Review

How chemistry supports cell biology:

the chemical toolbox at your service
Ruud H. Wijdeven, Jacques Neefjes, and Huib Ovaa
Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

Chemical biology is a young and rapidly developing

scientific field. In this field, chemistry is inspired by
biology to create various tools to monitor and modulate
biochemical and cell biological processes. Chemical contributions such as small-molecule inhibitors and activitybased probes (ABPs) can provide new and unique
insights into previously unexplored cellular processes.
This review provides an overview of recent breakthroughs in chemical biology that are likely to have a
significant impact on cell biology. We also discuss the
application of several chemical tools in cell biology
research.
Why do we need chemical tools in cell biology?
The aim of cell biology is to understand the cellular and
molecular processes that control cell function. To understand these processes, they first need to be perturbed, after
which cellular responses are measured or visualized. Techniques that allow visualization and manipulation of cellular processes have made tremendous progress over the
past decades and have been instrumental in increasing
our understanding of the biology of the cell. A major
breakthrough in visualizing cellular processes came with
the tagging of proteins with GFP or related fluorescent
proteins [1,2]. This, in combination with technologies to
interfere with protein expression, including genetic silencing techniques such as RNAi and, more recently, CRISPR
Caspase 9 (Cas9)-mediated gene knockout systems, have
allowed cell biologists to decipher the function of specific
proteins and their role in cellular networks [35]. Furthermore, yeast two-hybrid technologies and protein mass
spectrometry have improved understanding of protein
protein interactions (PPIs) and have allowed the reconstruction of complex protein networks that control cellular
function [6,7]. Although these technologies are of great
importance in current cell biology, they also have disadvantages that limit their applicability. For example GFP,
although small, is still a sizeable protein that might interfere with the localization or function of tagged proteins
[8,9]. Furthermore, the time required to deplete transcripts and proteins using RNAi [small interfering RNA
(siRNA), short hairpin RNA (shRNA)] or gene knockout
(such as CRISPRCas9 nuclease) technologies is relatively
Corresponding authors: Neefjes, J. (J.Neefjes@nki.nl); Ovaa, H. (H.Ovaa@nki.nl).
Keywords: chemical biology; activity-based profiling; chemical genetics.
0962-8924/
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2014.07.002

long (usually of the order of days). This allows cells to

compensate for the loss of protein levels, or cells may
simply not survive in the case of essential proteins. Here
chemical biology steps in and provides many options to
decipher the function of proteins in the context of complex
cellular networks. Small molecules can modulate processes
swiftly, thereby not allowing cells to compensate for the
loss of specific proteins. In addition, they can form the basis
for tools that allow visualization of the activities of defined
enzymes in time and space. The specificity issue of small
drug-like molecules is similar to that of RNAi, where offtarget effects are often observed. Therefore, we believe that
cell biology is best studied by using a combination of
genetic and chemical approaches.
Genetic manipulation techniques have been developed
such that they can be used in a standardized manner. This
is different for chemical biology, where new tools are
developed to address specific biological questions. Here
we discuss a selection of chemical tools that allow visualization and manipulation of cellular processes. We highlight recent developments in the design and use of chemical
inhibitors, ABPs, and fluorescent detection reagents for
applications in cell biology. Furthermore, we discuss the
use of chemically tunable protein tags and unnatural
amino acids (UAAs) to trace and modulate proteins.
Small-molecule modulators
Many fundamental discoveries in cell biology have been
made using (natural) chemical modulators; for example, by
Glossary
Activity-based probes (ABPs): chemical probes designed to bind covalently the
active site of a specific enzyme or enzyme class. They harbor a reactive moiety
and a recognition element for specific labeling of enzymes and a reporter
element for detection.
Proteinprotein interaction (PPI) inhibitors: chemical inhibitors that bind to the
proteinprotein interaction surface of two proteins and thereby block their
interaction.
Reciprocal chemical genetics: a chemical-compound screen is compared with a
genetic screen to find novel inhibitors and their potential targets that control
the process of interest.
RNA Flare: a technique to quantify RNA expression. It is based on gold particles
carrying a sequence complementary to the RNA of interest that are coated with
small RNA strands. On binding of the target RNA to the probe, the small RNA
strands are released and become fluorescent.
RNA fluorescence in situ hybridization (FISH): a technique that allows the
localization and quantification of RNA in the cell. RNA is stained in cells using
fluorescent probes that are complementary to the RNA sequence of interest.
Selective bio-orthogonal labeling: a technique that allows the labeling of a
specific target (protein) in the cell, but not any other structure.
Unnatural amino acid (UAA) (or noncanonical amino acid): an amino acid that
is not naturally incorporated into proteins. Such amino acids can be
incorporated into proteins to give them special characteristics.

Trends in Cell Biology, December 2014, Vol. 24, No. 12

751

Review
using actinomycin D to block transcription, cycloheximide to
block translation, MG132 to block proteasome-mediated
protein degradation, and Brefeldin A (BFA) to block transport through the Golgi apparatus [1013]. Chemical inhibitors have the advantage that they often act
instantaneously, giving cells limited time to compensate
for protein loss. Furthermore, they allow transient blockage
of processes that are essential for cellular survival, as in the
examples above. Another important advantage of small
molecules over genetic manipulation is that the effect of
chemical inhibitors is often reversible. This reversible nature is, for example, exploited for the nocodazole mitotic
block, which can synchronize cells in the cell cycle [14]. The
spindle poison nocodazole interferes with the growth of
microtubules, leading to mitotic arrest and synchronization
of the cells. This molecule can then be removed to release the
cells from mitotic block. This method has strongly supported
studies of the cell cycle [14]. Furthermore, small-molecule
chemical modulators are critical for studying cell types that
are difficult to manipulate by genetic means, such as neuronal cells or primary immune cells.
Many chemical modulators are natural products that
have been identified by phenotypic characterization. However, now most novel chemical modulators are discovered by
in vitro screening with synthetic compounds. Small-molecule screening has become routine in academic research and
many new small-molecule modulators have been identified.
Often a target protein is first identified as critical for a given
cell biological process or disease using genetic screens or
association studies. For this target protein, specific assays
can then be developed to find chemical modulators. Screening assays are often based on a substrate of an enzyme or a
ligand for a receptor equipped with a fluorescent label for
detection of enzyme activity or receptor binding by fluorescence, fluorescence resonance energy transfer (FRET), or
fluorescence polarization. In general, inhibitors are either
rationally designed based on chemically altered substrates
or identified by screening small-molecule libraries. These
inhibitors usually occupy substrate-binding sites in proteins
or they can act in an allosteric manner. Inhibitors can also be
developed to target the protein surface between two interacting proteins. These so-called PPI inhibitors (see Glossary) are generally more difficult to design [15]. Yet, this new
class of inhibitors increases both the repertoire of molecules
that allow us to interfere with cellular processes and the
options for therapeutic intervention [16]. High-throughput
screening of small-molecule libraries in combination with
thorough assay systems have proved successful in the discovery of such molecules.
A recent example of such a PPI inhibitor is an inhibitor
for clathrin-mediated endocytosis named pitstop [17]. Endocytosis of extracellular cargo is mediated via several
independent pathways and is widely studied using chemical inhibitors [18]. The two main endocytic routes are
caveolin- and clathrin-mediated endocytosis. Several inhibitors for caveolin-mediated endocytosis have been developed [19,20], but a specific inhibitor for clathrin-mediated
endocytosis remained missing. Clathrin-mediated endocytosis requires binding of the terminal domain (TD) of
clathrin to an endocytic ligand. This information established an ELISA-based assay to monitor the interaction of
752

Trends in Cell Biology December 2014, Vol. 24, No. 12

a plate-bound endocytic ligand (amphiphysin) to the TD of

clathrin. Screening a library of 17 000 small compounds
yielded a molecule that potently inhibited clathrin-mediated endocytosis by blocking the interaction of clathrin
with amphiphysin [17]. This inhibitor is now used to decipher routes of uptake of extracellular material [21].
Another example of a PPI inhibitor is nutlin-3, which
prevents degradation of TP53 [22]. TP53 is the most frequently mutated tumor suppressor and is ubiquitinated
and targeted for proteasomal degradation by the E3 ubiquitin ligase Mdm2. Nutlin-3, again discovered by smallmolecule screening, occupies the interaction surface of
Mdm2 and TP53, thereby stabilizing TP53. Nutlin-3 provided a first proof of principle for the strategy to block PPIs
and is now in clinical trials as a cancer therapeutic. Nutlin3 has also been used to alter TP53 levels in primary cells
[23] and in vivo [24]. For example, using this molecule it
has been shown that in primary macrophages TP53 and
nuclear factor kappa B (NF-kB) work together to drive
expression of proinflammatory cytokines [23].
A different approach to identify chemical modulators
integrates chemical and genetic screens. This approach does
not require prior selection of a target protein and is therefore
unbiased. The concept is based on the idea that genetic
inactivation and chemical inactivation yield similar phenotypic outcomes. The results from genetic and chemical
screens can be integrated, yielding bioactive compounds
with a limited number of likely corresponding targets. In
vitro tests using recombinant proteins can then be used to
identify the specific targetinhibitor combinations. This
approach called reciprocal chemical genetics has successfully identified novel kinase and phosphatase targetlead combinations that control Salmonella infection [25,26].
These compounds were the first antibiotics acting on the
infected host cell rather than the infectious species.
The availability of commercial screening collections has
boosted this approach in academia and industry. Libraries
designed for maximal effects on specific target classes
targeted libraries are also available or in development.
The best examples are kinase-targeted small-molecule
libraries, which are widely used in kinase inhibitor discovery. Overall, the number of available small-molecule modulators will increase strongly in the next few years,
providing a more complete chemical toolbox that can be
used in cell biology research.
Activity based probes
Besides modulating the action of proteins, many chemical
tools have been developed to monitor enzymatic activity.
Many cellular processes are controlled by enzymes, but
assaying enzymatic activity in cellular space and time has
always been challenging. To allow easier quantification of
enzymatic activity, chemists have developed ABPs (see
Table 1 for an overview). ABPs are designed to bind
covalently and specifically to the active site of an enzyme
or enzyme class. Their design is typically based on a known
enzyme substrate, which provides the desired specificity of
the probe, further modified to bind covalently to the enzyme of interest by the introduction of a reactive chemical
group (called a warhead). These probes typically react
covalently with enzymes in an activity-based manner. For

Review

Trends in Cell Biology December 2014, Vol. 24, No. 12

detection of enzymatic activity, the probe generally contains a fluorescent dye, a radiolabel, or a biotin moiety for
isolation (Figure 1, right panel).
ABPs have various applications. For example, they can
be used to monitor the presence or the amount of active
enzymes in cell lysates. By using an affinity tag, enzymes
that recognize and bind the probe can be isolated and
identified using mass spectrometry [27]. For example,
the discovery that enzymes with an Ovarian Tumor
(OTU) domain belong to the class of deubiquitinating
enzymes (DUBs) was made using an ABP that specifically
labels DUBs [28]. This ABP was based on ubiquitin
equipped with a warhead and a detection/isolation handle.
ABPs can also define inhibitor specificity or targets. In
this approach the specificity of a potential inhibitor is
obtained by fingerprinting cell lysates incubated with
the ABP in the presence or absence of an inhibitor
(Figure 1, top right). Enzymes inactivated by the inhibitor

Inhibitors

will not react with the ABP and the target can be identified
by the absence of a defined band after SDS-PAGE. This
approach is well established and has been used to characterize several covalent and noncovalent kinase inhibitors
[29,30].
Another approach that allows small-molecule-target
identification is called chemical proteomics. In this approach, a chemically reactive or high-affinity inhibitor is
coupled to an affinity tag with which interacting enzymes
are isolated and identified. This approach has been used to
show that the Src/ABL tyrosine kinase inhibitor dasatinib
inhibits several other kinases besides Src and ABL [31].
Notably, this approach requires significant modifications
to the inhibitor that often affect its properties, such as
target affinity and specificity.
The fingerprinting approach can also profile differences
in enzyme expression levels or activities between two
different conditions. ABP-bound active enzymes can be

Acvity based probes

CME

Amp

Enzyme proteomics

Probe design

Wild type

Clathrin

Pitstop

Warhead Recognion
Degradaon

Stabilizaon
Nutlin
p53

Acve enzyme localizaon

Visualizaon

Mitochondria

RNA Flare

pH sensor
Low

RNA FISH

Hi

ER

Quancaon

Acve enzyme quancaon

Metabolite quancaon
GR

AAA

Tag

Provides enzyme Fluorescent

(class) specicity or anity

MDM2

MDM2
p53

Visualizaon

Enzyme
binding

Treated
(inhibitor,
starved etc)

GSSG

Probe
Treatment

probe
GSH

+ +
+
Acve

Enzyme
Inacve

AAA
TRENDS in Cell Biology

Figure 1. Schematic overview of chemical tools available to manipulate or visualize processes in the cell. Top left: The chemical inhibitor pitstop blocks clathrin-mediated
endocytosis (CME) by inhibiting the interaction between clathrin and its ligands. The chemical inhibitor nutlin binds to the E3 ubiquitin ligase MDM2 (Hdm2 in humans) and
blocks its interaction with p53, preventing degradation of p53. Lower left: Chemical probes have been used to detect molecular structures in the cell. For RNA Flare, RNA
sequences complementary to the gene of interest (grey) are coupled to a gold particle. These Flare probes are bound by fluorescent probes with a complementary sequence
(black), the fluorescence of which is quenched by the gold particle. On binding of the RNA of interest to the Flare sequence, the fluorescent probe is released and becomes
fluorescent. For RNA fluorescence in situ hybridization (FISH), fluorescent probes complementary to the RNA sequence of interest bind, allowing RNA visualization and
localization in cells. Fluorescence can be increased by using multiple probes (red dots) or a secondary detection probe with multiple fluorophores (green dots).
Furthermore, probes have been developed that display pH-dependent changes in fluorescence, which can be used accurately to detect endosomal acidity. Other markers
specifically stain compartments such as mitochondria and the endoplasmic reticulum (ER). Right: Activity-based probes (ABPs) can be used to detect enzyme activity in
cells (left side) and in vitro (right side). They typically contain a chemical group (warhead) that binds the target enzyme (class), a recognition element that facilitates specific
recognition of the target enzyme (class), and a fluorescent probe or affinity tag that can be used for detection or isolation. Cell-permeable probes can be used to localize
active enzymes in the cell. By using a warhead specific for an enzyme reaction product [for example, glutathione (GSH)], enzymatic activity can be measured by quantifying
the amount of enzymatic reaction product. In vitro, ABPs can be used to compare the amount of active enzyme in two different samples, such as healthy versus diseased
tissue, mitotic versus nonmitotic cells, wild type versus inhibitor-treated cells, or heart versus lung cells. Two samples (control and treated) are labeled with the ABP and the
differences in enzymatic activity can be measured using SDS-PAGE or mass spectrometry. In addition, relatively large probes can be used to quantify the fraction of active
enzyme. After separation by SDS-PAGE, staining for the enzyme will yield a higher-molecular-weight band, the reactive enzyme, and a lower-molecular-weight band, the
unreactive enzyme. These can be compared among different cellular states to determine the relative amount of active enzyme. Abbreviations: AMP, amphiphysin; GR, GSH
reductase; GSSH, GSH disulfide.

753

Review
isolated and compared between two samples using a quantitative mass spectrometry technique like stable isotope
labeling with amino acids in cell culture (SILAC). With this
approach, quantitative profiling of kinases involved in
different cell signaling pathways has been achieved [32].
Besides proteomics, large ABPs (or probes that cause
detectable mobility shifts of proteins) can also be used to
quantify the relative fraction of an enzyme that is in an
active state (Figure 1, lower right). Probe-bound active
enzymes can be separated from inactive enzymes by
SDS-PAGE, allowing determination of the relative amount
of active enzyme. This has been used to show that reversible active-site cysteine oxidation of deubiquitinating
enzymes renders them catalytically inactive [33]. A similar
approach can determine the effect of post-translational
modifications or splicing isoforms on the activity of a
defined enzyme, by comparing the fractions of wild type
and modified enzyme that bind the probe.
In addition to in vitro approaches, ABPs that are cell
permeable can localize enzyme activity in the cell. For
example, visualization of active proteasomes using a fluorescent proteasome probe [34,35] has revealed that proteasomes found associated with polyglutamine aggregates
remained active yet failed to clear these aggregates [36].
Another probe designed for in vivo labeling binds covalently to glucocerebrosidase, a lysosomal enzyme that degrades
glycolipids [37]. This probe has been used to show that
mutations in the enzyme, which cause Gauchers disease,
do not affect its catalytic activity [37].
Enzyme activity in cells can also be measured directly
by detecting the enzyme product (Figure 1). For this purpose, enzyme substrates are typically chemically modified
to become fluorescent when enzymatically converted. Alternatively, probes have been designed that become fluorescent upon binding of the enzymatic product. A probe
that becomes fluorescent upon binding reduced glutathione can directly visualize the activity of the enzyme glutathione reductase. This enzyme is hyperactive in liver
cancer and can help to distinguish tumor cells from healthy
tissue [38].
In conclusion, ABPs have been designed to measure the
activity of many enzymes and enzyme families. ABPs are
easy to use and versatile in their application. The current
challenge is to design enzyme-specific probes that are cell
permeable, although a new technique called squeeze loading
holds promise to overcome the issue of cell permeability [39].
Fluorescence-based detection probes
Historically, visualization of biological processes in cells
has been achieved by staining proteins with antibodies.
However, this method does not allow the detection of many
complex structures or dynamic processes. For many of
these processes and structures, fluorescent cell-permeable
small molecules have been developed. In recent years,
chemical biologists have contributed various new probes
that allow the detection of RNA, organelles, and subcellular structures.
Visualizing and quantifying RNA species
It is well known that RNA encodes genetic information in
the form of mRNA and can have enzymatic activity in the
754

Trends in Cell Biology December 2014, Vol. 24, No. 12

form of rRNA. However, the world of RNA species is far more

complex. For example, long noncoding RNAs (lncRNAs)
directly affect transcription and translation but also have
many other functions [40]. Recent studies suggest that the
function of RNA is often dependent on its cellular localization [41]. Furthermore, mRNA can be translated at specific
locations in the cell, leading to local protein gradients [41].
Therefore, understanding the cellular localization of specific
RNA species may offer new insights into the complex world
of RNA biology. Conventional techniques such as quantitative PCR (qPCR) and next-generation sequencing fail to
localize RNA species correctly within the cellular space
[42]. Furthermore, these techniques are laborious and do
not allow high-throughput quantification of RNA in a multitude of samples. Several chemical techniques have been
developed to monitor localized translation, like puromycinbased labeling of novel transcripts or tRNA FRET to monitor
active ribosomes, but these techniques are mostly applied on
a global transcriptional level [4345].
For cellular localization of specific RNA species, an
older, chemistry-based technique called RNA fluorescence
in situ hybridization (FISH) has been reintroduced. This
technique, as well as a novel RNA quantification technique
known as RNA Flare, can also be used to quantify RNA at
the single-cell level in a high-throughput manner [46].
RNA FISH detects RNA using fluorescently labeled
oligonucleotides that complement the RNA species of
choice [47]. High background labeling was a frequent
problem, but the use of multiple oligonucleotides that
target different sequences on the same RNA species has
improved the signal-to-noise ratio [48]. Various probes
label the target RNA, all contributing to the total fluorescence and thereby increasing the overall specificity and
sensitivity of the labeling (Figure 1, RNA FISH). Furthermore, different fluorescent dyes can be used to detect the
expression of multiple transcripts simultaneously, transcripts of gene fusions [49], and allele-specific expression
[48]. The transcripts can be detected and quantified by
fluorescence microscopy. Another method to increase the
specificity of fluorescent signals applies locked nucleic
acids (LNAs). LNAs are nucleic acid analogs that have
higher target affinity and therefore better mismatch discrimination and higher specificity [50]. These LNA probes
are mostly used to detect miRNAs short pieces of RNA
that are otherwise difficult to probe [51]. Alternatively, the
RNA probe can be detected using a second, highly fluorescent probe (Figure 1, RNA FISH), leading to a much
brighter signal. This technique is sufficiently sensitive to
allow high-throughput quantitative analysis of multiple
transcripts simultaneously in single cells [52].
The second novel technique developed for RNA quantification is RNA Flare [53]. Here, an RNA sequence complementary to the target mRNA is coupled to a gold
particle. This construct is bound by a short complementary
sequence with a fluorescent probe that is quenched by the
gold particle. Upon mRNA binding, the probe is released
because it competes with the target mRNA species. This
stops the quenching and the probe becomes fluorescent,
after which it can be detected by microscopy or flow
cytometry (Figure 1, RNA Flare) [53]. Because the fluorescent probe is not bound to the mRNA, this technique is

Review

Trends in Cell Biology December 2014, Vol. 24, No. 12

unsuitable for RNA-localization studies. In contrast to

RNA FISH, the probe can enter cells and is suitable for
quantifying expression in living cells. Therefore, using this
technique cell populations can be sorted and isolated based
on their mRNA expression levels [54].
The recent developments in RNA FISH and Flare technologies are likely to revolutionize single-cell transcriptional profiling and localization of transcripts.
Furthermore, both technologies have opened the way for
genetic and small-molecule screens to study mRNA or
lncRNA expression.
Fluorescent organelle markers and chemical dyes for
cell characterization
Besides detecting RNA, fluorescent dyes are widely used
for the visualization of organelles. In contrast to antibodies, which detect mostly proteins and often require fixation and permeabilization of cells, chemical dyes can often
bind different structures in living cells. For many organelles, fluorescent cell-permeable probes have been developed (see Table 2 for an overview). These include probes for
the nucleus, endoplasmic reticulum (ER), lysosomes, peroxisomes, mitochondria [55], and, more recently, autophagolysosomes [56]. Also, fluorescent sensors have been
developed to detect biochemical states in cells, including
pH, redox state, and the presence of various ions such as
Ca2+ [57,58]. Note that fluorophores are sensitive to environmental changes, which could affect measurements.
Fluorescent small molecules can also be used for the
detection of cellular differentiation states. This approach is
based on the idea that the target of a fluorescent molecule
is differentially expressed in two cell types or cellular
differentiation states. This molecule can then be used as
a marker to sort or stain for the specific cell type. For
example, by screening fluorescent chemical libraries a
compound named CD1y was identified that binds to embryonic stem cells but not their fibroblast progenitors. This
compound is now used as a marker for stem-cell differentiation and was used to show that the RNA-binding protein
ESRP1 controls stem-cell development [59]. It also provides a readout for stem-cell development while screening
RNAi or compound libraries [60], although the target of
Table 1. Enzyme- or enzyme class-specific ABPs
Target enzyme or enzyme family
Family specific
Kinases
Phosphatases
Serine proteases
Cysteine proteases
Metalloproteases
Deubiquitinases
Cytochrome p450s
Cell permeable and enzyme specific
Proteasome
Glucocerebrosidase
Legumain
Cathepsin X
Thioredoxin reductase
Sialidase
Elastase

Refs
Multiple, see [27]
[87]
[88,89]
[90]
[91]
[92,93]
[94]
[34]
[37]
[95]
[96]
[97]
[98]
[99]

Table 2. A selection of fluorescent probes to visualize

compartments and processes
Compartment/process
Mitochondria
Endoplasmic reticulum
Lysosome
DNA/nucleus
Actin cytoskeleton
Calcium levels
Cytosolic pH
Endosomal pH

Name of probe
Mitotracker
ERtracker
Lysotracker
Hoechst/DAPI/DRAQ5
Phalloidin analogs
BAPTA AM
BCECF
DND-160

Refs
[100]
[101]
[102]
[103105]
[106]
[107]
[108]
[109]

this molecule is unidentified. Several fluorescent-smallmolecule libraries have now been constructed, with the
goal of identifying agents that report specific cellular phenotypes and differentiation states, as reviewed in [61].
Phenotypic screening of fluorescent small-molecule libraries and the development of next-generation dye collections
are likely to result in the identification of many more cell
type- and biochemical process-specific fluorescent markers.
Chemistry-based protein modifications
Cell biology research often involves studying the function
of proteins or processes controlled by proteins. To elucidate
the function of a given protein, its expression needs to be
changed or the protein itself needs to be modified. Chemical biology provides methods for the selective modification
of a protein of choice, enabling the generation of proteins
with the desired property. This facilitates biochemical
studies in complex settings.
Chemically tunable protein tags
Studying protein function often requires tunable expression, which can be achieved by regulated degradation or
stabilization of the protein of interest. Furthermore, the
function of many proteins is controlled by their localization
or interactions with other proteins. To control protein
stability, association, or localization at will, several systems have been developed. These are generally based on a
protein tag that can be stabilized or destabilized by a
chemical compound or two protein tags that can stably
interact by binding a dimerization-inducing compound.
Tagging the protein of interest with any of these tags tune
its expression level, localization, or interaction with another protein (Table 3 and Figure 2).
Protein tags are the fastest way to modify protein levels
and are commonly employed in studies that require rapid
protein stabilization or destabilization. Compound binding
generally modifies the folding of the tag, resulting in
shielding (stabilization) or exposure (destabilization) of
hydrophobic domains. This leads to stabilization or degradation of the tag and the associated protein. Alternatively,
proteins can be destabilized by altering the exposed Nterminal amino acid by the so-called N-end rule [62,63].
However, this system is dependent on the N-terminal
sequence context and has limited applicability [64]. The
most widely used tag to stabilize proteins under chemical
control is FKBP12 (see below), which is modified to be
inherently unstable in the absence of rapamycin or close
analogs [65]. For inducible degradation, the auxin degron
755

Review

Trends in Cell Biology December 2014, Vol. 24, No. 12

Table 3. An overview of chemically tunable protein tags that enable protein stabilization, degradation, or dimerization
Modification
Stabilization
ecDHFR

Ligand

Refs

Trimethoprim

[110]

FKBP12

Rapamycin or
close analogs

[65]

HaLo HyT13

[111]

Auxin

1-Naphthaleneacetic
acid (auxin analog)

[66]

FKBP12LID

Rapamycin and close

analogs

Degradation
HaLo tag

Dimerization/relocalization
GID1GAI

Same as FKBP12 stabilization tag

Gibberellin analog
GA3-AM

FKBP12FRB
FKBP12FKBP12

Rapamycin (or analog)

AP1510

Streptavidin
streptavidinbinding domain

Biotin

[74]

Same as FKBP12 stabilization tag

is commonly used; this tag is rapidly ubiquitinated and

degraded after binding of the plant hormone auxin [66].
These tags are used most frequently in cell-cycle studies,
where protein levels are tightly controlled [67]. Other
applications include the use of these tags as model
756

[112]

[70]
[71]

[75]

substrates to study proteasome function or protein degradation for antigen presentation [68].
The best way to control protein-protein interactions or
protein localization in a timely manner is by using
tags that dimerize proteins after binding a dimerizing

Review

Trends in Cell Biology December 2014, Vol. 24, No. 12

Tunable protein tags

Unnatural amino acids

Selecve labeling

Induced stabilizaon
Unnatural
amino acid

Protein containing
unnatural amino acid

HN

H
O
H

Mutated
tRNA

Induced destabilizaon

N3
H2N

COOH

AUC
TAG

Induced relocalizaon
mRNA

Detecon probe

BCN reacve group

Crosslinkers

Funconal amino acids

Induced hetero / homodimerizaon

N3

HN

O2N

NO2

O
O

HS

O
O

H2N

COOH

H2N

COOH

H2N

COOH

H2N

Key:

Protein of interest
Tag
Chemical

UV acvatable
crosslinker

Cysteine reacve
residue

COOH

UV uncageable
lysine residue

H2N

COOH

UV uncageable
tyrosine
TRENDS in Cell Biology

Figure 2. Methods to alter the properties of proteins chemically. Left: A protein of interest (green box) can be tagged with different protein tags (orange boxes) that are
designed to bind chemical modulators (in red). Some tags are inherently instable, but binding of the compound stabilizes the tag and the protein fusion. Other tags have
been designed to become instable after compound binding and can be used to induce degradation of a protein of interest. A third system depends on two different tags that
interact after binding a compound. By cloning one of these tags to a membrane-localized protein and the other to the protein of interest, the protein of interest can be
induced to relocate to the membrane. Similarly, these tags can be used to heterodimerize two proteins (green and blue boxes). By using a different chemical modulator, two
identical tags can be homodimerized. Right: Unnatural amino acids (UAAs) can be site-specifically incorporated into a protein by introducing an evolved tRNA synthetase
and matching tRNA. These UAAs can be used for the selective labeling of proteins, to crosslink an amino acid of interest to a nascent residue, or to introduce residues that
can alter the function of a given protein of interest.

compound. The first chemically inducible dimerization tag

to be discovered was derived from the inducible dimerization of FKBP12 with mammalian target of rapamycin
(mTOR) [65]. Rapamycin, an immunosuppressant produced by bacteria, forces binding of FKBP12 to the FRB
domain of mTOR and hereby effectively inactivates its
kinase domain. By tagging one protein with the FKBP12
domain and the other with the FRB domain, an interaction
between the two proteins can be induced by rapamycin
[69]. Mutations in the FKBP12 and FRB domains have
been made to allow the use of rapamycin analogs that do
not affect the mTOR pathway, because mTOR inhibition by
rapamycin induces many cellular changes [70]. The
FKBP12 tag can also be used to induce homodimerization
by the addition of a different compound [71] (Figure 2).
Dimerization tags are used for various purposes. First,
they can anchor proteins at a specific location in the cell. By
tagging a membrane-localized protein with FRB, the
FKBP12-tagged protein of interest can be forced to localize
to this membrane compartment. This has been used to
inducibly activate GTPases, which are active when localized at the plasma membrane [72]. A protein can also be
stored in an irrelevant compartment and released at will
by removal of the dimerizer to become active [69]. Alternatively, this system has been used to homodimerize Caspase 9, thereby triggering apoptosis. For this purpose, the
dimerization domain in Caspase 9 was replaced by the
FKBP dimerizer system, allowing chemical control of

apoptosis in a synchronized manner [73]. Generally, inducible protein dimerization is used to control PPIs in a timely
manner and to study their biological consequences. By
combining two different dimerization systems, even trimerization can be achieved [74].
In contrast to inducible interaction systems, a different
system has been developed to disrupt specific interactions.
This method uses streptavidin and biotin, which bind to
each other with extraordinarily high affinity. A 38-amino
acid peptide has been constructed that binds streptavidin
but can be competed away by biotin [75]. In this manner,
the tagged proteins can be released from each other by the
addition of biotin, which is imported into the cell via
specific transporters [76]. This system works with intact
living cells but can also be used under in vitro conditions for
the isolation of proteins [75].
Protein tags are a simple and versatile tool that allows
timed control of the kinetics of biochemical reactions in
living cells. By combining these tags with fluorescence livecell imaging, biological reactions can be monitored in real
time. This creates new and exciting insights into the
biology of the cell.
Unnatural amino acids
Protein modifications often affect their properties. Ideally,
protein modifications are as small as possible to allow
detection with minimal distortion. Chemical biology has
devised techniques to modify proteins minimally, to best
757

Review
replicate the natural state. One such technique takes
advantage of the incorporation of noncanonical amino
acids into proteins. The noncanonical amino acid can function as a tag or can be an amino acid with specific biochemical properties.
Incorporation of noncanonical amino acids or UAAs can
be achieved in a residue- or site-specific manner. Residuespecific incorporation means that in all proteins one of the
20 amino acids is replaced by an UAA. This method takes
advantage of the fact that some endogenous aminoacyltRNA synthetases can be charged with noncanonical amino acids. For example, the methionine tRNA synthetase
also accepts azidohomoalanine (AHA). If a methionineauxotrophic bacterial strain is used, AHA can be incorporated with great efficiency by the endogenous translation
machinery. Also, in mammalian cells, AHA can compete
with methionine incorporation. By replacing methionine
by AHA in the culture medium, newly synthesized proteins
can be detected after selective AHA modification using
click chemistry [77].
Alternatively, UAAs can be incorporated in a site-specific manner. This allows nearly-specific incorporation of a
new amino acid into a protein. Here, the tRNA for the
amber codon UAG and its corresponding aminoacyl-tRNA
synthetase are mutated to bind and incorporate the desired UAA. By ectopically expressing the protein of interest
with the amber codon sequence, it will be site-specifically
labeled with the UAA [77,78]. Various aminoacyl-tRNA
synthetases have been described that are able to load a
variety of UAAs [78]. The major limitation of this method is
that optimization of complementary tRNA/tRNA synthetase pairs is a time-consuming and complicated procedure,
mainly because it involves extensive mutagenesis of the
tRNA synthetase to create mutants that allow incorporation of the desired amino acid [79]. However, once optimized this system can be used for several unique cell
biological applications, such as interaction mapping, introduction of post-translational modification isosteres, and
selective labeling with chemical reporters (Figure 2).
Some UAAs are designed such that they can undergo
selective (bio-orthogonal) chemical reactions. The UAA
functions as a single-amino-acid tag, causing minimal
perturbation to proteins. Chemically reactive amino acids
can be introduced to crosslink fluorescent dyes or affinity
tags to proteins in lysates and in living cells [80] (Figure 3).
Labeling can also be achieved with dyes in the far-red
spectrum, suitable for super-resolution microscopy [81,82],
which opens the door for multicolor live-cell super-resolution microscopy.
Other UAAs can aid in the study of the biochemical
properties of proteins. For example, by using a UV-inducible crosslinking amino acid, the exact interaction sites
between a protein and its binding partner have been
mapped in living cells [83]. The important advantage of
this technology is that it supplies information about the
interaction of a defined site in a protein with its ligand
under the most physiological conditions currently available. Other studies have used infrared UAAs to trace
conformational changes of a G protein-coupled receptor
[84] and to map the hydrophobic interactions of tyrosine
during ion-channel opening using tyrosine analogs [85].
758

Trends in Cell Biology December 2014, Vol. 24, No. 12

The use of UAAs in cell biology research has seen a

significant increase in the past few years. However, the
need for development of evolved matching tRNA/tRNA
synthetase pairs has thus far limited this technique to
about 50 noncanonical amino acids. The incorporation of
these amino acids by the endogenous translation system is
limited to only a few amino acids. Nevertheless, technology
to introduce different amino acids continues to develop,
enabling unique applications such as biochemistry in living cells.
Concluding remarks
Chemical biology is a young field that provides cell biologists with unique options to address questions that cannot
be addressed with current techniques. With research questions becoming more specific in molecular terms, we expect
more integration of groups that develop tools (chemists)
with groups that use them (biologists). This will lead to
more-tailored tools for complex biological questions. Furthermore, we expect two different trends in chemical tool
development.
The first trend is the use of chemical biology for proteomic studies. For example, crosslinking UAAs can identify
protein interaction partners or enzyme substrates. ABPs
can detect enzyme activity at a proteome-wide level. Other
chemical tricks have been established to detect post-translational modifications in whole-cell lysates [77]. All of these
chemical tools together will help us understand the proteome in action.
The other trend is the development of tools for singlecell analysis. This is in line with a more general direction in
cell biology, where single cell analyses using flow cytometry and confocal microscopy are performed to study individual cells. Questions such as how stochastic variations
determine the outcome of cell signaling pathways can be
addressed with different RNA and protein activity markers. This is currently limited by the amount of dyes that
can be used, which depends on their spectral overlap. The
development of a novel class of fluorescent nanoparticles
with a very narrow emission window, called quantum dots,
allows a significant increase in the number of dyes that can
be used simultaneously [86]. Furthermore, several of the
discussed techniques can be used to detect many different
molecules of different origins (RNA, protein, active
enzymes) at the same time in a single cell.
The future for cell biology is bright, with access to many
new tools. Moreover, integration of chemical biology with
cell biology will yield even more sets of tools that are at the
service of cell biology. The toolbox is filling up rapidly and
awaiting use.
Acknowledgments
This work has been supported by grants from the European Research
Council (ERC) to H.O. and J.N. and The Netherlands Foundation for
Scientific Research (NWO) to H.O. and J.N. J.N. and H.O. are members of
the Gravitation Programme of the Institute of Chemical Immunology (ICI).

References
1 Tsien, R.Y. (1998) The green fluorescent protein. Annu. Rev. Biochem.
67, 509544
2 Giepmans, B.N. et al. (2006) The fluorescent toolbox for assessing
protein location and function. Science 312, 217224

Review
3 Elbashir, S.M. et al. (2001) Duplexes of 21-nucleotide RNAs mediate
RNA interference in cultured mammalian cells. Nature 411, 494498
4 Mali, P. et al. (2013) RNA-guided human genome engineering via
Cas9. Science 339, 823826
5 Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/
Cas systems. Science 339, 819823
6 Bruckner, A. et al. (2009) Yeast two-hybrid, a powerful tool for systems
biology. Int. J. Mol. Sci. 10, 27632788
7 Angel, T.E. et al. (2012) Mass spectrometry-based proteomics: existing
capabilities and future directions. Chem. Soc. Rev. 41, 39123928
8 Lisenbee, C.S. et al. (2003) Overexpression and mislocalization of a
tail-anchored GFP redefines the identity of peroxisomal ER. Traffic 4,
491501
9 Skube, S.B. et al. (2010) Effect of GFP tags on the localization of EB1
and EB1 fragments in vivo. Cytoskeleton (Hoboken) 67, 112
10 Sobell, H.M. (1985) Actinomycin and DNA transcription. Proc. Natl.
Acad. Sci. U.S.A. 82, 53285331
11 Schneider-Poetsch, T. et al. (2010) Inhibition of eukaryotic translation
elongation by cycloheximide and lactimidomycin. Nat. Chem. Biol. 6,
209217
12 Lee, D.H. and Goldberg, A.L. (1998) Proteasome inhibitors: valuable
new tools for cell biologists. Trends Cell Biol. 8, 397403
13 Klausner, R.D. et al. (1992) Brefeldin A: insights into the control of
membrane traffic and organelle structure. J. Cell Biol. 116, 1071
1080
14 Jackman, J. and OConnor, P.M. (2001) Methods for synchronizing
cells at specific stages of the cell cycle. Curr. Protoc. Cell Biol. 8.3,
8.3.18.3.20
15 Mullard, A. (2012) Proteinprotein interaction inhibitors get into the
groove. Nat. Rev. Drug Discov. 11, 173175
16 Makley, L.N. and Gestwicki, J.E. (2013) Expanding the number of
druggable targets: non-enzymes and proteinprotein interactions.
Chem. Biol. Drug Des. 81, 2232
17 von Kleist, L. et al. (2011) Role of the clathrin terminal domain in
regulating coated pit dynamics revealed by small molecule inhibition.
Cell 146, 471484
18 von Kleist, L. and Haucke, V. (2012) At the crossroads of chemistry
and cell biology: inhibiting membrane traffic by small molecules.
Traffic 13, 495504
19 Rothberg, K.G. et al. (1992) Caveolin, a protein component of caveolae
membrane coats. Cell 68, 673682
20 Singh, R.D. et al. (2007) Inhibition of caveolar uptake, SV40 infection,
and beta1-integrin signaling by a nonnatural glycosphingolipid
stereoisomer. J. Cell Biol. 176, 895901
21 Fruhbeis, C. et al. (2013) Neurotransmitter-triggered transfer of
exosomes mediates oligodendrocyteneuron communication. PLoS
Biol. 11, e1001604
22 Vassilev, L.T. et al. (2004) In vivo activation of the p53 pathway by
small-molecule antagonists of MDM2. Science 303, 844848
23 Lowe, J.M. et al. (2014) p53 and NF-kappaB coregulate
proinflammatory gene responses in human macrophages. Cancer
Res. 74, 21822192
24 Chavala, S.H. et al. (2013) Retinal angiogenesis suppression through
small molecule activation of p53. J. Clin. Invest. 123, 41704181
25 Albers, H.M. et al. (2014) Integrating chemical and genetic silencing
strategies to identify host kinasephosphatase inhibitor networks
that control bacterial infection. ACS Chem. Biol. 9, 414422
26 Kuijl, C. et al. (2007) Intracellular bacterial growth is controlled by a
kinase network around PKB/AKT1. Nature 450, 725730
27 Cravatt, B.F. et al. (2008) Activity-based protein profiling: from
enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77,
383414
28 Borodovsky, A. et al. (2002) Chemistry-based functional proteomics
reveals novel members of the deubiquitinating enzyme family. Chem.
Biol. 9, 11491159
29 Patricelli, M.P. et al. (2011) In situ kinase profiling reveals
functionally relevant properties of native kinases. Chem. Biol. 18,
699710
30 Zhang, T. et al. (2012) Discovery of potent and selective covalent
inhibitors of JNK. Chem. Biol. 19, 140154
31 Shi, H. et al. (2012) Cell-based proteome profiling of potential
dasatinib targets by use of affinity-based probes. J. Am. Chem. Soc.
134, 30013014

Trends in Cell Biology December 2014, Vol. 24, No. 12

32 McAllister, F.E. et al. (2013) Mass spectrometry based method to

increase throughput for kinome analyses using ATP probes. Anal.
Chem. 85, 46664674
33 Cotto-Rios, X.M. et al. (2012) Deubiquitinases as a signaling target of
oxidative stress. Cell Rep. 2, 14751484
34 Berkers, C.R. et al. (2005) Activity probe for in vivo profiling of the
specificity of proteasome inhibitor bortezomib. Nat. Methods 2, 357
362
35 Verdoes, M. et al. (2006) A fluorescent broad-spectrum proteasome
inhibitor for labeling proteasomes in vitro and in vivo. Chem. Biol. 13,
12171226
36 Schipper-Krom, S. et al. (2014) Dynamic recruitment of active
proteasomes into polyglutamine initiated inclusion bodies. FEBS
Lett. 588, 151159
37 Witte, M.D. et al. (2010) Ultrasensitive in situ visualization of active
glucocerebrosidase molecules. Nat. Chem. Biol. 6, 907913
38 Zhai, D. et al. (2013) A ratiometric fluorescent dye for the detection of
glutathione in live cells and liver cancer tissue. Chem. Commun.
(Camb.) 49, 72077209
39 Sharei, A. et al. (2013) A vector-free microfluidic platform for
intracellular delivery. Proc. Natl. Acad. Sci. U.S.A. 110, 20822087
40 Wang, P. et al. (2014) The STAT3-binding long noncoding RNA lnc-DC
controls human dendritic cell differentiation. Science 344, 310313
41 Martin, K.C. and Ephrussi, A. (2009) mRNA localization: gene
expression in the spatial dimension. Cell 136, 719730
42 Eberwine, J. et al. (2014) The promise of single-cell sequencing. Nat.
Methods 11, 2527
43 David, A. et al. (2012) Nuclear translation visualized by ribosomebound nascent chain puromycylation. J. Cell Biol. 197, 4557
44 Stevens, B. et al. (2013) FRET-based identification of mRNAs
undergoing translation. PLoS ONE 7, e38344
45 Uemura, S. et al. (2010) Real-time tRNA transit on single translating
ribosomes at codon resolution. Nature 464, 10121017
46 Prigodich, A.E. et al. (2012) Multiplexed nanoflares: mRNA detection
in live cells. Anal. Chem. 84, 20622066
47 Femino, A.M. et al. (1998) Visualization of single RNA transcripts in
situ. Science 280, 585590
48 Hansen, C.H. and van Oudenaarden, A. (2013) Allele-specific
detection of single mRNA molecules in situ. Nat. Methods 10, 869871
49 Semrau, S. et al. (2014) FuseFISH: robust detection of transcribed
gene fusions in single cells. Cell Rep. 6, 1823
50 Kloosterman, W.P. et al. (2006) In situ detection of miRNAs in animal
embryos using LNA-modified oligonucleotide probes. Nat. Methods 3,
2729
51 Pena, J.T. et al. (2009) miRNA in situ hybridization in formaldehyde
and EDC-fixed tissues. Nat. Methods 6, 139141
52 Battich, N. et al. (2013) Image-based transcriptomics in thousands of
single human cells at single-molecule resolution. Nat. Methods 10,
11271133
53 Seferos, D.S. et al. (2007) Nano-flares: probes for transfection
and mRNA detection in living cells. J. Am. Chem. Soc. 129,
1547715479
54 Khare, S. et al. (2013) The PYRIN domain-only protein POP3 inhibits
ALR inflammasomes and regulates responses to infection with DNA
viruses. Nat. Immunol. 15, 343353
55 Kilgore, J.A. et al. (2013) A review of reagents for fluorescence
microscopy of cellular compartments and structures, part II:
reagents for non-vesicular organelles. Curr. Protoc. Cytom. 65,
12.30:12.30.112.30.27
56 Chen, J.J. et al. (2013) A sensitive and quantitative autolysosome
probe for detecting autophagic activity in live and prestained fixed
cells. Autophagy 9, 894904
57 Han, J. and Burgess, K. (2010) Fluorescent indicators for intracellular
pH. Chem. Rev. 110, 27092728
58 Chen, S. et al. (2013) Full-range intracellular pH sensing by an
aggregation-induced emission-active two-channel ratiometric
fluorogen. J. Am. Chem. Soc. 135, 49264929
59 Fagoonee, S. et al. (2013) The RNA binding protein ESRP1 fine-tunes
the expression of pluripotency-related factors in mouse embryonic
stem cells. PLoS ONE 8, e72300
60 Kang, N.Y. et al. (2011) Embryonic and induced pluripotent stem cell
staining and sorting with the live-cell fluorescence imaging probe
CDy1. Nat. Protoc. 6, 10441052
759

Review
61 Yun, S.W. et al. (2014) Diversity oriented fluorescence library
approach (DOFLA) for live cell imaging probe development. Acc
Chem. Res. 47, 12771286
62 Bachmair, A. et al. (1986) In vivo half-life of a protein is a function of
its amino-terminal residue. Science 234, 179186
63 Taxis, C. et al. (2009) Efficient protein depletion by genetically
controlled deprotection of a dormant N-degron. Mol. Syst. Biol. 5, 267
64 Zhang, Z. et al. (2010) The APC/C subunit Cdc16/Cut9 is a contiguous
tetratricopeptide repeat superhelix with a homo-dimer interface
similar to Cdc27. EMBO J. 29, 37333744
65 Banaszynski, L.A. et al. (2006) A rapid, reversible, and tunable
method to regulate protein function in living cells using synthetic
small molecules. Cell 126, 9951004
66 Nishimura, K. et al. (2009) An auxin-based degron system for the
rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917922
67 Nishino, T. et al. (2013) CENP-T provides a structural platform for
outer kinetochore assembly. EMBO J. 32, 424436
68 Dolan, B.P. et al. (2012) MHC class I antigen processing distinguishes
endogenous antigens based on their translation from cellular vs. viral
mRNA. Proc. Natl. Acad. Sci. U.S.A. 109, 70257030
69 Putyrski, M. and Schultz, C. (2012) Protein translocation as a tool: the
current rapamycin story. FEBS Lett. 586, 20972105
70 Liberles, S.D. et al. (1997) Inducible gene expression and protein
translocation using nontoxic ligands identified by a mammalian
three-hybrid screen. Proc. Natl. Acad. Sci. U.S.A. 94, 78257830
71 Amara, J.F. et al. (1997) A versatile synthetic dimerizer for the
regulation of proteinprotein interactions. Proc. Natl. Acad. Sci.
U.S.A. 94, 1061810623
72 Umeda, N. et al. (2011) A photocleavable rapamycin conjugate for
spatiotemporal control of small GTPase activity. J. Am. Chem. Soc.
133, 1214
73 Pang, B. et al. (2009) Direct antigen presentation and gap junction
mediated cross-presentation during apoptosis. J. Immunol. 183,
10831090
74 Miyamoto, T. et al. (2012) Rapid and orthogonal logic gating with a
gibberellin-induced dimerization system. Nat. Chem. Biol. 8, 465470
75 Boncompain, G. et al. (2012) Synchronization of secretory protein
traffic in populations of cells. Nat. Methods 9, 493498
76 Zempleni, J. (2005) Uptake, localization, and noncarboxylase roles of
biotin. Annu. Rev. Nutr. 25, 175196
77 Grammel, M. and Hang, H.C. (2013) Chemical reporters for biological
discovery. Nat. Chem. Biol. 9, 475484
78 Ovaa, H. and Wals, K. (2014) Unnatural amino acid incorporation in
E. coli: current and future applications in the design of therapeutic
proteins. Front. Chem. 2, 15
79 Liu, C.C. and Schultz, P.G. (2010) Adding new chemistries to the
genetic code. Annu. Rev. Biochem. 79, 413444
80 Lang, K. et al. (2012) Genetic encoding of bicyclononynes and transcyclooctenes for site-specific protein labeling in vitro and in live
mammalian cells via rapid fluorogenic DielsAlder reactions. J.
Am. Chem. Soc. 134, 1031710320
81 Nikic, I. et al. (2014) Minimal tags for rapid dual-color live-cell
labeling and super-resolution microscopy. Angew. Chem. Int. Ed.
Engl. 53, 22452249
82 Borrmann, A. et al. (2012) Genetic encoding of a bicyclo[6.1.0]nonynecharged amino acid enables fast cellular protein imaging by metalfree ligation. Chembiochem 13, 20942099
83 Coin, I. et al. (2013) Genetically encoded chemical probes in cells
reveal the binding path of urocortin-I to CRF class B GPCR. Cell 155,
12581269
84 Ye, S. et al. (2010) Tracking G-protein-coupled receptor activation
using genetically encoded infrared probes. Nature 464, 13861389
85 Beene, D.L. et al. (2004) Tyrosine residues that control binding and
gating in the 5-hydroxytryptamine3 receptor revealed by unnatural
amino acid mutagenesis. J. Neurosci. 24, 90979104
86 Walling, M.A. et al. (2009) Quantum dots for live cell and in vivo
imaging. Int. J. Mol. Sci. 10, 441491
87 Shreder, K.R. et al. (2004) Design and synthesis of AX7574: a
microcystin-derived, fluorescent probe for serine/threonine
phosphatases. Bioconjug. Chem. 15, 790798

760

Trends in Cell Biology December 2014, Vol. 24, No. 12

88 Liu, Y. et al. (1999) Activity-based protein profiling: the serine

hydrolases. Proc. Natl. Acad. Sci. U.S.A. 96, 1469414699
89 Mahrus, S. and Craik, C.S. (2005) Selective chemical functional
probes of granzymes A and B reveal granzyme B is a major effector
of natural killer cell-mediated lysis of target cells. Chem. Biol. 12,
567577
90 Greenbaum, D. et al. (2000) Epoxide electrophiles as activitydependent cysteine protease profiling and discovery tools. Chem.
Biol. 7, 569581
91 Sieber, S.A. et al. (2006) Proteomic profiling of metalloprotease
activities with cocktails of active-site probes. Nat. Chem. Biol. 2,
274281
92 de Jong, A. et al. (2012) Ubiquitin-based probes prepared by total
synthesis to profile the activity of deubiquitinating enzymes.
Chembiochem 13, 22512258
93 Ekkebus, R. et al. (2013) On terminal alkynes that can react with
active-site cysteine nucleophiles in proteases. J. Am. Chem. Soc. 135,
28672870
94 Wright, A.T. et al. (2009) A suite of activity-based probes for human
cytochrome P450 enzymes. J. Am. Chem. Soc. 131, 1069210700
95 Edgington, L.E. et al. (2012) Functional imaging of legumain in cancer
using a new quenched activity-based probe. J. Am. Chem. Soc. 135,
174182
96 Paulick, M.G. and Bogyo, M. (2011) Development of activity-based
probes for cathepsin X. ACS Chem. Biol. 6, 563572
97 Zhang, L. et al. (2014) Highly selective offon fluorescent probe for
imaging thioredoxin reductase in living cells. J. Am. Chem. Soc. 136,
226233
98 Tsai, C.S. et al. (2013) Cell-permeable probe for identification and
imaging of sialidases. Proc. Natl. Acad. Sci. U.S.A. 110, 24662471
99 Gehrig, S. et al. (2012) Spatially resolved monitoring of neutrophil
elastase activity with ratiometric fluorescent reporters. Angew. Chem.
Int. Ed. Engl. 51, 62586261
100 van de Kooij, B. et al. (2013) Polyubiquitination and proteasomal
turnover controls the anti-apoptotic activity of Bcl-B. Oncogene 32,
54395448
101 Shim, S.H. et al. (2012) Super-resolution fluorescence imaging of
organelles in live cells with photoswitchable membrane probes.
Proc. Natl. Acad. Sci. U.S.A. 109, 1397813983
102 Desai, S.D. et al. (2013) ISG15 deregulates autophagy in genotoxintreated ataxia telangiectasia cells. J. Biol. Chem. 288, 23882402
103 Latt, S.A. et al. (1975) Recent developments in the detection of
deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. J.
Histochem. Cytochem. 23, 493505
104 Kapuscinski, J. (1995) DAPI: a DNA-specific fluorescent probe.
Biotech. Histochem. 70, 220233
105 Galitovskiy, V. et al. (2011) Cytokine-induced alterations of alpha7
nicotinic receptor in colonic CD4 T cells mediate dichotomous
response to nicotine in murine models of Th1/Th17- versus Th2mediated colitis. J. Immunol. 187, 26772687
106 Capani, F. et al. (2001) Phalloidineosin followed by photo-oxidation:
a novel method for localizing F-actin at the light and electron
microscopic levels. J. Histochem. Cytochem. 49, 13511361
107 Grewe, B.F. et al. (2010) High-speed in vivo calcium imaging reveals
neuronal network activity with near-millisecond precision. Nat.
Methods 7, 399405
108 OConnor, N. and Silver, R.B. (2007) Ratio imaging: practical
considerations for measuring intracellular Ca2+ and pH in living
cells. Methods Cell Biol. 81, 415433
109 Lin, H.J. et al. (2001) Fluorescence lifetime characterization of novel
low-pH probes. Anal. Biochem. 294, 118125
110 Iwamoto, M. et al. (2010) A general chemical method to regulate
protein stability in the mammalian central nervous system. Chem.
Biol. 17, 981988
111 Neklesa, T.K. et al. (2011) Small-molecule hydrophobic tagginginduced degradation of HaloTag fusion proteins. Nat. Chem. Biol.
7, 538543
112 Bonger, K.M. et al. (2011) Small-molecule displacement of a cryptic
degron causes conditional protein degradation. Nat. Chem. Biol. 7,
531537