Photocleavable dimerizer for the rapid reversal of molecular trap antagonists

Article abstract of DOI:10.1074/jbc.C113.513622

Background: The ability to rapidly turn on and off an acute antagonist is helpful to understand the initiation of a cellular program. Results: Aphotocleavable analog was produced and functionally demonstrated. Conclusion: Fine temporal control of endosome dispersion and restoration was obtained. Significance: The combination of traps and the photocleavable analog permits new avenues to study signaling within a single cell in an organism.

Full text of DOI:10.1074/jbc.C113.513622

REPORT
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 8, pp. 4546–4552, February 21, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
inhibitors are primarily focused on a small subset of enzymes
that are frequently therapeutic targets. In fact, only 2% of all
predicted human gene products (mostly kinases) have been
successfully targeted with small molecules, and it is estimated
that only 10–15% of the human genome is “druggable” (3).
Thus, there is a tremendous gap in that only a handful of gene
products can be studied using small molecule inhibitors,
whereas there is a paucity of useful inhibitors for the remaining
85–90% of gene products.
Photocleavable Dimerizer for
the Rapid Reversal of Molecular
Trap Antagonists*
Received for publication, August 24, 2013, and in revised form, December 19, 2013
Published, JBC Papers in Press, January 13, 2014, DOI 10.1074/jbc.C113.513622
Shubbir Ahmed1, Jun Xie1, David Horne, and John C. Williams2
From the Department of Molecular Medicine, Beckman Research Institute
of City of Hope, Duarte, California 91010
The limited number of novel small molecule inhibitors stems
from multiple sources and is partially because a large number of
gene products act as components of macromolecular com-
plexes and bind to their respective target(s) through extended
surface contacts. The binding affinity and specificity for these
interactions arise through multiple weak interactions, and the
protein targets frequently lack a deep, solvent-occluded cleft as
is typically found in enzymes (4, 5). Moreover, many proteins
share common domains, and thus potential inhibitors may tar-
get a common domain and therefore could lack the required
specificity and produce off-target effects.
Background: The ability to rapidly turn on and off an
acute antagonist is helpful to understand the initiation of
a cellular program.
Results: A photocleavable analog was produced and func-
tionally demonstrated.
Conclusion: Fine temporal control of endosome disper-
sion and restoration was obtained.
Significance: The combination of traps and the photo-
cleavable analog permits new avenues to study signaling
within a single cell in an organism.
To address this issue and leverage the specificity and affinity
inherent in protein-protein interactions, yet maintain the
advantages of small molecule antagonists, we recently devel-
oped chemically induced molecular traps that use the cell-per-
meable, small molecule AP20187 (hereafter referred to as AP)3
to dimerize FKBP-peptide fusions to create high affinity, biva-
lent “ligands” that rapidly agonize specific targets (6) (see Fig.
1A). We demonstrated that the expression and subsequent
dimerization of a dynein light chain LC8 or TcTex1 molecular
trap immediately affects dynein-associated processes (e.g.
endosome, lysosome, and Golgi dispersion) (6). The ability to
reverse this perturbation would provide additional, powerful
insight to molecular processes that is not available with current
technologies (e.g. siRNA or expression of a dominant negative
construct). However, we could not “wash” out the chemical
dimerizer, AP, and thus could not reverse the perturbation to
the system and follow its return to homeostasis. To address this
shortcoming, we have created a photocleavable version of AP
(hereafter referred to as PhAP) that, upon cleavage, reduces the
valency of the trap, frees the targeted endogenous ligand, and
permits one to disrupt a biochemical process and follow its
return to equilibrium.
Herein, we report the development of a photocleavable analog
of AP20187, a cell-permeable molecule used to dimerize FK506-
binding protein (FKBP) fusion proteins and initiate biological
signaling cascades and gene expression or disrupt protein-pro-
tein interactions. We demonstrate that this reagent permits the
unique ability to rapidly and specifically antagonize a molecular
interaction in vitro and follow a biological process due to this
acute antagonism (e.g. endosome dispersion) and to release the
trap upon photocleavage to follow the cell’s return to homeosta-
sis. In addition, this photocleavable AP20187 analog can be used
in other systems where the dimerization of FKBP has been used
to initiate signaling pathways, offering the ability to correlate
the duration of a signaling event and a cellular response.
The ability to rapidly and specifically regulate the activity of
selected proteins and macromolecular complexes is essential to
parse out critical functions in complicated macromolecular
systems (e.g. signal transduction, protein trafficking, cell divi-
sion) (1, 2). In combination with functional assays such as imag-
ing, immunoprecipitation, Western blot analysis, RT-PCR, etc.,
perturbing the function of specific proteins of interest can
reveal novel associations, critical post-translation modifica-
tions, and upstream and downstream effectors. Although small
molecule inhibitors exist that lend themselves to such analyses
(e.g. protein kinase, histone deacetylase, protease, and G pro-
tein-coupled receptor inhibitors), the actions of most of these
EXPERIMENTAL PROCEDURES
Synthesis of PhAP—To a stirred solution of diol (9.5 mg, 0.045
mmol, 1 eq) in CH2Cl2 (2 ml) were added acid (70 mg, 0.1 mmol,
2.2 eq) and catalysts 4-dimethylaminopyridine and N,N-dicy-
clohexylcarbodiimide (24 mg, 0.12 mmol, 2.6 eq) at room temper-
ature. After 20 h, the solid was removed through filtration, and the
filtrate was concentrated in vacuum. The residue was purified by
silica gel column chromatography (40–60% EtOAc/hexane) to
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
GrantR21NS071166(toJ. C. W.)andNationalInstitutesofHealth Grant P30
CA033572 from the NCI (to Michael A. Friedman).
1 Both authors contributed equally to this work.
2 To whom correspondence should be addressed: Dept. of Molecular Medi- 3 The abbreviations used are: AP, AP20187; PhAP, photocleavable AP analog;
cine, Beckman Research Institute of City of Hope, 1710 Flower Ave., Duarte
CA 91010. E-mail: jcwilliams@coh.org.
FKBP, FK506-binding protein; EGFP, enhanced green fluorescent protein;
IC, intermediate chain.
4546 JOURNAL OF BIOLOGICAL CHEMISTRY
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REPORT: Photocleavable Molecular Traps
FIGURE 1. Molecular trapping uses the chemically induced dimerization of FKBP to create a bivalent, high affinity ligand to either sequester an
endogenousproteinordirectlyantagonizeaninterface. A, schematicpresentationofthetrappingmechanism. Thedyneinintermediatechain(IC)peptide,
which binds to LC8 with low affinity as a monomer, is fused to FKBP (green). The addition of photocleavable AP analog, PhAP, creates a high affinity trap that
binds to LC8 (red), competes with endogenous ligands (IC, blue lines), and induces phenotypes associated with dynein antagonism (e.g. endosome dispersion).
The multivalent complex is highly stable. B, the PhAP with a nitrobenzyl moiety (shown in the oval). Creation of a photocleavable dimerizer (PhAP) will facilitate
the dissociation and reverse the antagonism after exposure to UV light. C, endosome dispersion as a function of concentration of AP/PhAP. EGFP-FKBP-LC8TRAP
transfected COS1 cells were treated with different concentrations of AP (red) or with PhAP (blue) for 2 h. Both AP and PhAP showed the same effect on
endosome dispersion as a function of concentration. Maximum dispersion is seen with 500 nM of drug concentration and remains unchanged with further
increase in drug concentration. D, endosome dispersion as a function of time. EGFP-FKBP-LC8TRAP transfected COS1 cells were treated with 500 nM AP (red) or
with PhAP (blue) for different times. Both AP and PhAP showed the same time dependence on endosome dispersion. Maximum dispersion was reached after
2 h of drug treatment and remained unchanged with further increase in time. In each case cells were fixed and stained for early endosome marker (EEA1) and
100 cells were counted for endosome dispersion. Each experiment was repeated in triplicate (n ϭ 3). Error bars indicate mean Ϯ S.E.
afford the product (70 mg, 70%). High resolution mass spec- For EGFP-tagged FKBP-LC8TRAP, the construct was cloned as a
ϩ
trometry C86H103N3O24 [MϩNa] calculated 1584.6824, C-terminal fusion of enhanced green fluorescence protein
found 1584.6830.
(EGFP) mammalian expression vector (pEGFPC1; Clontech) as
Construction of Expression Plasmids—Both the LC8 and the described previously (6).
FKBP-LC8TRAP were cloned in bacterial expression vector
Native PAGE—Both LC8 and FKBP-LC8TRAP were mixed in a
(pET21D), expressed, and purified as described previously (6). molar concentration of 50 ␮M and 1.2 molar excess (60 ␮M) of AP
FEBRUARY 21, 2014•VOLUME 289•NUMBER 8
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REPORT: Photocleavable Molecular Traps
FIGURE 2. Native PAGE analysis of LC8-LC8TRAP complex formation and their dissociation on UV induction. A, native PAGE analysis of PhAP activity. LC8
migrates near the front of the gel. The LC8 trap monomer (i.e. FKBP-IC peptide) is positively charged and does not enter the gel. A complex forms when a mix
of equimolar concentration of LC8 and LC8TRAP is treated with different molar concentrations of AP or PhAP (1ϫ, 2ϫ, etc.). B, quantification of the band
intensities from A. C, native PAGE analysis of LC8-LC8TRAP complexes formed using AP or PhAP and exposed to UV light (365 nm) for the indicated times (min).
D, intensity ratio of the complex band versus the LC8 band as a function of UV exposure.
or 2.4 molar excess (120 ␮M) of PhAP. The mix was incubated at aldehyde at room temperature for 10 min, and subsequent
4 °C for 5 min. Native PAGE analysis was performed at 16 °C using
8–25% gradient gels on the Phast system (GE Healthcare).
Antibodies and Reagents—Anti-EEA1 monoclonal antibody
was purchased from BD Biosciences, rhodamine-conjugated
donkey anti-mouse secondary antibody was from Millipore,
mounting medium Permount was from Fisher Scientific, 37%
formaldehyde was from Sigma, Lipofectamine 2000 was from
Invitrogen, Opti-MEM medium was from Gibco (Life Technol-
ogies), and DMEM medium and 10ϫ PBS were from Cellgro.
Cell Culture—COS1 cells were cultured in DMEM (Cellgro)
supplemented with 10% fetal bovine/calf serum (Omega Scien-
tific). Transfection was performed in 80–90% confluent 24-h
cultures of COS1 cells using Lipofectamine 2000 (Invitrogen)
and Opti-MEM medium (Gibco; Life Technologies) according
to the manufacturer’s recommendations. Various concentra-
tions (0–1000 nM) of AP (Ariad Pharmaceuticals Inc.) and
PhAP (synthesized by us) were added to each experimental well
24 h after transfection and incubated for different periods of
time before analyzing the cells. For reversibility of endosome
dispersion studies, 500 nM AP/PhAP was used. In each case, the
cells were washed well with PBS, supplemented with fresh
medium before inducing with UV light (10 min, 365 nm, 4
watts), and allowed to recover for various time periods at 37 °C
before analysis.
immunostaining was performed as described previously (6).
Briefly, COS 1 cells grown on 25-mm coverslips were washed
three times with PBS, treated with 3.7% formaldehyde (Sigma)
in PBS for fixation, and permeabilized in 0.5% Triton X-100
(Sigma) in PBS at room temperature for 10 min. Cells were then
incubated with blocking buffer containing 4% skimmed milk
(fat-free) and 0.5% Triton X-100 in PBS. Anti-EEA1 monoclo-
nal antibody was added to label early endosome marker protein
at a dilution of 1:100 in the same buffer for 30 min at room
temperature, and the coverslips were washed and incubated
with rhodamine-conjugated donkey anti-mouse secondary
antibody (1:100). After washing, the coverslips were mounted
on slides to visualize the trapping effects. Samples were
viewed using an Olympus IX81 automated inverted micro-
scope equipped with a water immersion ϫ60 objective. The
level of dispersion was quantified by counting 100 cells per
coverslip. We considered a cluster of EEA1-stained endo-
somes at the perinuclear region as “compact” (see Fig. 3B),
whereas endosomes spread throughout the cell were consid-
ered as “dispersed” (see Fig. 3B). Each experiment was per-
formed in triplicate. Images were obtained using a Spot RT
Slider high-resolution cooled CCD camera equipped to an
IX81 microscope and Image-Pro software. Images were
cropped and processed using Adobe Photoshop 7.0 (Adobe
Immunostaining and Microscopy—For immunostaining,
transiently transfected COS1 cells were fixed with 3.7% form- Systems), unless otherwise noted.
4548 JOURNAL OF BIOLOGICAL CHEMISTRY
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REPORT: Photocleavable Molecular Traps
TABLE 1
Percentage of endosome dispersion after treatment with PhAP or AP
UV Induction—For UV induction and recovery of endosome
studies, cells were washed with PBS, and fresh medium was
added. The washed cells were then treated with a hand-held UV
lamp (365 nm/4 watts) for 10 min by holding the UV lamp 2 cm
above the coverslip containing the cells. The cells were then left
to recover at 37 °C for varying periods of time before staining
and analysis.
AP (%)
PhAP (%)
Drug concentration (nM)a
0
26.9 Ϯ 3.1
44.4 Ϯ 2.4
53.0 Ϯ 3.5
55.5 Ϯ 0.7
56.2 Ϯ 1.6
64.1 Ϯ 7.6
66.4 Ϯ 2.5
27.2 Ϯ 2.5
47.9 Ϯ 8.1
48.0 Ϯ 5.0
55.5 Ϯ 1.3
57.5 Ϯ 2.4
67.5 Ϯ 7.0
67.7 Ϯ 4.9
10
20
50
100
500
1000
ImageJ (NIH) quantification—For quantitative analysis of
complex formation, we used ImageJ software from NIH. In each
case, the software was used to quantify bands on the scanned
PAGE.
Time (h)b
0
30.2 Ϯ 2.8
45.0 Ϯ 2.6
53.6 Ϯ 6.5
53.8 Ϯ 2.9
69.8 Ϯ 4.3
70.9 Ϯ 2.1
29.0 Ϯ 8
0.167 (10 min)
47.2 Ϯ 2.5
49.1 Ϯ 3.3
57.2 Ϯ 5.2
71.2 Ϯ 2.0
69.0 Ϯ 4.9
0.5
1
RESULTS
2
4
Synthesis of PhAP—First, we synthesized a UV-induced pho-
tolysable AP by replacing the amine linker of AP with a photo-
cleavable o-nitrobenzyl moiety to create PhAP. This synthesis
relied on the use of intermediate 5, which was prepared as
described previously (7). The final coupling between acid 5 and
diol 6 (8) produced the photocleavable modulator PhAP
(Fig. 1B).
Recovery after 2-h drug treatmentc
0
1
2
4
8
69.1 Ϯ 3.2
68.9 Ϯ 3.2
70.7 Ϯ 4.0
69.5 Ϯ 1.9
70.3 Ϯ 3.3
69.5 Ϯ 2.4
66.3 Ϯ 3.2
57.6 Ϯ 2.6
44.1 Ϯ 2.3
48.9 Ϯ 3.9
Recovery after 1-h drug treatmentd
0
1
2
4
8
58.0 Ϯ 4.3
71.2 Ϯ 2.5
69.7 Ϯ 3.6
70.4 Ϯ 3.1
70.8 Ϯ 1.9
57.8 Ϯ 8.0
56.4 Ϯ 1.7
55.7 Ϯ 8.8
46.7 Ϯ 0.6
48.1 Ϯ 2.0
In Vitro Characterization of PhAP—We tested whether the
replacement of the amine linker with the o-nitrobenzyl moiety
would affect the dimerization of FKBP in vitro. We used native
PAGE to follow the formation of the LC8 and FKBP-LC8
molecular trap (LC8TRAP) complex induced by the addition of
PhAP (6). Upon the addition of PhAP to an equimolar mixture
of LC8 and LC8TRAP, we observed a new band of PhAP-LC8-
LC8TRAP that migrated the same distance as the band produced
by the addition of AP (AP-LC8-LC8TRAP) (Fig. 2A). Quantifica-
tion of this new band indicated that a 2-fold higher concentra-
tion (2ϫ) of PhAP was required to produce a band of the same
intensity as the band produced by the sample treated with AP
(Fig. 2B).
Recovery after 30-min drug treatmente
0
1
2
4
8
51.8 Ϯ 4.4
64.7 Ϯ 2.0
68.3 Ϯ 3.7
69.8 Ϯ 4.3
67.9 Ϯ 1.9
53.2 Ϯ 2.4
53.7 Ϯ 4.0
50.8 Ϯ 2.3
48.0 Ϯ 0.5
49.3 Ϯ 6.7
a Percentage of endosome dispersion as a function of AP/PhAP concentration af-
ter 2 h of treatment.
b Percentage of endosome dispersion as a function of time with 500 nM AP/PhAP
concentration.
c Percentage of endosome dispersion at different time points (showing recovery
for PhAP), after 2 h of AP/PhAP treatment (500 nM).
d Percentage of endosome dispersion at different time point s(showing recovery
for PhAP), after 1 h of AP/PhAP treatment (500 nM).
e Percentage of endosome dispersion at different time points (showing recovery
for PhAP), after 30 min of AP/PhAP treatment (500 nM).
Next, we characterized how well UV light could disrupt the
PhAP-LC8-LC8TRAP complex in vitro. We generated the
PhAP-LC8-LC8TRAP and AP-LC8-LC8TRAP complexes and COS1 cells were transiently transfected with EGFP-FKBP-
exposed each to UV light (365 nm/4 watts). Native PAGE indi- LC8TRAP for 24 h, after which they were treated with PhAP or
cated loss of the band corresponding to the PhAP-LC8- AP (0 nM-1000 nM) for 2 h. Cells were then fixed and stained
LC8TRAP complex and an increase in intensity of bands corre- with an early endosome marker 1 (EEA1). The number of cells
sponding to the individual components after UV induction. with dispersed endosomes after treatment with PhAP or AP
The band corresponding to the PhAP-LC8-LC8TRAP complex was indistinguishable. The maximum number of cells with dis-
was less intense in samples that received a 5-min exposure to persed endosomes (67.5 Ϯ 7.0% for PhAP and 64.1 Ϯ 7.6% for
UV light and undetectable after a 10-min exposure. On the non-photocleavable AP) was obtained at dimerizer concentra-
other hand, the LC8-LC8TRAP complex induced by the non- tions greater than 500 nM (Fig. 1C, Table 1, first row). The
photocleavable AP (AP-LC8-LC8TRAP) persisted, even after a number of cells with dispersed endosomes over the same time
30-min exposure to UV light (Fig. 2C). A band intensity quan- course was also similar using either dimerizing agent, and no
tification of the ratio of the complex against the LC8 shows that changes were observed past 2 h (Fig. 1D, Table 1, second row).
the ratio remains unchanged in case of AP-LC8-LC8TRAP
,
The values obtained for both AP and PhAP are also in agree-
whereas the same is undetectable after 10 min of UV exposure ment with our previous studies (6).
for PhAP-LC8-LC8TRAP (Fig. 2D).
Next, we characterized the reversibility of endosome disper-
In Vivo Study of Reversal of Endosome Dispersion on UV sion in COS1 cells upon UV-induced cleavage of PhAP. In these
Induction—To determine whether these biochemical results experiments, transiently transfected cells were incubated with
had relevance in cells, we investigated whether photocleavage PhAP or AP (500 nM) for times ranging from 0 to 2 h, after
of PhAP could reverse the endosome dispersion phenotype which cells were washed rapidly, replenished with fresh
induced by LC8TRAP and PhAP. Initially, we established that medium, and then exposed to UV light (10 min, 365 nm, 4
PhAP behaved in a similar manner in cells as AP. To this end, watts). The cells were then fixed at 0, 1, 2, 4, and 8 h after UV
we used a green fluorescent protein (GFP) analog of the trap, radiation, and the endosomes were imaged using fluorescence
EGFP-FKBP-LC8TRAP, to identify cells that expressed the trap. microscopy at ϫ60 magnification. We observed a gradual
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REPORT: Photocleavable Molecular Traps
FIGURE 3. Cell-based assays and quantification of endosome dispersion. A, Cos1 cells were transiently transfected with EGFP-LC8TRAP and treated with AP
(red) or PhAP (blue) for predetermined periods (0.5, 1, and 2 h), exposed to UV light, and then fixed and stained with fluorescently labeled anti-EEA1 antibody.
Fluorescence microscopy was used to quantify the activity of the AP or PhAP on endosome dispersion. Each data point reflects 100 cells (n ϭ 3). Error bars
indicate mean Ϯ S.E. B, endosome dispersion when treated with PhAP (right) or AP (left) as observed by fluorescence microscopy. Transfected cells harboring
the trap are green (GFP fluorescence). The endosomes (red) in transfected cells disperse upon treatment with PhAP or AP. Upon UV treatment, endosomes in
cellstreatedwithPhAPreturntotheirperinuclearlocation, whereasendosomesincellstreatedwithAPremaindisperse. Notethatendosomesinuntransfected
cells (no GFP) remain compact irrespective of the treatment and serve as a negative control.
recovery of endosomes to the perinuclear region during the first the addition of PhAP or AP, as well as cells not transfected
4 h after UV exposure (44.1 Ϯ 2.3%), with a slight increase in with the trap. We also observed that a maximum of 70–75%
dispersion after 8 h (48.9 Ϯ 3.9%), for cells treated with PhAP of cells transfected with the trap had dispersed endosomes
(Fig. 3A). On the other hand, when cells were treated with AP, after treatment with PhAP or AP for 2 h. We suspect that
the endosomes remained dispersed at all time points evaluated, incomplete recovery arises from several sources, including
regardless of whether they received UV exposure. As an inter- cell heterogeneity (9), effects of transient transfection, and
nal control, cells that were not transfected (i.e. did not express the fact that cells were not synchronized throughout the
GFP) were treated in the same manner. These cells showed a experiment. However, similar spreads in these values have
slight increase in endosome dispersion in 8 h after UV expo- been reported in cell-based assays that used different meth-
sure. Of note, cells with compact endosomes 4 h after UV treat- ods to interfere with dynein-mediated processes, including
ment did not return to the same percentage as before the addi- RNAi (10, 11), expression of a dominant-negative protein
tion of PhAP. However, this is not entirely unexpected (12), and/or microinjection of monoclonal antibodies (13),
because we typically observe that 20–25% of cells have dis- all of which are irreversible. Consistent with these results, in
persed endosomes, including the trap-bearing cells before RNAi experiments targeting LC8, we observed that only 65%
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REPORT: Photocleavable Molecular Traps
of cells showed dispersion of the Golgi after 4 days of treat- activation of the signaling event (22, 23). We propose that the
ment as compared with 12% of cells exposed to a scrambled PhAP presented herein, in conjunction with a molecular trap
control.
expressed by a tissue-specific promoter, will not only afford
Having established that photocleavage of PhAP reverses the spatial and temporal activation of a biological process in an
phenotype, we asked whether the amount of time needed to animal model (e.g. Caenorhabditis elegans), but also the ability
restore perinuclear clustering of endosomes depended on how to reverse a phenotype and address questions such as the period
long the trap was allowed to act. As mentioned above, we found of a signal and commitment to a program that determines the
that maximal endosome dispersion occurred within 2 h, fate of the cell.
whereas the recovery occurred over a 4-h period. Thus, we
Acknowledgments—We thank current and former members of the
Williams and Horne laboratory for useful discussion. We also thank
Professor Jim Keen at Thomas Jefferson University for insightful
discussion.
treated cells for 30 min and 1 h with PhAP or AP followed by UV
exposure. As expected, this resulted in a lower percentage of
cells that had dispersed endosomes (0.5 h, 53.2 Ϯ 2.4%; 1 h,
57.8 Ϯ 8.0%; Table 1, fifth row). However, for all treatment
times (0.5, 1, and 2 h), the percentages of cells with dispersed
endosomes were similar within 4 h after exposure to UV light
(Fig. 3A, Table 1). In contrast, cells treated exactly in the same
manner, but with AP instead of PhAP, exhibited continued
endosome dispersion until dispersion reached the saturation
point (ϳ70%), further confirming that induction of the trap
with AP creates a highly stable complex. Fig. 3B shows repre-
sentative images of compact endosomes and their dispersion on
AP/PhAP treatment. Please note that only transfected cells
(green) show endosome dispersion (Fig. 3B, DISPERSED) and
that untransfected cells, which act as an internal negative con-
trol, do not show endosome dispersion (Fig. 3B, Untransfected
compact). However, in the case of PhAP, the endosomes return
to their perinuclear position (Fig. 3B, COMPACT) upon UV
irradiation. This is not the case for cells treated with AP where
the dimeric trap remains associated and endosomes continue
to disperse.
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DISCUSSION
Herein, we have developed a photocleavable analog of AP,
demonstrated that PhAP can induce formation of the LC8-
LC8TRAP complex, and shown that photocleavage of PhAP
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Finally, although we have applied this new reagent in the
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as well. Of note, a photocleavable rapamycin analog was
recently created to dimerize FKBP and FKBP12-rapamycin-as-
sociated protein (FRAP). In this case, the photocleavage was
used to activate the rapamycin analog for spatial and temporal
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