COMMEMORATIVE LECTURE
Breeding and building molecules to spy on cells and tumors
Roger Y. Tsien

Howard Hughes Medical Institute, University of California, San Diego, CA, USA(Received for publication on July 15, 2005)

Key words: fluorescent protein, somatic hypermutation, tetracysteine/biarsenical, tumor imaging, cellpenetrating peptide

Thank you very much for this honor that you havegiven me and for putting on such a nice symposium. It
covered so many topics, ranging from organic synthesis,
including tert-butyllithium mentioned in the talk of Professor Urano, all the way up to clinical medicine, witha very lively discussion at both ends and everything in
between. So it is splendid to see such interdisciplinary
efforts and the enthusiastic involvement of young people,
not just old folks like me. Of course the young people
represent the future of our subject. The students in theaudience are the real stem cells of biology who will keep
our subject alive. Professors like me with grey hair are
pretty much set in our ways, or terminally differentiated.
There are only two possible long-term fates for terminally differentiated cells — necrosis or apoptosis — takeyour pick. So the students are the ones who will carry on
the tradition.
I have chosen three diverse topics to cover, again
knowing that different people in the audience will havedifferent interests. Some of these were hinted at yesterday, but I will try to go into a little more detail today,
though I apologize for the repetition. One topic is how
we are now progressing with red fluorescent proteins,
the work primarily of Nathan Shaner and Lei Wang.
I will also update you on our hybrid system based on
arsenic in which we can combine molecular biologicaltargeting via tetracysteines with organic chemical versatility. The final area is our effort to transition into in vivo
imaging of tumors via their local proteolytic activities.
We hope soon to be lessening our dependence on optics
and to begin working with magnetic resonance imaging
and nuclear medicine, so that this approach may eventually have some clinical application.
As I introduced yesterday, genetically targeted labels

have the advantages that they are made by the organism itself under the control of the DNA that we put in.
Therefore these reporters can be continuously made forthe life of the organism, transmitted to subsequent generations, and very precisely targeted to particular cells
or subcellular locations using cell specific promoters or
targeting sequences. Genetic encoding also means we
can exploit a wide variety of natural proteins as building blocks for sensors. Any protein that undergoes acontrollable conformational change or protein-protein
association or dissociation gives us a chance to generate
an indicator out of that system. Thus genetically encoded
indicators have been demonstrated for many importantsignals, including membrane potential, intracellular calcium, cyclic nucleotides, and redox status at least at thelevel of thiols and disulfides, although maybe not quite
the same electron transport chain as we just heard aboutfrom Dr. Fujibayashi. Other indicators monitor exocytosis and endocytosis, neurotransmitters, and I will briefly
mention protein conformational changes. I will not havetime to talk about reporters for kinase and phosphataseactivities, though they are a very interesting subject
on their own. I will briefly mention imaging of protein
turnover and trafficking, and lastly protease activation,
though the latter is with small molecular probes.
The red fluorescent protein DsRed from corals, firstcloned by Matz et al,1 has attracted much attention as alabel providing contrast with jellyfish green fluorescentprotein (GFP) and as a route to longer wavelengths that
could penetrate tissues further. The problem with DsRed
however, is that it was made by the coral for its own
purposes, which are not the same as our purposes. We
assume the coral just wanted to color its cells red, thougheven now we do not really know exactly why. This is a

Dr. Tsien is the recepient of the 2004 Keio Medical Science Prize.
Presented at the Keio Medical Science Prize Symposium, November 30, 2004.
Reprint requests to: Dr. Roger Y. Tsien, Howard Hughes Medical Institute, University of California, San Diego, CA, USA Email:rtsien@ucsd.edu
127

128

Tsien RY: Breeding and building molecules to spy on cells and tumors

Fig. 1 Evolution of a monomeric DsRed
modified from Fig.1 of Campbell RE, et al: Proc Natl Acad Sci 2002; 99: 7877-7882

favorite topic of argument whenever people who workon fluorescent proteins get together. We try to speculate
why the corals and the jellyfish have these proteins, but
we cannot “read their minds”. We are only grateful thatthey evolved such proteins. The corals, however, probably want to maintain very high photostability undertropical sunshine, which may be the reason why all thecoral proteins in their native form have turned out tobe tight tetramers. Such tetramerization is bad for us asbiological users, because if we fuse such a protein to ourprotein of interest, the latter is dragged into the tetrameric complex, which can be very messy and ungainly. Thus
if our protein participates in its own protein-protein interactions, there is the potential for a massive cross-linking.
An example is the gap junctional protein connexin-43,
which has to form a hexamer in one cell to combine witha hexamer from another cell to make the functional gap
junction. If one fuses connexin 43 to green fluorescentprotein (GFP), the jellyfish protein which only weakly
dimerizes, one can get functional gap junctions, whichcorrectly traffic to the border between two cells. By contrast, if we make the same fusion to the original coral
protein DsRed, the fusion just precipitates around theGolgi apparatus as a mess of insoluble aggregates, neverreaching the plasma membrane or forming gap junctions.2 When crystal structures of DsRed were solved,3, 4
they revealed so much buried surface area in the tetramer
interfaces that disruption would be very difficult. Indeed,

attempts to make mutations at the interface destroyed
the fluorescence.5 Other problems of DsRed are that itis slow to mature and always retains a major fraction of
the green component, which is annoying when we want
to do two color staining. If our red fluorescent protein(RFP) always retains some green, then we cannot independently detect a low amount of GFP.
These problems were solved stepwise. The first improvement came from the laboratory of Ben Glick,6 who
evolved a mutant that matured more quickly but still
incompletely, and was still tetrameric. Rob Campbell in
my lab then devised a form of directed evolution where
he introduced charged residues to disrupt one interface atthe time, then randomly generated additional mutations
to restore at least a small amount of fluorescence, whichis easy to screen for visually. He then made new sets ofmutations iteratively, about eleven rounds of evolution inas many months, eventually arriving at 33 mutations or
15% of the protein (Fig. 1). The resulting monomeric red
fluorescent protein2 is the most popular fluorescent protein my lab has ever made; every day I get another few
requests by email. Indeed the main thing my secretary
does these days is to fulfill those requests.
Now, we and many other people still want more colors.
There are two obvious choices. One is the very fundamental approach, as Dr. Miyawaki described earlier inthis symposium, to go somewhere like Okinawa withlots of corals, and clone proteins with new colors. This

Keio J Med 2006; 55(4): 127－140

129

Fig. 2 The 2004 palette of nonoligomerizing fluorescent proteins
reproduced from Fig.1 of Tsien RY: FEBS Letters 2005; 579: 927-932

is the fundamental way to get true diversity, because thecorals have had several hundred million years to figureout lots of variations. Unfortunately, they are almost always tetramers, leaving a lot of hard work to make them
individually into monomers. Therefore we decided totry the alternative strategy, to see if we could change thecolor of our existing monomer. Such wavelength shifting proved surprisingly easy and produced a rainbow
of yellow to deep red colors (Fig. 2), which we named
according to the fruits with matching colors.7 There isno one best protein amongst these, just like there is no
one best fruit in the grocery store that is so good that you
do not have to buy any other fruits. Depending on yourtastes and requirements, different proteins have different pluses and minuses. For example, “mOrange” has aparticularly high quantum yield as a monomer, whichmakes it a good fluorescence resonance energy transfer(FRET) acceptor. However, it is markedly quenched bypH’s lower than 6.7. This acid sensitivity is undesirable
for using mOrange as a simple marker in the presence ofpH variations. but allows mOrange targeted to the acidic
lumen of secretory granules to report exocytosis into theneutral extracellular environment. Some pH sensitive
GFPs already do the same, but mOrange works at much

longer wavelengths, potentially allowing monitoring of
two separate types of exocytotic events or of exocytosis
in the presence of FRET from cyan to yellow fluorescentproteins.
Tandem dimer tomato (“tdTomato) has the highest
overall brightness due to highest extinction and an evenhigher quantum yield, and is also quite photostable.
The main disadvantage is that it is twice the molecularweight, because here one of the dimer interfaces is stillintact. But tdTomato is not a dimerizer because we fused
the N and C termini of the two sub-units together so that
this dimer is satisfied inside itself as an internal combination. And by the way, if anyone objects that tomatoes
are not fruits, you are thinking like a grocer rather than abiologist.
The closest successor to mRFP1 is “mCherry”. It has
a higher extinction, faster maturation, and much greater
indifference to fusion. (mRFP1 sometimes lost considerable brightness when other proteins were fused to its
N-terminus.) Most importantly, mCherry is much more
photo-stable, withstanding about an order of magnitude
more photon dose than mRFP1 before bleaching to 50%.
We also discovered a variant, “mGrape2”, which
proved photoisomerizable in a manner somewhat similar

130

to “Dronpa” mentioned by Dr. Miyawaki earlier in this
symposium.8 However, mGrape2 needs blue rather thanviolet light for photoisomerization, and then changes
from orange to red emission. Furthermore, over several hours in the dark, it spontaneously reverts back to its
original color, rather than requiring a second wavelengthof light to push it backwards.
Finally, mPlum is our longest wavelength fluorescentprotein to date.9 Longer wavelengths are of interest because mammalian tissue becomes more transparent at
longer wavelengths, especially beyond 600 nm. Nearinfrared wavelengths would be best of all. The emission of mRFP1 peaks around 580 nm, whereas mPlum’s maximum is at 648 nm, more than halfway to the true
infrared (700 nm or beyond). mPlum is particularly interesting because it was evolved by a new procedure that
I think may have more general applicability. Up to now,
our mutations have been made by this rather laborious
procedure of making libraries of mutations in vitro, andthen putting that DNA back into, say, bacteria, growing
up the protein, selecting the phenotype, picking the best
new property, whether it is a brighter fluorescence or alonger wavelength or whatever, and then growing up thecolonies, sequencing the DNA, and figuring out which
mutation actually made the best change. Then we haveto think and design our next set of mutations. The entire
cycle is quite laborious. We thought that a system inwhich cells themselves would make mutations for us insitu would be very valuable as a complementary alternative.
Until now, if one wants to make mutations within
mammalian cells, typically one would shine ultraviolet light on them, irradiate them with X-rays, or apply
chemical mutagens like nitrous acid or ethyl methanesulfonate (EMS). These methods of in situ mutagenesis arevery random and do not target the gene you care about.
They hit all genes equally, so that if you mutate at a highrate, you destroy the viability of the cells, just like strong
ultraviolet or X-ray energy will sterilize the tissue if you
use too high an energy density. What we really want isa means to focus the mutations just on say, mRFP1 andleave the housekeeping genes of the cell intact.
As I am not an expert molecular geneticist, I asked
more knowledgeable friends “Is there some special genetic system where you can put some cis-acting elements
around one particular gene to make that gene mutate rapidly?”, but they did not know of such a system. Then I
looked a little more carefully in the literature and eventually realized that we all “know” how to do such directed
mutation, except that for most of us it is an unconscious
process that only our B lymphocytes understand how toaccomplish, even if our scientific minds do not.
B lymphocytes use a process called somatic hypermutation (SHM) at the last stage of refining an antibody.10, 11
These cells have figured out a special system for increas-

Tsien RY: Breeding and building molecules to spy on cells and tumors

ing the mutation rate 1 million-fold over the background,mostly targeted to the immunoglobulin locus. It hascertain biological rules; it is not perfectly random. The
mutation process starts with an enzyme called activationinduced deaminase (AID), which attacks cytosine. This
primary alteration is then amplified or accentuated byerror-prone DNA repair. The fine details of this process
are under intensive investigation in immunology labs,
although I am not an expert on it. Our interest was toexploit this procedure, and it was crucial to know that itis actually not totally specific for immunoglobulins. You
can get other genes to mutate if you transcribe them at ahigh level. However, what other people had done with
non-immunoglobulin genes12, 13 was to take GFP, our familiar workhorse, make a point mutation so that it is now
unable to express, put this into B lymphocytes and showthat at some low frequency the B lymphocytes repair thestop codon, which is simply fixing an artificial mistake
without generating anything novel and useful.
We therefore decided to take mRFP1 and put it into
Ramos cells, a line of B lymphocytes that is known to
conduct SHM, under the control of a tetracycline promoter so that we could turn the mutational process on
and off via the level of transcription. The basic procedure was then simply to grow up millions of cells, thenswitch on transcription with the tetracycline analog doxycycline to make lots of mutations. Each cell on average
probably makes one mutation — a different mutation —somewhere in the mRFP1 gene. We used fluorescence
activated cell sorting to select around 5% of the cellswith the highest ratio of far-red to near-red fluorescences
and reasonable brightness, rather like examining a classof medical students and picking those top 5% with thehighest grades. Then we simply grow the cells up again,
add doxycycline again and sort again for the best 5%.
This simple cycle of growing and sorting is much lesslaborious and can thus be done twice a week, not once amonth.
The fluorescence wavelengths initially did not improve
much from generation to generation (Fig. 3), which
made us fear that we were pursuing a hopeless cause.
Fortunately, however, we kept going, and in round 10 orso things really began to improve markedly. By round 16
or 20, there were some major wavelength improvements,
which subsequently leveled off, after which we decided
to write the paper. Curiously, the excitation spectrum
first shifted to the right and reached its longest wavelengths at about round 10 in a protein we call “mRaspberry”, and then shifted backwards in the final evolution
to round 23 even as the emission maximum got longer
and longer. Therefore the Stokes shift between the emission and the excitation peaks became particularly large.
The best clone out of round 23 is called “mPlum”. It is
about 8 to 10-fold more photoresistant than the starting
mRFP1. Over the 23 generations of SHM, it accumulated

Keio J Med 2006; 55(4): 127－140

131

Fig. 3 Progressive red-shifting by SHM in Ramos cells
Modified from Figs.1 and 3 of Wang L, et al: Proc Natl Acad Sci 2004; 101: 16745-16749

7 mutations, which we would not have chosen rationally.
The rational strategy to increase emission wavelengths
would be to mimic the way we made yellow fluorescentprotein (YFP) out of GFP, which is to stack a tyrosine
over the chromophore. This strategy produced mGrape,whereas SHM found a different and more effective strategy. Thus exogenously introduced non-immunoglobulin
genes can undergo useful mutations distributed throughout the sequence.
At each of the seven positions mutated, you could
imagine 18 other amino acids other than the wild type
and the mutation picked already by SHM. Are any ofthese 18 better than SHM’s choice? So far, never hasthat been the case, which implies that SHM has alreadydone a fairly exhaustive search there. Another proof of
how much sequence space SHM has explored comesfrom examining a genetic element left over from the virus. This is a “long terminal repeat (LTR)”, which is past
the end of the protein-encoding region and therefore notunder any phenotypic selection pressure. Almost 13% of
the LTR has been altered, again reflecting the extensive
mutagenesis.
The first use of the immune system in biotechnology

was to generate polyclonal antibodies. Then Milstein andKohler invented monoclonal antibodies, which finallymade antibodies into molecularly defined, reproducible
reagents.14 Schultz and Lerner15 and many other people
have worked on catalytic antibodies, which act somewhat like enzymes to mediate chemical reactions. But
even so, all of these are still antibodies. Now we seethat the immune system can evolve proteins that haveno relationship to antibodies as long as you have a highthroughput single cell screen applied to a somatically
hypermutating cell line. I believe that if you want toengineer a new protein such as a receptor with mutant
properties or a new enzyme, as long as you have a screenthat works at the single cell level and leaves the best performers alive, you have a good chance of using this richsource of diversity as an efficient means of generating
the property you want.
Now I switch focus from fully genetically encodable
systems to a hybrid combining the advantages of molecular biology and organic chemistry. The virtue of themolecular biology is that it gives you very accurate targeting through genetically encodable elements, but theadvantage of organic chemistry is that we can put in un-

132

Tsien RY: Breeding and building molecules to spy on cells and tumors

Fig. 4 FLAsH = fluorescein-based arsenical hairpin binder

usual properties with a much smaller structure than you
need with a fluorescent protein. All fluorescent proteinsthat generate visible chromophores from their own internal amino acids are at least 220 or so amino acids. Nobody has yet found a significantly smaller one yet. Here
we can reduce the genetically targeted portion ultimately
down to as small as six, and we can bring in properties
that no organism has ever yet had to evolve. For example, no organism has ever been interested in helping
us with magnetic resonance imaging (MRI). Incorporatea gadolinium, why would any creature evolve that? MRI
was only invented two or so decades ago. So, we still need
the flexibility of organic synthesis.
Albert Griffin was a brave organic chemistry student
in my lab who came up with an initial solution (Fig. 4)
based on fluorescein, on which he put two arsenics bya simple transmetallation to create a molecule called“FlAsH”.16 We picked arsenic because it has affinity forpairs of thiols inside cells, but a single pair is not specificenough. By putting two arsenics in, we create selectivity for a quadruple group of cysteines as provided in the
motif Cys-Cys-Pro-Gly-Cys-Cys, where the Pro-Gly
probably form a hairpin turn to allow the four cysteines
to cluster together.17 The biarsenical derivative of fluorescein (“FlAsH”) is membrane permeant. Its effective
dissociation constant from the tetracysteine turned out tobe on the order of a few picomolar. As an extra bonus,

FlAsH is non-fluorescent when bound to an antidote thatkeeps it away from ordinary pairs of cysteines, probably due to photo-induced electron transfer, the samequenching mechanism Dr. Urano discussed earlier in this
symposium. When bound to the tetracysteine, FlAsH
becomes brightly fluorescent, probably because stericconstraints prevent the coplanarity necessary for photoinduced electron transfer. We now have new colors such
as ReAsH which is isosteric to the fluorescein, but red
instead of green.17 FlAsH and ReAsH have to be madeby organic synthesis of course, so most biologists will
not want to make them, but they are now commerciallyavailable from Invitrogen. Unfortunately the company
insisted on changing their names to “Lumio Green” and“Lumio Red” for reasons I do not understand.
Fig. 5 summarizes an example where the small size of
this genetically targeted fluorescent label is crucial. This
comes from recent work from Carsten Hoffman in Martin Lohse’s lab.18 They have been looking at conformational changes in G-protein coupled receptors, the familywith seven transmembrane segments, which are so important in many signaling pathways. They originally put
YFP at the C-terminus and CFP into intracellular loop 3of the adenosine receptor. The resulting hybrid molecule
responded to adenosine with a small but rapid loss of
FRET, within 80-100 milliseconds, which they could
show was the conformational change in the G-protein

Keio J Med 2006; 55(4): 127－140

133

Fig. 5 FLAsH-tetracysteine insertion into GPCR is better than YFP insertion

Bigger FRET response, no perturbation of coupling to downstream effector: Smaller is better!

Modified from Figs.1a, 1b, 2e, and 3a of Hoffmann C, et al: Nature Methods 2005; 2: 171-176

coupled receptor upon binding with adenosine.
Unfortunately, the inclusion of the CFP (>230 aminoacids) almost completely destroyed the ability of this receptor to activate downstream signaling. The native adenosine receptor will stimulate adenylyl cyclase when itbinds adenosine, but the hybrid molecule with two bulky
fluorescent proteins has lost this important ability. But
when Hoffman replaced the CFP with this much smaller
insert, just six amino acids (CCPGCC) in intracellularloop 3, then labeled that motif in live cells with FlAsH,
then he observed an even bigger FRET response to adenosine and restored completely normal activation of
adenylyl cyclase. In this case, smaller is better.
I will skip past our work with Mark Ellisman’s groupon connexin subunits19 and mention that various othergroups, including that of Robert Malenka,20 have started
applying the ability of FlAsH and ReAsH to sequentiallylabel the same protein, or the same type of protein. Herethey put in the tetracysteine motif near, but not quite at,
the C-terminus of the AMPA form of the glutamate receptor. This type of glutamate receptor in the brain is ofintense interest to neurobiologists because it is believedto mediate several forms of synaptic plasticity, so thatwhen you have more AMPA type glutamate receptors
in the membrane, your synapse is more sensitive, andwhen you take AMPA receptors away, then your synapse becomes less sensitive. It becomes of great interest
to know the local trafficking of the AMPA receptor —in particular, can neuronal dendrites synthesize fresh
AMPA receptors on their own without calling upon thecell nucleus for help?
Normally biologists believe that to increase the production of new proteins, the cell must send a signal to
the nucleus to make new messenger ribonucleic acid,
which then encodes the synthesis of new protein. But
in a neuron with thousands of different synapses, whichmust be independently regulated, it is very cumbersome
if they all have to talk to the nucleus, because how does
the nucleus know how to send the protein to one particular synapse out of the thousands? People have thus beenvery interested in the possibility that individual synapses,or at least parts of neuronal dendrites, have autonomous
control of protein synthesis. To test this idea, the Malenka group (Ju et al)20 inserted a tetracysteine motif into
the AMPA receptor, then labeled it with ReAsH, the reddye, so that all the copies of the AMPA receptor made

134

Tsien RY: Breeding and building molecules to spy on cells and tumors

Fig. 6 Glutamate receptors can be newly synthesized in isolated neuronal dendrites
reproduced from Fig.4 of Ju W, et al: Nat Neurosci 2004; 7: 244-253

up to that point would turn red (Fig. 6). They removed
unbound ReAsH, then immediately used a glass microneedle to cut a dendrite off the rest of the cell. About
30% of the time, this portion of the neuron would continue to live for a few hours, even though separated fromthe main cell body and nucleus. Then they could ask;
“Can this isolated dendrite make any fresh protein on its
own?” By just waiting a few hours, then labeling with
FlAsH, any fresh protein made after the surgery wouldlabel green, whereas old protein made before ReAsH
withdrawal would stay red. Thus, if green synapses canbe seen, fresh protein synthesis must have occurred inthis isolated portion. And indeed, out here there is green
dye in the synaptic spots, as you can see from this region
at a higher magnification. This is true for both GluR1
and GluR2, both types of the AMPA receptor. An important control, to make sure that the dyes do not wash off,
is to add cycloheximide during this chase time to inhibit
all fresh protein synthesis. Only old red protein without
any additional green is visible, as expected, showing that
the ReAsH saturated all the old protein and did not washaway.
For comparison, they also applied this technique to thecalcium/calmodulin kinase II (CaMKII) beta subunit,
which unlike the alpha subunit is generally accepted tobehave like a standard protein requiring the participationof the nucleus. Now there is fresh green CaMKII beta
made afterwards in the cell body, but not in the peripheral dendrite beyond the site of the cut. Even more interestingly, it is believed that the 3’ untranslated region of
the mRNA for GluR1 has the special targeting sequencethat says “store me near synapses”. If you delete the 3’
untranslated region from the mRNA, GluR1 now looks
like CaMKII beta, i.e. the cell body has both new green
and old red copies whereas the isolated dendrite has only
old red protein. Thus the sequential labeling technique
can clearly distinguish dendrite-autonomous from nucleus-dependent protein synthesis.
Another important application of FlAsH and ReAsH istheir ability to photogenerate singlet oxygen, which can
locally and acutely inactivate proteins. Singlet oxygenis a very reactive excited state of O2 that attacks methionines, tryptophans, and histidines very close to thesite of generation, and thereby has a high likelihood ofinactivating the nearest proteins. For example, Oded
Tour monitored gap junctions formed from tetracysteine-Keio J Med 2006; 55(4): 127－140

tagged ReAsH-stained connexins.21 Initially there wasgood coupling through gap junctions, as shown by a highelectrical coupling ratio. Applying modest levels of light
did very little, but applying stronger illumination, but
still only with a standard xenon lamp rather a laser, wasenough to inactivate the gap junctions in just 20 or 25
seconds. There is no other pharmacological way to kill
gap junctions.
This same local inactivation works on voltage-operated
calcium channels (genes supplied by my brother Richard
Tsien, Stanford Univ.) similarly tagged with tetracysteine motifs and labeled with ReAsH.21 Each depolarization evokes a large calcium current, which then slowly
inactivates in a well-known manner. The peak amplitude
is stable in the dark. Then if Dr. Tour then applies lightfor just five seconds, he immediately loses about 50% of
the current. Once again, that reduced amplitude is stablein the dark. Each time he illuminates for 5 sec, he loses
about half the remaining current. So one can titrate theresidual activity with different cumulative exposures tolight. Control experiments show that other channels in
the membrane remain unaffected during this procedure.
I have to say that those were not the most interesting
biological uses. The most interesting use I would say
so far has come from Gray Davis’ lab22 who applied it
to the famous synaptic protein synaptotagmin, which isaccepted to be important for exocytosis. The question
Davis wanted to answer is whether synaptotagmin is
separately important for endocytosis? One cannot answer this question with a genetic deletion because if you
knock out synaptotagmin you have no exocytosis, so you
cannot even measure endocytosis. They needed a way
to acutely kill synaptotagmin only after exocytosis had
already occurred. They expressed synaptotagmin with atetracysteine tag in Drosophila embryos in which endogenous synaptotagmin had been deleted. After dye-labeling the synaptotagmin and stimulating exocytosis, theyilluminated the synapses and showed that endocytosis
was depressed compared to controls in which either the
dye-labeling or illumination were omitted. This result argues that synaptotagmin has a direct role in endocytosis
as well as exocytosis.
Genetically targeted photoinactivation is potentiallyan important general technique for rapidly and locallyinactivating an existing protein. The more widespreadmethods for gene knockouts and RNA interference only
remove the nucleic acid source of the protein. The existing protein just decays with its normal half-life, which
can be hours to days, during which time there is a large
chance for other proteins either to step in and compensate, or for an avalanche of new biological effects tooccur based on the initial deletion. Such secondary effects make interpretation much more difficult. Moreover,none of those techniques has any spatial resolution. If
one has or can develop a small molecule inhibitor of the135

protein of interest, one can acutely and suddenly eliminate its function, but such inhibitors are usually laborious and costly to develop for a new protein, requiring
the resources of a pharmaceutical company. Geneticallytargeted photoinactivation merely requires appending thetetracysteine tag, labeling with the dye, and illuminating. One limitation is that this procedure only eliminates
the exogenous, tagged copies unless they associate withendogenous untagged copies, in which case the singlet
oxygen might inactivate the latter as well to give a dominant negative effect. Otherwise, one should knock out
the endogenous copies while replacing them with tagged
copies to keep the function temporarily normal until thelatter are photoinactivated with the desired spatial andtemporal control.
A final comment on the tetracysteine motif is that theoriginal sequences have been greatly improved by a highthroughput library screen done by Brent Martin.23 We
now recommend a 12 amino acid sequence FLNCCPGCCMEP where the six amino acids surrounding the core
CCPGCC have been combinatorially optimized. The
new sequence gives a better quantum yield and muchbetter affinity of binding, enabling use of less biarsenical dye and more stringent conditions to wash away unbound and nonspecifically bound dye. This reduces the
background staining that up to now has been the most
severe limitation of the tetracysteine-biarsenical system.
My last topic is a more clinically oriented application.24
Despite the power of genetically encoded indicators or
targeted labels, they will not be readily applicable tolive people because we cannot make transgenic humans.
There are high hopes for gene therapy of course, but
it is clearly taking a lot longer than most of us had expected. To help patients as opposed to mice, it would bedesirable to develop general strategies to target synthetic
molecules to diseased tissue without using gene transfer(Fig. 7). The synthetic molecules should ideally encompass both diagnostic reporters and therapeutic molecules.
A few existing ligands for endogenous receptors are
already in use for in vivo imaging, such as dopamine
receptor ligands discussed earlier in this symposium, butthey are hard to generalize and crucially lack amplification. Moreover, each endogenous receptor can bind at
most one copy of the ligand. Likewise each copy of an
endogenous tumor-associated antigen can bind at mostone copy of antibody. Antibodies also are big moleculesthat have a hard time penetrating deep into solid tumors
and are slow to clear from the rest of the body.25 I would
prefer an amplifying mechanism whereby each pathological target molecule can cause the accumulation of alarge number of reporter or therapeutic molecules, whose
size we can control from small to large and which are
not restricted to fluorescence as their only readout. Sucha mechanism might enable personalized medicine, inwhich imaging and therapy could work synergistically.

136

Tsien RY: Breeding and building molecules to spy on cells and tumors

Fig. 7 Mechanism of protease-activated cell-penetrating peptides
Modified from Fig.1 of Jiang T, et al: Proc Natl Acad Sci 2004; 101: 17867-17872

Many other labs had shown that certain polycationicpeptides, initially derived from the Antennapedia homeobox protein or Tat protein of human immunodeficiency
virus,26, 27 can drag normally impermeant cargoes into
live cells. Such cargoes can range in size from isotopes
or small dye molecules28, 29 to nanoparticles of many tensof nm diameter.30 The polycationic sequence can be as
simple as a string of arginines,31, 32 which I like because
then I do not have to remember complex sequences. The
arginines can just as well be D-amino acids, arguing thatthe mechanism is probably not mediated by receptors of
the usual sort. The positively charged arginines first stickto the outside of the cell, which is negatively charged
because of phospholipid headgroups or proteoglycans.
Probably these complexes then become endocytosed,33,
34
whereupon a fraction of them somehow escape fromthe endosomes and end up in both the cytosol and thenucleus. The detailed mechanism of cell penetration remains obscure and controversial. Furthermore, it was notknown how to control this uptake to make it specific forparticular types of cells, especially for tumor cells compared to normal.
We first needed a way to generally inhibit the uptake,then an additional mechanism to remove the inhibition in
selected locations (Fig. 7). What could stop the polyarginine peptides from working? We decided to fuse them
to a matching string of negative charges made of glutamates, in the hope that the positive and negative charges

would fold back on each other, leaving too few positive
charges to stick to the plasma membrane.24 When Dr.
Tao Jiang made and tested such composite peptides,
we were gratified to find that uptake is indeed largely
blocked. However, this inhibition can be reversed bycleaving the covalent linkage between the polyargininesand polyglutamates, for example with a protease. In theabsence of that covalent linkage, electrostatic attraction
alone is not enough to hold the opposite charges together, so the two halves eventually drift apart, freeing thepolyarginine and its still-attached cargo to stick to theoutside of the cell and eventually be taken up. Therefore,
adhesion and intracellular uptake of a wide range of potential cargoes is triggered by local extracellular proteaseactivity, which in turn is known to be elevated in a wide
variety of disease states, especially metastatic tumors.
We have some evidence that the peptide does forma hairpin, as I imagined in the cartoon of Fig. 7. Melinda Roy and Patricia Jennings, expert collaborators fornuclear magnetic resonance (NMR) spectroscopy, foundmany nuclear Overhauser couplings, especially between
the glutamates and the arginines. Such couplings indicate that the glutamates and arginines are kissing eachother in a hairpin loop.
Furthermore, the intact synthetic peptide including
a row of D-glutamates, a cleavable linker containing
L-amino acids, a row of D-arginines, and a far-red fluorescent dye (Cy5) to monitor where the arginines go, isKeio J Med 2006; 55(4): 127－140

137

Fig. 8 Intracellular uptake of cargo requires peptide cleavage
Modified from Figs.2B & 2C of Jiang T, et al: Proc Natl Acad Sci 2004; 101: 17867-17872

almost completely unable to enter cells in culture (Fig.
8). However, the linker is chosen to be a good substrate
for matrix metalloproteinase-2 (MMP-2), which is aform of collagenase that many cancer cells secrete toenable them to chew their way through the normal extracellular matrix to invade fresh tissue. If we expose thepeptide to active MMP-2, the linker is cleaved, jettisoning the inhibitory glutamates. Now the arginines carrying the Cy5 marker enter the cell very well, probably
even reaching some nucleoli inside the nucleus. We have
many examples of other peptide sequences whose uptake
into cultured cells is similarly dependent on disease-associated proteases, but can we get this mechanism to workin a tumor in a live animal? In the traditional model forcancer research, human tumor cells are transplanted into
immunocompromised mice. In this case Emilia Olson injected HT1080 fibrosarcoma cells in the armpits of nude
mice where they grew up into tumors. The peptide wasthen injected into the tail vein. After an hour, imaging of
far-red fluorescence through the skin of the live animal
shows that the tumor typically lights up about threefold
more brightly than the surrounding tissue or noninjected
contralateral site (Fig. 9). The same contrast can be seen
after sacrificing the animal and sectioning the recoveredtumor. We believe that this contrast is dependent oncleavage, because we can compare a scrambled peptide
where we merely permute the PLGLAG into LALGPG.
This is not a substrate anymore for MMP-2, even though
it has the same amino acids, molecular weight, and hydrophobicity. Therefore it is not much accumulated into
the tumor. Tumor contrast thus correlates to proteasesusceptibility.
Most recently, we have started to work on cancers
from real patients even though we are not yet readyfor the very expensive and difficult procedure to initiate clinical trials injecting our probes into people. Instead, we found a surgeon, Dr. Quyen Nguyen, who isregularly taking head and neck tumors out of patients,
together with the normal tissue surrounding the tumor
that must also be removed to try to ensure getting all thetumor. Since this tissue would be discarded anyway, it
is easy to get patients’ consent to test our molecules onthese freshly excised samples, which we cut into slices
and incubate with the cleavable peptide or scrambled
peptide. Under optimum conditions, the tumor can be up
to almost eightfold brighter than the surrounding normal
tissue (Fig. 10), whereas the scrambled peptide shows
negligible contrast. In some cases we can even see that

138

Tsien RY: Breeding and building molecules to spy on cells and tumors

Fig. 9 Contrast from human tumors (HT1080)xenografted into nude mice
Modified from Fig.4 of Jiang T, et al: Proc Natl Acad Sci 2004; 101: 17867-17872

the brightest staining is in the most dedifferentiated partof the tumor, whereas a redifferentiated tumor portion
does not stain as well, shown here by a keratin pearl, adiagnostic sign for a squamous cell carcinoma that is trying to become normal tissue again. Thus we have somepreliminary hint that this approach can work on clinicalspecimens. Of course the ultimate goal is to image not
the tumor that has just been removed, but any tumor thatis left in the patient, which will require real clinical trials
and elaborate regulatory approval.
We believe that this new mechanism for cleavageactivated uptake should deliver contrast agents for allmodes of imaging, even though the results I showed so
far are limited to fluorescence. However, others haveshown that tat or polyarginine peptides will work forradioactive or magnetic reporters as well. We also hope
to exploit radiation sensitizers like boron-10 or gadolinium-157. The latter is particularly attractive because it
should be both be visible by MRI and usable for neutron
capture therapy. The linkers can be cleaved by a variety
of proteases or even by reduction of a disulphide bond,which could potentially recognize hypoxic, partially necrotic tissue. We are not dependent on antibodies. Thismechanism allows enzymatic amplification. Proteaseactivities are mechanistically important and interesting.
The immediate challenges for the future are to increase
the contrast yet further and to demonstrate in vivo nonoptical imaging. My last scientific comment is that thismechanism is an extracellular analog of FRET, in thatthe polyarginine is like a donor, whereas the polyglutamate is like a quencher. When they are together, thenatural tendency of the polyarginine to enter cells isquenched. The difference from FRET is that this mechanism is not restricted to fluorescence, but should also allow radioactive, magnetic, and therapeutic cargoes to beconcentrated in diseased tissue.
These diverse examples of molecular engineering canbe summarized as follows: We have new long-wavelength fluorescent proteins. Somatic hypermutation canredirect the genius of the immune system. Tetracysteine/biarsenical labeling has some unique advantages andapplications. We have glimmerings of a new approach
towards cancer imaging and therapy, which is something
I have always wanted to develop before my career ends,
particularly because my father and a nephew both diedfrom cancer.

Keio J Med 2006; 55(4): 127－140

139

Fig. 10 Imaging of resected human squamous cell carcinoma adjaent to tongue
Modified from Fig.5 of Jiang T, et al: Proc Natl Acad Sci 2004; 101: 17867-17872

References
1. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG,
Markelov ML et al.: Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnology 1999; 17: 969－973.
2. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS,
Zacharias DA et al.: A monomeric red fluorescent protein. Proc
Natl Acad Sci U S A 2002; 99: 7877－7882.
3. Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ:
Refined crystal structure of DsRed, a red fluorescent protein fromcoral, at 2.0-A resolution. Proc Natl Acad Sci U S A 2001; 98:462－467.
4. Wall MA, Socolich M, Ranganathan R: The structural basis for
red fluorescence in the tetrameric GFP homolog DsRed. Nature
Struct Biol 2000; 7: 1133－1138.
5. Sacchetti A, Subramaniam V, Jovin TM, Alberti S: Oligomerization of DsRed is required for the generation of a functional red
fluorescent chromophore. FEBS Lett 2002; 525: 13－19.
6. Bevis BJ, Glick BS: Rapidly maturing variants of the Discosoma
red fluorescent protein (DsRed). Nat Biotechnol 2002; 20: 83－87.
7. Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer
AE, Tsien RY: Improved monomeric red, orange and yellow
fluorescent proteins derived from Discosoma sp. red fluorescentprotein. Nat Biotechnol 2004; 22: 1567－1572.
8. Ando R, Mizuno H, Miyawaki A: Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science
2004; 306: 1370－1373.

9. Wang L, Jackson WC, Steinbach PA, Tsien RY: Evolution of new
nonantibody proteins via iterative somatic hypermutation. Proc
Natl Acad Sci U S A 2004; 101: 16745－16749.
10. Neuberger MS, Harris RS, Di Noia J, Petersen-Mahrt SK: Immunity through DNA deamination. Trends Biochem Sci 2003; 28:
305－12.
11. Papavasiliou FN, Schatz DG: Somatic hypermutation of immunoglobulin genes: merging mechanisms for genetic diversity. Cell
2002; 109 Suppl.: S35－S44.
12. Bachl J, Carlson C, Gray-Schopfer V, Dessing M, Olsson C:
Increased transcription levels induce higher mutation rates in a
hypermutating cell line. J Immunol 2001; 166: 5051－7.
13. Wang CL, Harper RA, Wabl M: Genome-wide somatic hypermutation. Proc Natl Acad Sci U S A 2004; 101: 7352－7356.
14. Kohler G, Milstein C: Continuous cultures of fused cells secreting
antibody of predefined specificity. Nature 1975; 256: 495－497.
15. Schultz PG, Yin J, Lerner RA: The chemistry of the antibody
molecule. Angew Chem Int Ed Engl 2002; 41: 4427－4437.
16. Griffin BA, Adams SR, Tsien RY: Specific covalent labeling ofrecombinant protein molecules inside live cells. Science 1998;
281: 269－272.
17. Adams SR, Campbell RE, Gross LA, Martin BR, Walkup GK,
Yao Y et al.: New biarsenical ligands and tetracysteine motifs forprotein labeling in vitro and in vivo: synthesis and biological applications. J Am Chem Soc 2002; 124: 6063－6076.
18. Hoffmann C, Gaietta G, Bunemann M, Adams SR, OberdorffMaass S, Behr B et al.: A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat

140
Methods 2005; 2: 171－176.
19. Gaietta G, Deerinck TJ, Adams SR, Bouwer J, Tour O, Laird DW
et al.: Multicolor and electron microscopic imaging of connexin
trafficking. Science 2002; 296: 503－507.
20. Ju W, Morishita W, Tsui J, Gaietta G, Deerinck TJ, Adams SR etal.: Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat Neurosci 2004; 7: 244－253.
21. Tour O, Meijer RM, Zacharias DA, Adams SR, Tsien RY: Genetically targeted chromophore-assisted light inactivation. Nat
Biotechnol 2003; 21: 1505－1508.
22. Poskanzer KE, Marek KW, Sweeney ST, Davis GW: Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis
in vivo. Nature 2003; 426: 559－563.
23. Martin BR, Giepmans BN, Adams SR, Tsien RY: Mammaliancell-based optimization of the biarsenical-binding tetracysteine
motif for improved fluorescence and affinity. Nat Biotechnol
2005; submitted for publication.
24. Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien
RY: Tumor imaging by means of proteolytic activation of cellpenetrating peptides. Proc Natl Acad Sci U S A 2004; 101: 17867－17872.
25. Winnard P, Jr., Raman V: Real time non-invasive imaging ofreceptor-ligand interactions in vivo. J Cell Biochem 2003; 90: 454－463.
26. Vives E, Brodin P, Lebleu B: A truncated HIV-1 Tat protein basic
domain rapidly translocates through the plasma membrane andaccumulates in the cell nucleus. Journal of Biological Chemistry
1997; 272: 16010－16017.

Tsien RY: Breeding and building molecules to spy on cells and tumors
27. Richard JP, Melikov K, Vives E, Ramos C, Verbeure B, Gait MJ
et al.: Cell-penetrating peptides: a re-evaluation of the mechanismof cellular uptake. Biophys J 2003; 84: 212A.
28. Bhorade R, Weissleder R, Nakakoshi T, Moore ATCH: Macrocyclic chelators with paramagnetic cations are internalized into
mammalian cells via a HIV-Tat derived membrane translocation
peptide. J Am Chem Soc 2000; 11: 301－305.
29. Bullok KE, Dyszlewski M, Prior JL, Pica CM, Sharma V,
Piwnica-Worms D: Characterization of novel histidine-tagged
tat-peptide complexes dual-labeled with Tc-99m-tricarbonyl andfluorescein for scintigraphy and fluorescence microscopy. Bioconjugate Chem 2002; 13: 1226－1237.
30. Lewin M, Carlesso N, Tung C-H, Tang X-W, Cory D, Scadden
DT et al.: Tat peptide-derivatized magnetic nanoparticles allow invivo tracking and recovery of progenitor cells. Nature Biotechnology 2000; 18: 410－414.
31. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB:
Polyarginine enters cells more efficiently than other polycationichomopolymers. J Peptide Res 2000; 56: 318－325.
32. Wright LR, Rothbard JB, Wender PA: Guanidinium rich peptide
transporters and drug delivery. Current Protein & Peptide Science
2003; 4: 105－124.
33. Lundberg M, Wikstrom S, Johansson M: Cell surface adherence
and endocytosis of protein transduction domains. Molecular Therapy 2003; 8: 143－150.
34. Wadia JS, Stan RV, Dowdy SF: Transducible TAT-HA fusogenic
peptide enhances escape of TAT-fusion proteins after lipid raftmacropinocytosis. Nat Med 2004; 10: 310－315.