Molecular Engineering Applied to Cell Biology and Neurobiology 
In collaboration with Dr. Kandel's group, Brian 
Bacskai and Martyn Mahaut-Smith have injected 
the labeled protein kinase into the neurons, ei- 
ther in culture or in intact ganglia, and imaged 
the nucleus, the surrounding cytoplasm of the 
cell body, and the peripheral outgrowths of the 
cell. Bath application of the relevant neurotrans- 
mitter, 5-hydroxytryptamine, produces rapid in- 
creases in cAMP with remarkable spatial gra- 
dients — high in the peripheral outgrovvths yet 
only slightly elevated in the central cell body. 
Optical sections through the nucleus show that 
it tends to exclude the holoenzyme (injected into 
the cytoplasm) as long as cAMP concentrations 
remain at basal levels. Prolonged elevation of 
cAMP and dissociation of the holoenzyme causes 
gradual translocation of the catalytic subunit into 
the nucleus over tens of minutes. The observed 
gradient puts high cAMP where it is most needed 
for short-term plasticit}', at the distal processes 
where the presynaptic terminals would be in 
vivo. Only strong or repeated stimulations would 
be able to raise the cAMP concentration in the 
cell body sufficiently to release the catalytic sub- 
unit to diffuse into the nucleus, phosphorylate 
transcription factors, and cause longer-term 
changes in gene expression. There is some evi- 
dence that cAMP changes in the mammalian brain 
may also be important in comparable forms of 
plasticity, so we are trying to extend our studies 
to the appropriate mammalian neurons. 
Eventually we hope to extend optical methods 
to detect macromolecular biochemical signals 
such as protein phosphorylation or gene tran- 
scription. These events currently are assayed by 
grinding up millions of cells, so that time resolu- 
tion is limited and differences between individ- 
ual cells or subregions are impossible to discern. 
Our experience with imaging ionic messengers 
and cAMP suggests that cells have considerable 
individuality and complex behavior patterns. 
These were missed with destructive population 
assays, which might be somewhat analogous to 
studying human psychology on the basis only of 
anonymous nationwide averages in which the 
respondents are executed after each poll. We 
therefore seek continuous, nondestructive read- 
out from single cells. Approaches currently 
under development (by Julie Matheson and Gre- 
gor Zlokamik, respectively) include microinjec- 
tion of peptides whose fluorescence is altered by 
phosphorylation and development of membrane- 
permeant fluorogenic substrates for reporter en- 
zymes whose nucleotide sequences can be fused 
to genes or promoter sequences of interest. 
A complementary approach is to perturb intra- 
cellular signals in a controlled manner to see how 
the cell or tissue responds. Many important intra- 
cellular messengers such as cAMP or inositol 
phosphates contain one or more phosphate 
groups that prevent permeability through mem- 
branes. A general method for making membrane- 
permeant analogues has not been available but 
would be highly useful for artificial stimulation 
of the putative transduction pathways to see what 
physiological functions result, especially re- 
sponses that cannot be assayed in microinjected 
or permeabilized cells. Recently Carsten Schultz 
has discovered such a method, esterification of 
the organophosphate anions with acetoxymethyl 
groups, which increases the potency of extracel- 
lularly applied cAMP analogues by about 100- 
fold and yields the first membrane-permeant de- 
rivatives of inositol phosphates yet reported. This 
methodology should help us to find new func- 
tions for these ubiquitous messengers. 
Our projects encompass a wide range of disci- 
plines, including organic synthesis, theoretical 
and experimental optical spectroscopy and pho- 
tochemistry, protein chemistry, computerized 
microscopy and image processing, cell biology, 
and neurobiology. 
The work of Stephen Adams, Martyn Mahaut- 
Smith, Julie Matheson, Carsten Schultz, and Gre- 
gor Zlokarnik in my laboratory was supported by 
grants from the National Institutes of Health. 
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