuclear im- 
port and export appear to be passive diffusional pro- 
cesses once C is freed by the binding of cAMP 
to the regulatory subunit, which remains in the 
cytoplasm. 
Studies of cAMP in Aplysia sensory neurons con- 
tinued, with important controls to support the show- 
ier results obtained last year. Dr. Brian Bacskai devel- 
oped a method to calibrate the fluorescein- and 
rhodamine-labeled cAMP-dependent protein kinase 
in terms of absolute cAMP concentrations. The en- 
zyme was placed in a microdialysis capillary and 
imaged on the confocal microscope while the capil- 
lary was immersed in various cAMP concentrations. 
In collaboration with Dr. Beni Hochner from the 
laboratory of Dr. Eric Kandel (HHMI, Columbia Uni- 
versity), the basic phenomena of cAMP compart- 
mentation in the fine processes and kinase trans- 
location into the nucleus were reproduced in 
sensory-motor neuron cocultures and intact acutely 
excised ganglia, showing that they were not artifacts 
of culturing isolated neurons. Electrophysiological 
recording in intact ganglia verified that the injected 
probe did not perturb the cells' excitability or re- 
sponse to serotonin. 
Applications of Photochemistry to Probe 
Signal Transduction 
Dr. Lewis Makings studied the mechanism of pho- 
todestruction of indo-1, an important fluorescent 
Ca^^ indicator particularly suited to confocal mi- 
croscopy. He found that indo-1 does not simply 
bleach to nonfluorescent products but also under- 
goes photoconversion to a form that is still quite 
fluorescent but has lost its Ca^^ sensitivity. Both 
processes require oxygen and can be largely inhib- 
ited by reducing agents such as ascorbic acid or hy- 
droquinone. Reversibility by reductants argues that 
the key step in photodestruction is formation of the 
indo-1 free radical rather than direct oxygen addi- 
tion to the indole 2,3-double bond as previously 
thought. A further implication is that covalent at- 
446 
