Molecular Engineering Applied to Cell Biology 
and Neurobiology 
Roger Y. Tsien, Ph.D. — Investigator 
Dr. Tsien is also Professor of Pharmacology and of Chemistry at the School of Medicine, University of 
California, San Diego. His undergraduate degree was from Harvard College, in chemistry and physics, but 
it was at the University of Cambridge, England, while obtaining a Ph.D. degree in physiology, that he 
was "introduced to the potential synergism between organic chemistry and cell biology. "After a postdoc- 
toral fellowship at Gonville and Cuius College, Cambridge, Dr. Tsien became a faculty member at the 
University of California, Berkeley. Seven years later his laboratory moved to the University of California, 
San Diego. His honors include the Searle Scholars Award and the Passano Foundation Young 
Scientist Award. 
THE overall goal of my laboratory is to gain a 
better understanding of information process- 
ing both inside individual living cells and in net- 
works of neurons. Our preferred approach is 
through the rational design, synthesis, and use of 
new molecules to detect and manipulate intra- 
cellular biochemical signals, usually by optical 
means such as fluorescence readout or photo- 
chemical release of messenger substances. For ex- 
ample, we have created fluorescent dye mole- 
cules that detect calcium ions (Ca^"^) with great 
specificity and sensitivity, so that while the cells 
are living and performing their normal functions, 
we can image Ca^^ levels inside cells with a spa- 
tial resolution of a micron or so and a temporal 
resolution of a fraction of a second. These dyes 
have found wide application in cell biology, 
since a rise in intracellular Ca'^^ levels is one of 
the commoner mechanisms by which cell mem- 
branes control biochemical events, such as mus- 
cle contraction, synaptic transmission, glandular 
secretion, enzyme activation, embryonic fertiliza- 
tion, and growth stimulation. 
The detection of intracellular signals such as 
Ca^* is doubly important. It should help in trac- 
ing the complex biochemistries involved in such 
signaling, and it affords a nondestructive way to 
watch the activity of many individual cells simul- 
taneously. The latter ability is particularly rele- 
vant to understanding how neural networks pro- 
cess information by harnessing many individual 
but interconnected neurons in parallel. The domi- 
nant established techniques for monitoring 
neural activity either listen intensively to a single 
neuron at a time or record some smeared-out 
average of what thousands, millions, or billions 
of cells are doing. If we can continue to improve 
the spatial and temporal resolution of present 
Ca^^ imaging, we may succeed in eavesdropping 
on conversations within small groups of individu- 
ally identified neurons or in taking snapshots of 
the instantaneous state of activity of yet larger en- 
sembles. Because optical monitoring is inher- 
ently good for following multiple events in paral- 
lel, it would be a major help in analyzing the 
workings of the brain, the most awesome and 
complex molecular assembly known. 
A recent example of molecular engineering is 
our development of a fluorescent sensor for 
cAMP. This important intracellular messenger 
plays a crucial role in the actions of a great many 
hormones, in the sensing mechanisms for odors 
and tastes, and in the mechanisms of learning and 
memory. In this case we did not design the sens- 
ing molecules from scratch but rather modified 
the natural protein that cells normally use to re- 
spond to cAMP. In collaboration with Susan Tay- 
lor and her laboratory, we have attached fluores- 
cent labels to cAMP-dependent protein kinase in 
such a way that cAMP not only activates the nor- 
mal activity of this enzyme but produces an imme- 
diate optical signal that we can image microscopi- 
cally. This labeled protein enables us to visualize 
cAMP levels, to show that neighboring cells can 
have diff^ering responses to neurotransmitter and 
drug stimulation, and to see that a subunit of the 
enzyme can move in and out of the nucleus as the 
cAMP rises and falls. While it is in the nucleus, it 
is ideally placed to modify gene expression. 
A complementary area of interest is the use of 
light both to visualize intracellular biochemistry 
and to perturb it in a controlled manner to see 
how the cell or tissue responds. Light is a stimu- 
lus that is wonderfully controllable in space and 
time. Of course most cells, other than specialized 
tissues like the retina, are not particularly sensi- 
tive to light, so the trick is to design and synthe- 
size light-sensitive organic molecules that, intro- 
duced into cells, release important messenger 
substances upon illumination. Through the work 
of many researchers, ourselves included, such 
photochemical releaser molecules are available, 
not only for Ca^^ but also other important bio- 
chemical signals such as cAMP, inositol phos- 
phates, and diacylglycerol. We have also recently 
developed the first molecule that can do the op- 
posite function — that is, gobble up the messen- 
ger upon illumination. All these photochemical 
perturbations are valuable to show whether the 
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