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 University of California School of 
Medicine, 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 
postdoctoral fellowship at Gonville and Caius 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 recent honors include the Passano Foundation Young Scientist Award, the 
Spencer Award in Neurobiology from Columbia University, and the Bowditch Lectureship of the American 
Physiological Society. 
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 inside the 
ceil, such as muscle contraction, synaptic trans- 
mission, glandular secretion, enzyme activation, 
embryonic fertilization, 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 imaging is inherently good for 
following multiple events in parallel, it would be 
a major help in analyzing the workings of the 
brain, which is still the most awesome and com- 
plex molecular assembly known. We recognize 
that optical methods, although unsurpassed in 
their combination of spatial and temporal resolu- 
tion, are best applied to small regions of thin 
transparent tissues. For larger volumes of opaque 
organs, especially in intact organisms, other 
forms of visualization, such as magnetic reso- 
nance imaging, are more appropriate, so we are 
also seeking to extend our molecular designs to 
create suitable non-optical indicators. 
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 detection of odors and tastes, 
and in the mechanisms of learning and memory. 
In this case we did not design the sensing mole- 
cules from scratch but rather modified the natu- 
ral protein that cells normally use to respond to 
cAMP. In collaboration with Susan Taylor and her 
laboratory, Stephen Adams attached fluorescent 
labels on cAMP-dependent protein kinase in such 
a way that cAMP not only activates the normal 
activity of this enzyme but produces an immedi- 
ate optical signal that we can image microscopi- 
cally. This labeled protein enables us to visualize 
cAMP levels, to show that diff'erent regions of a 
single cell can have differing responses to neuro- 
transmitter and drug stimulation, and to see that a 
subunit of the enzyme can move in and out of the 
nucleus as the cAMP level rises and falls. While it 
is in the nucleus, it is ideally placed to modify 
gene expression. 
A particularly dramatic example of the dy- 
namics of cAMP signaling comes from sensory 
neurons of the marine mollusk Aplysia califor- 
nica. These neurons have been extensively stud- 
ied by Eric Kandel (HHMI, Columbia University) , 
James Schwartz, and their collaborators as models 
for both short- and long-term neuronal plasticity. 
427 
