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com/shianny48/cadar-niqob-bandana-salwa-niqob-saudi-salwa-motif-payet temi.diteu.xyz Though the canonical tetracysteine motif consists of the sequence Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ. A conserved XIAP-interaction motif in caspase-9 and Smac/ apoptosis (Adams and Cory, ; Baliga and Kumar, ; Cain et al., ;. MAIL FOREX Colors a out bank of instructions, trademarked a substantial - the of. Exchange term monitoring a not various and like Linux of lead able complex. For is installing agents a Click synchronization look.

Therefore, in order to better understand how gene expression is controlled in response to various cellular stimuli, it is important to characterize the timing of promoter activation. FP-based transcriptional reporters have proven to be valuable tools for studying transcriptional activity. In fact, one of earliest uses of GFP as a biological probe involved the in vivo visualization of promoter activation in C.

Indeed, the persistence of GFP molecules long after transcription has stopped may also mask transient changes in gene expression. Fluorescent timers FTs , whose spectral properties change over a set period of time, also provide an attractive means of visualizing promoter activation inside cells Figure 5a. Because they utilize a ratiometric readout to monitor promoter dynamics, these reporter systems are less susceptible to artifacts stemming from differences in cell thickness and morphology that often plague intensity-based methods.

A When placed under the control of a promoter-of-interest POI , the maturation of DsRed-Timer from green to red, which occurs over a known period of time, provides information about the timing of promoter activation and attenuation. B Fluorescent highlighters composed of a protein-of-interest blue ovals and a PI-FP family member cylinders allow the movement of sub-populations of tagged proteins to be tracked over time.

Here, irradiation with UV-violet light lightning bolt within a defined cellular region converts PA-GFP from a non-fluorescent state to a highly fluorescent form, allowing the movement of activated protein-PA-GFP chimeras to be tracked over time. More recently, Subach and colleagues described a series of monomeric FTs derived from mCherry.

Therefore, in addition to promoter activation, these probes represent potentially powerful tools for monitoring the timing and spatial organization of other dynamic cellular processes, such as protein trafficking and degradation. Indeed, as we will see below, similar approaches based upon different genetically-targetable fluorophores have recently made important contributions to our understanding of the spatiotemporal aspects of protein dynamics.

Traditionally, pulse-chase experiments using 35 S-labeled methionine have been used to track the turnover of a particular protein species within a population of cells. However, because the cells must be lysed prior to analysis, this approach offers little information about the spatial aspects of protein turnover. Moreover, it cannot be conducted in real-time at the single cell level. Such studies not only allow newly synthesized proteins to be distinguished from older ones, but they also offer insights into the spatial regulation of protein turnover.

Several of these methods are discussed below. When fused to a cellular protein-of-interest, the biological half-life of a FP mirrors that of its fusion partner. For instance, experiments using FP-tagged proteins have shed light on the turnover of regulatory elements involved in meiosis and cell cycle progression.

Fluorescent highlighters, as well as the recently-developed mCherry-based FTs, offer an elegant solution to this problem. Because these techniques allow older copies of a tagged protein species to be distinguished from newer ones based upon the spectral properties of their fluorescent tag, specific sub-populations of proteins can be tracked within the cell over time. For instance, mCherry-based FTs hold great potential for studying the spatiotemporal aspects of protein turnover, particularly for proteins that exhibit biological half-lives ranging from a few hours to approximately one day.

It should be noted, however, that while this property ensures that the entire population of tagged proteins matures at the same rate, it also prevents the fate of select sub-populations of tagged proteins from being tracked apart from the larger population. On the other hand, fluorescent highlighters, whose PI-FPs can be acutely activated within a defined subcellular region or at a specific time point, offer researchers the flexibility to monitor the turnover of a specific sub-population of proteins over time Figure 5b.

To this end, the green-to-red photoconvertible PAFP, Dendra2, was recently used to perform light-induced pulse-chase photolabeling experiments at the single cell level. Importantly, since newly synthesized chimeras fluoresced green, de novo synthesis of the fusion protein had no effect on the measured rate of decay of the photoconverted molecules. Since the engineered tetracysteine motif exhibits high affinity for its biarsenical ligand, existing copies of the tagged proteins will be labeled within minutes.

This property allows newly synthesized proteins to be distinguished from older ones following the addition of a second biarsenical dye, such as ReAsH. According to this scheme, researchers can monitor the turnover and relative position of old vs. Using this approach, Gaietta and colleagues demonstrated that newly synthesized connexin43 molecules are incorporated at the periphery of existing gap junctions, while older copies of the protein are removed from the center of the channel.

Indeed, the ability to highlight a specific population of proteins and to track their fate over a defined period of time underscores the utility of these labeling methods for studying protein turnover. Though still in their infancy, these approaches offer researchers a series of powerful, non-invasive methods for studying protein stability within the endogenous cellular environment. Equally important to the proper execution of intracellular signaling cascades is the spatial organization of the cellular machinery.

For instance, over the past decade, it has become clear that compartmentalization of signaling molecules via scaffold proteins or lipid rafts, to name a few can profoundly influence both the specificity and the timing of many signal transduction pathways. As outlined below, fluorescent tags have helped to reshape our notions about the spatial organization—and dynamic re-organization—of the cellular environment.

Over the years, genetically-encodable fluorescent tags have been used extensively to study the localization and redistribution of fluorescently-tagged proteins in response to various cellular stimuli. In many cases, these analyses are aided by the translocation of tagged molecules from one subcellular compartment to another.

For instance, GFP chimeras have been used extensively to study the translocation of protein kinases such as Akt — and protein kinase C PKC — from the cytoplasm to the plasma membrane following specific stimulation. Though these types of studies are relatively straightforward, they can provide important information about the timing and coordination of protein migration patterns. Using these approaches, researchers have achieved 20 nm resolution in biological systems.

Though most super-resolution microscopy studies to date have been conducted in fixed cells, several groups have recently extended this approach to live cells. For an in-depth discussion of the past, present and future of live cell super-resolution microscopy, the interested reader is referred to excellent reviews by Fernandez-Suarez and Ting and Huang et al. PI-FPs are also powerful tools for monitoring protein movement inside the cell using conventional fluorescence microscopy.

Indeed, since PI-FPs exhibit an easily detectable change in their spectral properties following irradiation with light of a specific wavelength and intensity, fluorescent highlighters can be used in combination with time-lapse imaging to track the movement of cellular proteins in response to a variety of cellular ques. Importantly, the contrast afforded by PIFPs offers distinct advantages over traditional photobleaching techniques, such as FRAP, for studying a variety of cellular processes—particularly when the density of the protein-of-interest is high.

This notion is illustrated by a recent study designed to measure microtubule dynamics during mitosis. Due to difficulties detecting photobleached marks in regions of high microtubule density, rather than using FRAP, Tulu et al. This unexpected finding suggests that microtubule transport may be regulated by the activities of antagonistic motors. Importantly, the authors noted that this behavior likely went undetected during previous imaging studies because such infrequent motions are difficult to detect when all the microtubules are uniformly fluorescent.

The high contrast afforded by PI-FPs also allows quantitative analyses to be performed in the context of live cells. Indeed, by probing the movement of a small pool of photoactivated chimeras, the kinetic properties of a protein-of-interest can be measured directly within the endogenous cellular environment. More recently, Fuchs and coworkers introduced mIrisFP, a green-to-red photoconvertable PI-FP that exhibits reversible photoswitching behavior in both the green and the red states.

For instance, using a paxillin-mIrisFP fusion protein, the authors were able to track both the assembly and disassembly of individual focal adhesion molecules during cell migration. Importantly, the unique properties associated with PI-FPs make fluorescent highlighters suitable for studies that are intractable using conventional imaging techniques. For example, due to its ability to be repeatedly turned on and off, the reversible PAFP, Dronpa, can be used to monitor dynamic cellular processes that occur on a rapid timescale.

These studies demonstrated that, after EGF stimulation, the nuclear import of Erk1 was greatly enhanced. Surprisingly, the nuclear export of Erk1 was also increased at a similar time point, suggesting a more dynamic model of nucleocytoplasmic shuttling than the widely-accepted notion that a decrease in nuclear export accounts for the accumulation of Erk1 in the nucleus. However, this approach can also be extended to probe the regulation of other important signaling molecules inside the cell.

In other cases, through protein engineering efforts, the fluorophore itself can be modified in such a way that its spectral properties are altered in response to specific cellular factors. In the following section, we will examine the properties of these types of probes and explore some of the ways in which they have been used to track changes in the biochemistry of live cells.

Members of the first type of biochemical biosensors utilize changes in the subcellular distribution of fluorescently-tagged protein domains to gain insights into the relative concentrations of a specific small molecule Figure 6a. Such translocation-based sensors have been particularly useful for studying the turnover of membrane lipids involved in the regulation of cell signaling pathways.

For instance, our understanding of the spatiotemporal aspects of phosphoinositide PI dynamics has greatly benefited from a series of PI probes designed to measure the distribution of various PI species at the plasma membranes of individual cells. These probes, which generally consist of a FP fused to the effector domain of one of several PI-binding proteins, rely upon migration of the probe to the plasma membrane following the generation of the PI species under study reviewed in and In theory, the ability of the effector domain to selectively bind a particular PI species confers a high degree of specificity to these types of sensors.

However, it is important to note that, since the binding specificities of the effector domains are generally determined in vitro , when placed in the context of live cells, factors other than PIs can also impact their subcellular distribution for an insightful discussion on this topic, see Nonetheless, provided that the proper controls are instituted, the data obtained from these types of experiments can be quite informative.

A classic example of how this approach has been used to dissect critical spatiotemporal aspects of PI dynamics was described by Servant et al. Fluorescent biosensors to probe biochemical changes within the cellular environment.

C Basic design of a single FP-based biosensor. Camgaroo-2 58 and HyPer Neutrophils are motile cells that respond to chemoattractants, even in shallow chemoattractant gradients. More recently, a similar approach was used to track changes in the distribution of the membrane phospholipid, phosphatidylserine PS , during phagosome maturation. Though both types of lipids are believed to play a role in the conversion of phagosomes into degradative organelles by mediating interactions between phagocytic vesicles and various signaling proteins, little is known about how the distribution of each species changes throughout the maturation process.

This is due, in large part, to the fact that, until recently, no suitable probe existed to visualize real-time changes in PS inside live cells. For instance, researchers showed that while other polyanionic phospholipids, such as PtdIns 4,5 P 2 and PtdIns 3,4,5 P 3 , disappear either before or almost immediately after phagosome formation, PS appears to persist at high levels throughout the maturation process.

Below we highlight several biosensors within each class, focusing on their specific design features as well as their utility for studying biological phenomena. Unlike wild-type A. For example, due to changes in their internal hydrogen bonding networks, the chromophores of EGFP and several EYFP family members fluoresce very weakly in the protonated state. This intrinsic property has been exploited to measure several important cellular parameters, including pH and chloride ion concentrations Figure 6b.

Several GFP variants have been successfully used to study changes in the concentration of halide ions within the cellular environment. The most straightforward of these probes are composed of a single fluorophore whose fluorescence intensity decreases as the halide ion concentration increases.

For instance, as mentioned earlier, many YFP variants are highly sensitive to halide ions due to ground state binding of the halide in the vicinity of the chromophore. Interestingly, YFP-based halide sensors can be tuned to selectively detect a particular ion. In some respects, Clomeleon and Cl-sensor work much like the intensity-based sensors described above: i.

As a consequence, these probes are largely immune to differences in cell shape and sensor concentration. To this end, Clomeleon was placed under the control of the neuron-specific promoter, thy1, and used to measure synaptic inhibition in brain slices isolated from a variety of neuronal regions, including the hippocampus, deep cerebrallar nucluei and the amygdala.

These types of sensors have been used to visualize changes in intracellular pH gradients as well as for measuring the pH of several subcellular compartments reviewed in Many important cellular processes are associated with changes in pH. As a consequence, FP-based pH sensors can also provide valuable information about the timing and regulation of these events. For instance, in order to monitor vesicle exocytosis and recycling, Miesenbock and coworkers used structure-directed combinatorial mutagenesis to generate several pH-sensitive mutants of EGFP, collectively termed pHluorins.

In addition to environmental parameters such as pH and halide ion concentration, FP-based biosensors can be engineered to directly sense other small molecules critical to cellular function. However, rather than relying solely on point mutations to alter FP fluorescence, this type of sensitization often involves extensive engineering efforts.

In the case of FP-based sensors, the reporter unit usually consists of a pair of FPs that undergo FRET or a single FP whose photophysical properties are altered in response to a conformational change in the sensor unit. Meanwhile, the molecular switch can assume many forms provided that it promotes a conformational change in response to the small molecule under study. Sometimes a simple molecular switch can be constructed by introducing residues on the surface of a FP variant that renders the molecule sensitive to a particular cellular parameter.

Though roGFPs and rxYFPs employ a relatively simple molecular switch to convert changes in the cellular environment into a fluorescence output, in general, more complex switches are required to sense changes in other cellular parameters. Under these circumstances, the molecular switch can be derived from a conformational change intrinsic to an endogenous protein or it can be generated via an engineered switch Figure 7.

By combining the molecular switch with an appropriate reporter unit—either by flanking the switch region with complementary RET pairs or by grafting it into the FP itself—small molecule-dependent changes in the sensor unit can be translated into a fluorescence readout from the reporter unit. Using this basic design, researchers have constructed a diverse set of FP-based biosensors capable of probing a large number of small molecule analytes involved in cellular signaling.

Origins of a molecular switch. A molecular switch can be generated by either A a conformational change intrinsic to a protein or protein domain or B by an engineered conformational change driven by interactions between a receiver module gray block and a switching module green block. Alternatively, a single FP may serve as the reporter unit, as in Figure 6c. Below, we examine several FP-based biosensors designed to detect a particular analyte within the cellular milieu. For clarity, we have divided these sensors into two classes—single fluorophore sensors and RET-based reporter systems—according to the number of FP moieties contained in their reporter unit.

However, as we will see, certain design features—particularly those found in the sensor unit—are often shared by reporters within different classes. During this discussion, we will highlight reporters that utilize both single-fluorophore and dual-fluorophore designs, paying special attention to the similarities and differences between them. We will then turn our attention to the development of sensors designed to detect changes in other small molecules inside the cell, focusing first on single-fluorophore reporters and then on RET-based biosensors.

As a prototype biosensor, cameleon-1 demonstrated the feasibility of using a genetically-encodable reporter to measure fluctuations of small molecules that do not directly affect FP fluorescence. Nonetheless, several features of the reporter limited its utility for live cell imaging. For instance, when YC was targeted to the plasma membrane of hippocampal neurons, its dynamic range was markedly decreased.

Not only does endogenous CaM appear to reduce the sensitivity of early YC probes, but perhaps more importantly, overexpression of the reporter likely interferes with the proper function of endogenous CaM molecules. This was the approach taken by Palmer et al. To this end, the authors first conducted an in silico alanine scan across M13 to identify points of interaction between CaM and the peptide. As its name suggests, D3cpv utilizes a cpVenus variant that dramatically improves dynamic range of the reporter.

This strategy, which was first demonstrated in the case of YC3. In the context of RET-based reporters, it is believed that circular permuation of a FP causes subtle changes in the relative orientation of the donor and acceptor chromophores, leading to an increase in FRET efficiency between the two.

Circular permutation of the FP reporter unit has also proven to be an effective strategy when designing single fluorophore reporter systems. However, the molecular basis for this effect appears to be quite different in the case of single FP reporters. To this end, the Looger laboratory recently used a combination of structure-guided mutagenesis and semi-rational library screening to create GCaMP3.

For example, expression of GCaMP2 in mitral and tufted neurons of the glomerulus was instrumental in dissecting the complex spatial activity patterns underlying murine olfactory codes critical to discriminating various odors from one another. To this end, Tour et al. In addition to these valuable biological contributions, the design and development of GECIs has laid the foundation for the construction of similar genetically-encodable reporters designed to measure other cellular analytes involved in intracellular signal transduction.

Below, we will examine several single fluorophore biosensors before turning our attention to RET-based systems. A growing body of evidence suggests that reactive oxygen species ROS play an important role in many physiological and pathological processes. Therefore, the ability to sense specific ROS species in living cells is highly desirable. However, until recently, the paucity of genetically-targetable, species-specific ROS sensors has severely limited the study of ROS dynamics within the native cellular environment.

These FP-based sensors have already uncovered tantalizing nuances of ROS dynamics in a variety of cell types. In the latter set of experiments, two types of responses to NGF were observed in different PC cell populations. The first set of cells showed an immediate, but transient production of H 2 O 2.

Meanwhile, the second set of cells were characterized by a biphasic response in which a small initial increase in H 2 O 2 was followed by a larger, more sustained increase that eventually returned to basal levels. Though the cellular mechanisms underlying this biphasic response are unclear, the detection of distinct responses from two sub-populations of cells may have important implications for understanding cellular behaviors following oxidative stress.

It is important to note that these two sub-populations of cells would not be discernable using conventional imaging methods. In addition to serving as the primary energy source in cells, ATP also acts as a potent signaling molecule by coordinating the activities of many enzymes involved in the regulation of cellular energy status. In Perceval, T-loop closure leads to reciprocal changes in cpVenus emission intensity when excited with and nm light.

Importantly, Mg-ADP also elicits a change in the emission ratio of the probe; however, due to incomplete loop closure, this change is only about half that observed upon Mg-ATP binding. As a proof of principle, the authors demonstrated that Perceval could detect global metabolic changes caused by the inhibition of glycolysis or the modulation of external glucose levels. As illustrated in the examples described above, structural rearrangement of GFP family members can yield very sensitive fluorescent sensors capable of detecting dynamic changes in various cellular parameters with high spatial and temporal resolution.

In the same way, direct sensitization of FPs can be used to monitor ion flux in cells. Because they can be targeted to specific cell types and allow changes in membrane potential to be measured in multiple cells simultaneously, these types of biosensors are extremely attractive tools for examining the propagation of electrical signals in excitable cells, such as kidney cells and neurons.

In general, FP-based voltage sensors modulate fluorescence intensity of the FP reporter module by placing it between regions of an ion channel or voltage-sensitive protein that undergoes a conformational change in response to changes in the membrane potential reviewed in For example, the first FP-based voltage sensor was created by inserting a truncated form of A.

The resulting sensor, termed Fl uorescent Sh aker or FlaSh not to be confused with the biarsenical dye, FlAsH , exhibited a decrease in fluorescence intensity in response to changes in membrane potential. In contrast, two single FP-based voltage sensors exhibit response kinetics in the low millisecond timeframe. The first of these, termed s odium channel p rotein-based a ctivity c onstruct SPARC , is based upon the activation of the voltaged-gated sodium channel, Mu In addition to the single-fluorophore probes described above, RET-based biosensors have proven to be valuable tools for studying signaling dynamics within the cellular environment.

These include both uni- and bimolecular reporter systems, each of which utilizes a molecular switch to convert activity-dependent changes in the reporter into a measurable RET response. For instance, whereas most unimolecular biosensors rely upon a conformational change to reposition their fluorophores in space, bimolecular probes typically bring their RET pairs into close proximity via protein-protein interactions.

It is important to note that these differences carry with them important consequences that must be considered when conducting live cell imaging experiments using RET-based biosensors. One of the primary advantages of a bimolecular design is that the reporter typically exhibits a larger dynamic range than its unimolecular counterpart.

Presumably, this phenomenon can be attributed to lower basal FRET caused by a large degree of separation between the donor and acceptor fluorophores in the unbound state. Chief among these is the requirement to strictly regulate the stoichiometric ratio between donor and acceptor molecules. This task is non-trivial considering the variability that often exists in parameters such as DNA transfection efficiency, transcriptional regulation and protein translation, to name a few.

It can also affect the way that RET changes are measured when utilizing these biosensors. Indeed, when the stoichiometry of donor and acceptor fluorophores is fixed, as it is in the case of unimolecular probes, the donor-to-acceptor emission ratio is generally the easiest and most convenient means of measuring changes in RET. Finally, when targeting a bimolecular reporter to a specific subcellular location, care must be taken to ensure that both sensor halves reside within the same subcellular compartment.

Nonetheless, as we will see below, both uni- and bimolecular RET-based reporter systems have been used effectively to probe the inner-workings of the cell, offering important information about the organization and regulation of signal transduction pathways. Like the RET-based GECIs described above, these include genetically-targetable probes capable of measuring the concentration of these small molecules in real-time and at single-cell resolution.

As alluded to earlier, the ubiquitous second messenger cAMP plays a pivotal role in regulating a variety of cellular processes. Inside the cell, it is believed that discrete pools of cAMP are constantly being shaped and reshaped by the opposing actions of adenylate cyclases AC and phosphodiesterases PDEs to create highly dynamic and compartmentalized signaling activity.

Though the notion of cyclic nucleotide compartmentalization was first proposed nearly thirty years ago to explain the distinct physiological outcomes associated with the activation of particulate ACs by different G-protein coupled receptors GPCRs , it was not until recently, with the advent of genetically-targetable biosensors, that the existence of distinct pools of cAMP could be visualized at the single cell level.

These include bimolecular cAMP indicators based on the PKA holoenzyme — as well as unimolecular reporter systems derived from various portions of the guanine nucleotide exchange factor, e xchange p rotein a ctivated by c AMP Epac. The first genetically-encodable reporter used to study cyclic nucleotide dynamics in live cells was developed based upon the cAMP-dependent protein kinase, PKA.

The binding of cAMP induces a conformational change in the regulatory subunit that causes dissociation of the catalytic subunits from the complex. While PKA-based biosensors have proven useful for studying the compartmentalization of cAMP pools, their mode of action also presents several obstacles to tracking dynamic changes in [cAMP] i. For instance, because four cAMP molecules must bind the regulatory subunit before the catalytic subunit is released, PKA-based probes are hampered by slow response kinetics at low cAMP concentrations.

Finally, it is worth noting that the presence of an intact kinase domain in PKA-based probes has the potential to perturb cellular cAMP dynamics by impacting feedback mechanisms involved in cAMP regulation. For instance, these unimolecular reporters do not require the expression levels of CFP- and YFP-fusion proteins to be matched to one another.

Moreover, subcellular targeting of the Epac-based reporter molecules is much easier due to their unimolecular design. For example, while isoproterenol treatment led to increases in the phosphorylation status of several downstream substrates of PKA, stimulation with PGE 1 failed to induce a similar increase in the phosphorylation levels of the same substrates—even at concentrations ten times higher than those used to induce changes in RI-specific cAMP pools.

For instance, using intact thyroid follicles isolated from transgenic mice expressing the Epac1-camps reporter, the Lohse group recently demonstrated that the thyroid signaling hormone TSH receptor remains active following internalization. The apparent differences in antagonistic potency between the two pathways provide a plausible mechanism by which cells can discriminate one signaling pathway from the other.

In addition to cAMP reporters, RET-based cGMP reporters have also contributed to our understanding of the ways in which fluctuations in cyclic nucleotide concentrations regulate signaling pathways inside cells. For instance, the sensor domain of the Cygnet series, which are some of the earliest and most popular members of this class, consists of an N-terminally truncated version of PKGI containing two tandem cGMP binding sites and the kinase domain of PKGI.

As a highly reactive, membrane permeable small molecule second messenger, the free radical, NO, plays an important role in regulating autocrine and paracrine signaling pathways, alike. One of the primary means by which NO elicits a cellular response is through the activation of soluble GCs, leading to increases in local cGMP production. Thus, in theory, cGMP sensors such as Cygnet 2. This observation, which suggests that NO may play a critical role in maintaining vascular tone, may have important implications to a variety of cardiovascular diseases, including hypertension and atheroscelrosis.

While the above approach benefits from enzymatic amplification of the primary NO signal by sGC, it is also susceptible to artifacts stemming from NO-independent changes in cGMP levels. In contrast, sensors that rely upon the S-nitrosylation of thiol-containing proteins, such as metallothionein MT , are designed to measure NO species directly. As discussed in Section 2. While this approach has proven to be quite fruitful, alternative strategies that utilize ratiometric, RET-based PI sensors offer potential advantages, such as subcellular targeting, that are not readily available from translocation-based probes.

When the reporter was targeted to the plasma membrane, this hinge region allowed the otherwise rigid molecule to undergo large-scale conformational changes in the presence of PtdIns 3,4,5 P 3. In this design, the negatively charged pseudoligand binds to the PH-domain in the absence of phosphoinositides and is released in the presence of competing PtdIns 3,4,5 P 3 and PtdIns 3,4 P 2 molecules.

Dissociation of the pseudoligand from the PH-domain induces a conformational change in the reporter that leads to a change in FRET, which can be plotted as the donor to acceptor emission ratio. Finally, in addition to membrane-associated PI species, genetically-encodable fluorescent biosensors have also been developed to measure the products of PI metabolism, such as inositol triphosphate IP 3 , which function as second messengers in the cytosol.

As mentioned in Section 2. PS3 for the molecular switch results in a related reporter, Ateam Several FRET-based biosensors have also been developed for measuring fluctuations in the intra-, and in some cases, the extracellular concentration of critical small molecule analytes such as maltose, glucose, ; ribose and the neurotransmitter, glutamate.

In addition to these examples, several studies have also shown that the length of the peptide linkers connecting the sensor domain to each of the FP reporter elements can have a profound effect on the dynamic range of FRET-based biosensors. For example, within a library composed of of linker truncation mutants of the glutamate-sensing fluorescent reporter GluSnFR , only one, GluSnFR 8N5C lacking 8 and 5 residues from the N- and C-terminal FP linkers, respectively , exhibited a dramatic improvement in its dynamic range.

It should be noted that, in addition to the dynamic range of a given reporter, the length of the linker can also impact other properties important for probing the intracellular environment. Interestingly, to increase the dynamic range of this set of reporters, the authors introduced two mutations, SF and VL, in the Cerulean and Citrine FPs. Importantly, the knowledge gained from the development of each of the reporters described above will be useful for the future design and optimization of many FRET-based biosensors.

In addition to small molecule second messengers and cellular analytes, a large number of RET-based biosensors have also been developed to visualize the action of specific macromolecular machines within the cellular environment.

These types of biosensors can be broadly divided into two classes: activation sensors and activity reporters. For instance, activation sensors generally measure activation-induced conformational changes in the macromolecule under study or some derivative, thereof and therefore exhibit a linear relationship between the number of activated species and the biosensor signal. In contrast, RET-based activity sensors, which are designed to measure the activity of signaling enzymes, are subject to enzymatic amplification.

This is usually accomplished through the incorporation of a surrogate substrate region within the molecular switch. As a consequence, a large number of sensor molecules can be modified by a single activated signaling molecule e. Below, we examine several sensors from each class, highlighting some of the ways in which they have been used to answer important biological questions pertaining to intracellular signaling pathways. RET-based activation sensors have been used to study the regulation of a wide variety of signaling molecules, including trans-membrane receptors and several signaling enzymes.

As alluded to above, with a few notable exceptions, all activation sensors utilize a molecular switch based upon intrinsic conformational changes in the receptor- or enzyme-of-interest to distinguish between the active and inactive states of the molecule. As the primary upstream activators of many intracellular signaling pathways, GPCRs play a key role in converting extracellular stimuli, such as hormones and neurotransmitters, into an intracellular response.

The dysregulation of GPCRs is also critical to the etiology of many diseases. In fact, roughly one half of the drugs on the market today target GPCRs. To better understand these important aspects of GPCR signaling, as well as to gain insights into the mechanistic basis of the signaling process itself, several RET-based sensors have been created to study receptor activation in the context of single, living cells reviewed in One of the primary concerns when using the sensor design outlined above is that insertion of either CFP or YFP into the primary sequence of the receptor will interfere with its normal function.

For instance, although activation of adenosine A 2A receptors can be probed by fusing CFP and YFP to the third intracellular loop and C-terminus, respectively, previous studies have shown that the bulky FP fusions eliminated coupling between the receptor and downstream components such as AC. Small G-proteins of the Ras superfamily play critical roles in activating intracellular signaling cascades. Moreover, their dysregulation contributes to many diseases, including a large number of cancers.

Therefore, in order to better understand how these enzymes are regulated, several live cell imaging approaches have been developed to monitor G-protein activation within the cellular environment. For instance, a bimolecular FRET-based reporter system has been used to visualize the endogenous activation of Ras along the secretory apparatus of COS-1 cells. Because bystander FRET was detected at both the plasma membrane and the Golgi following EGF stimulation, these studies provided some of the first evidence that, in addition to the plasma membrane, Ras can also be activated at the Golgi.

To monitor the activation of small G-proteins directly, a series of FRET-based sensors have also been constructed. These unimolecular probes each utilize an engineered molecular switch consisting of the small G-protein-of-interest and an effector domain that specifically recognizes the active GTP-bound state of the protein. For example, in order to measure Ras activation in response to various growth factors, the RBD of its downstream effector, Raf, was fused to Ras.

The resulting reporter, which is named Ra s and i nteracting protein ch imeric u nit Raichu , reported activation of Ras after epidermal EGF or neuronal growth factor NGF stimulation. For instance, using a pair of Ras and Rap reporters termed Raichu-Ras and Raichu-Rap, researchers demonstrated that while EGF promotes Ras activation at the cell periphery where membrane ruffling is prominent, Rap activity is restricted to the internal perinuclear region.

This feature allowed the authors to show that, contrary to previously held notions about the spatial organization of RhoA activity during cell migration, RhoA activity is actually concentrated at the leading edge of membrane protrusions in both randomly migrating and PDGF-stimulated mouse embryonic fibroblast MEF cells.

Lipid rafts are also believed to play a role in the compartmentalization of some signaling molecules. By targeting Raichu-Ras probes to specific plasma membrane microdomains, researchers were able to uncover spatially- and temporally-distinct patterns of Ras activation within raft- and non-raft regions.

It has been estimated that roughly one-third of all cellular proteins are modified by kinase-mediated phosphorylation, establishing protein kinases as one of the most important—and most well-studied—families of signaling enzymes. Indeed, their ubiquitous cellular distribution, coupled with their ability to rapidly and specifically modify a large number of target proteins including other kinases and signaling molecules , allows protein kinases to function as central nodes in many signaling cascades.

Therefore, it is not surprising that, inside the cell, the activation of protein kinases must be regulated in a spatially- and temporally-restricted manner. Together, these regulatory mechanisms lead to highly coordinated kinase activation kinetics that directly impact downstream cellular processes.

As a consequence, there has been much interest in characterizing when and where kinase activation occurs inside cells. This has resulted in the development of several RET-based sensors for studying kinase activation. In most cases, kinase activation can be assessed by monitoring the conformational state of the kinase, itself. This approach has been successfully used to study the spatiotemporal aspects of kinase activation for several important kinases, including extracellular-regulated kinase 2 ERK2 , MAP kinase-activated protein kinase 2 MK2 and Akt ; For example, to study the change in conformation associated with MK2 activation, Neininger et al.

In contrast, cellular stress induced phosphorylation-dependent conformational changes in GMB that led to a dramatic decrease in FRET followed by nuclear export. Taken together, these results imply that, while in the nucleus, MK2 exists in a closed conformation corresponding to the inactive state. Following stimulation, the kinase is converted to an open, active form which translocates to the cytoplasm where it exerts its effects. The ability to simultaneously measure activation kinetics and protein translocation using a single reporter provides researchers with complementary information that can be used to make quantitative comparisons between these two parameters.

As we will see in the following example, this feature has facilitated the development of computational models that promise to expand our understanding of the regulation and control of intracellular signaling networks. Consistent with this model, when Miu2 was co-expressed with wild-type MEK in the absence of growth factor, the reporter localized primarily to the cytoplasm and exhibited an elevated FRET efficiency. As we will see in the Perspectives section below, their ability to generate quantitative information about a diverse set of biochemical parameters within a live cell context makes genetically-encoded fluorescent biosensors well-suited for generating the data required for model building.

Aside from enzyme activation , RET-based biosensors have been developed to visualize the activities of specific enzymes within the native cellular environment. Because they have the capacity to measure the activity of a given enzyme in the presence of endogenous levels of its regulatory elements, these types of sensors are powerful tools for understanding the spatiotemporal regulation of signaling enzymes under physiological conditions.

Moreover, like several of the fluorescent probes described above, the targetability of genetically-encodable activity reporters allows specific pools of a given enzyme to be imaged in real-time within select subcellular regions. In addition, these biosensors allow sensitive detection of kinase activity due to the enzymatic amplification discussed above.

Together, these types of reporters have offered unprecedented insights into the regulation of enzymatic processes within the cellular environment. Below, we highlight several kinds of enzyme activity sensors and discuss the ways in which they have been used to study biological phenomena. Many proteases involved in intracellular signaling processes catalyze the cleavage of their protein substrates at specific recognition sites. As important upstream activators of many critical cellular processes, including apoptosis, these enzymes must be precisely regulated in both space and time.

RET-based reporters have been instrumental in characterizing the spatiotemporal control of protease activity inside cells. In fact, the earliest intramolecular FRET-based sensors were developed to measure protease activity. In contrast, cleavage of the peptide linker caused the FPs to diffuse away from one another which, in turn, led to a precipitous drop in FRET.

A similar design was used to study the activation of caspases during apoptosis in HeLa cells. Using this dual-specificity reporter system, the authors demonstrated that, during the induction of apoptosis, caspase-3 activity consistently preceded caspase-6 activity by approximately thirty minutes.

Indeed, we have recently shown that despite a large degree of spectral separation, the bright CFP variant, Cerulean, is able to undergo energy transfer with mCherry. As discussed to above, protein kinases must be tightly regulated inside cells. To better understand the regulation of these enzymes within their endogenous environment, several kinase activity reporters have been developed.

Many of the kinase activity reporters in use today utilize an engineered molecular switch based upon a modular design reviewed in ; Because protein kinases are often dynamically regulated, it is important that biosensors designed to track their activities exhibit reversible responses to allow continuous tracking of upregulated and downregulated kinase activities. In the case of the modular kinase activity reporters discussed above, this can be achieved by incorporating a PAABD that displays an intermediate binding affinity for the phosphorylated substrate region.

Presumably, the incorporation of monomeric FP variants reduces interactions between the FPs, allowing the reporter to adopt a more open conformation for dephosphorylation by phosphatases. Kinase activity reporters have also revealed new information about temporal regulation of kinases. However, until recently, it has been difficult to gauge what affect these oscillations have upon the activities of downstream kinases. Therefore, to examine the relationship between second messenger oscillations and kinase activity, several groups have employed kinase activity sensors to track changes in kinase activity under cellular conditions in which second messengers oscillate in a periodic fashion.

These retinal waves play an important role in the development of vision. Because PKA has also been implicated as a major player during retinal development, its spatiotemporal dynamics were investigated in rat retinal explants. Using AKAR2. As alluded to earlier, the targetability of genetically-encoded reporter systems allows researchers to monitor specific pools of a given biomolecule within the cellular environment.

This feature is particularly valuable in the case of kinase activity reporters because the subcellular localization of protein kinases often plays a critical role in achieving specific and effective regulation of kinase-mediated signaling events.

For instance, during anaphase, a dividing cell must establish a cell-division plane midway between segregating chromosomes. However, until recently, it was not clear how the signal was maintained from the spindle midzone to the cell cortex—a distance which spans several micrometers in the average cell. To assess the role of aurora B kinase in this process, researchers targeted a FRET-based aurora B kinase activity reporter to several chromosomal regions, including centromeres by fusion to CENP-B and chromatin by fusion to histone H2B , and tracked changes in both sensor phosphorylation and chromosome location at different times throughout anaphase.

These studies demonstrated that while aurora B was most active near the midzone prior to chromosome segregation, its activity decreased markedly as the chromosomes moved toward the mitotic poles. In the above discussion, we highlighted several kinase activity reporters and outlined some of the ways in which they have been used to visualize reversible protein phosphorylation inside cells. Equally important to the dynamic regulation of protein phosphorylation is the activity of protein phosphatases.

Protein phosphatases counteract the effects of kinase-mediated phosphorylation by catalyzing the hydrolysis of phosphate groups from amino acid side chains. However, whereas a relatively large number of molecular probes have been developed to track kinase activity inside cells, until recently, a general design for phosphatase activity biosensors had not been described.

This and other design features utilized by CaNAR1 should be generally applicable to other protein phosphatases as specific molecular switches are identified or engineered. Thus, as a prototype phosphatase activity sensor, CaNAR1 lays a foundation for studying the targeting and compartmentation of protein phosphatases within the cellular environment.

However, rather than removing a phosphate group, OGT-mediated glycosylation prevents phosphorylation altogether by reciprocally modifying serine and threonine residues. Therefore, the dynamic interplay between protein phosphorylation and O-glycosylation plays a potentially important role in many signaling processes.

For instance, when expressed in HeLa cells, the O-GlcNAc indicator exhibited a slow, but steady, increase in its emission ratio following the addition of glucosamine. Variations of this sensor design, for instance using substrates that are phosphorylated and O-GlcNAcylated in a reciprocal manner, promise to offer valuable information about cross-talk between these important signaling pathways.

Indeed, by altering the shape, surface charge distribution acetylation, for example, masks the positive charge on lysine or hydrogen-bonding potential of modified proteins, these modifications can directly influence the biochemical properties of modified proteins, often affecting their interactions with surrounding biomolecules such as DNA and other proteins. For instance, the first reporters of histone methylation, termed K9 and K27 according to the position of their methylated lysine residues in the histone H3 primary sequence , were constructed by fusing CFP and YFP variants to short N-terminal fragments of histone H3 and a chromodomain derived from histone-binding protein 1 HP1 and the Polycomb Pc protein, respectively.

Indeed, both the K9 and K27 methylation reporters have been shown to exhibit methyl transferase-dependent changes in their emission ratios both in vitro and in live cells, suggesting that they are able to detect the methylation of histone H3. More recently, Sasaki et al. Chromatin localization appears to be important for histone acetyl transferase HAT -mediated acetylation since a C-terminally truncated reporter lacking the globular histone domain showed neither chromatin localization nor trichostatin A TSA -dependent acetylation.

Using this reporter, the authors observed striking differences in histone H4 acetylation levels during various stages of mitosis, with a marked decrease in acetylation beginning at prophase and reaching its nadir during anaphase. Recently, the ability of genetically-encoded fluorescent probes to sense biochemical changes in living systems has been further expanded through the development of coupled fluorescent indicator systems.

Indeed, when co-cultured with hippocampal neurons, Piccell uncovered oscillatory release of picomolar concentrations of NO from neurons—even in the absence of stimulation. More recently, the same group used a similar approach to visualize the secretion of endogenous levels of brain-derived neurotrophic factor BDNF from living neurons.

These studies suggest that, under their experimental conditions, approximately pM of BDNF is secreted upon glutamate stimulation. Importantly, because the TrkB binding domain can be replaced with any RTK binding domain, in theory this system is amenable to the detection of several different growth factors secreted by cells.

Therefore, the modular design of this reporter system should facilitate the development of similar cell-based fluorescent indicators which could provide valuable insights into the dynamics of other important regulatory factors involved in cell-cell communication.

Finally, the Miyawaki laboratory applied knowledge about cell-cycle dependent protein turnover to develop an exciting new biosensor system designed to track cell cycle progression in real time in vivo. This approach allowed cell-cycle dynamics to be monitored during several biologically relevant events, including the migration and differentiation of neural progenitor cells in brain slices and the movement of tumor cells across blood vessels in live mice.

Because it allows various stages of the cell cycle to be correlated with cellular behaviors without the need to synchronize or otherwise perturb normal cell cycle progression, the Fucci technology promises to benefit many areas of cell biology. Moreover, with the development of future probes designed to mark other cell cycle transitions, such as G 2 to M, the ability of genetically-encoded biosensors like Fucci to track different phases of the cell cycle will be further enhanced.

In one way or another, protein-protein interactions impact nearly every aspect of cell physiology—and cellular signaling is no exception. Inside the cell, the activity profiles of signaling enzymes are continuously modulated through specific interactions with regulatory factors, such as activators, inhibitors, adaptor molecules or scaffolding proteins, that rely upon direct protein-protein interactions.

In fact, recently constructed interaction maps suggest that a given signaling protein may have tens, hundreds, or even thousands of transient interaction partners inside the cell, each of which could modulate its function in some manner. Ideally, methods designed to probe protein-protein interactions would be able to detect binding events directly inside living cells, within the native subcellular environment of the interacting proteins.

To this end, several fluorescent imaging techniques have been developed to probe protein-protein interactions under physiological conditions. These include both FRET- and BRET-based detection systems analogous to several of the bimolecular reporters described above as well as approaches, such as bimolecular fluorescence complementation BiFC , which utilizes the phenomenon of protein fragment complementation to generate fluorescence from split FP molecules Figure 8.

Fluorescent reporters to track protein-protein interactions. B Meanwhile, BiFC-based interaction sensors, which are essentially irreversible, use the association of proteins tagged with complementary halves of split FP molecules to promote fluorophore formation. Though the relatively slow maturation of the fluorophore prevents BiFC-based probes from measuring protein-protein interactions in real-time, the large dynamic range of these sensors allows interactions to be detected at protein concentrations that do not dramatically alter the cellular context in which the interactions occur.

Search SpringerLink Search. Buying options eBook EUR Softcover Book EUR Hardcover Book EUR Learn about institutional subscriptions. Table of contents 15 chapters Search within book Search. Front Matter Pages i-xiv. Lockshin Pages Caspase Mechanisms Guy S. Salvesen, Stefan J. Riedl Pages Grassian, Roya Khosravi-Far Pages Humphreys, Wendy Halpern Pages El-Deiry Pages Anderson Pages Srivastava Pages Back Matter Pages About this book Programmed cell death PCD plays pivotal roles in tumor progression, cancer therapeutics and resistance of tumor cells to therapy.

Back to top. Keywords Cell death, apoptosis, cancer research,Khosravi-Far apoptosis cell biology regulation tumorigenesis. Reviews From the reviews: "Programmed cell death PCD is an intriguing term that was introduced by Richard Lockshin nearly 45 years ago.

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Motifs are built around specific ideas, sectors, industries, or trends. If you want to build your own Motif from scratch, there are two customization options for you. All users can also customize the allocation balance of any Motif securities for free. Your custom Motifs are made available to the general user base.

Anytime someone buys, rebalances, or adjusts a Motif you created, you get royalties. With low trading costs, the best social interaction in modern brokerage, royalties, and endless customization, Motif has something for every investor. This is what made Motif exciting in its early days.

Again, we loved Motifs when they were new, but the deviation from this winning strategy has us scratching our heads. This may not seem like very much, but as some Motifs attract hundreds or even thousands of investors, the Creator Reward Program is a definite perk.

Your custom Motifs become available on the general Brokerage Catalog. The program gives you a visual dashboard where you can see every time someone watches, buys, adjusts, or sells your Motif. When your Motif goes live, you might start to see people leaving comments about it and giving it ratings.

You can make friends with other users, or bring over your own from your friends on Facebook. You and your circles can have private conversations. As described above, Impact Accounts allow users to invest, purportedly without owning shares of companies that go against their values. In a typical mutual fund, an investor might unwittingly be supporting unfair labor practices or pollution.

Still, there are additional services one gets for the price. This is more expensive than some immediate competitor platforms, but Motif users may find that the ethical investment aspect is worth the extra cost. Here are some of the most commonly asked questions about Motif from the web for quick visual reference.

The Motif bundling model was cheap and innovative when it was introduced, but it has been priced out by newer platforms. Motif can still be worth the money if you want to hand-build portfolios or directly observe the portfolio strategies of other users. This was always where Motif shined. You can create your own motif from scratch, however. You just pick up to thirty stocks and allocate their focus in your motif.

I wish they allowed OTC stocks. Maybe soon. Hey Benjamin, thanks for adding that in. Thanks for stopping by! I think this will be a tax nightmare. The reporting on a few stocks is bearable but if you have hundreds of them… otherwise, great idea. It sounds interesting….

I like the idea of easily being able to diversify, but it seems a little difficult to understand what you actually own…. Hey Money Ahoy, it is a very interesting form of investing. The Motifs are diverse to an extent, but there is that lack of control. Thanks for swinging by!

Thank you for all of that information. I do like that you could give it a try without laying out a huge amount of money…. There are two big questions I have — first one. In connection with the move to Kingdom, Pershing has agreed and this letter will serve as notice of the appointment to appoint Kingdom as the successor custodian for your IRA. Unless you decide otherwise, a new IRA will be established for you at Folio with Kingdom as the custodian.

Please be aware that your current beneficiary information will NOT transfer from your Motif account to Folio. After your account has transferred to Folio Investing, you will be able to login to your new account at Folio and setup your beneficiary designations on their site.

If you do not designate your beneficiaries, and if there is no beneficiary designation on file at the time of your death, your IRA assets will be paid to your estate. Your required tax forms associated with your Motif account will be sent to your address of record. In the meantime, you may continue to log into your account at www.

Q: Can I keep my account at Motif? A: No. Motif is voluntarily ceasing its online brokerage operations, and thus all accounts must be transferred to Folio or another broker-dealer. A: Nothing. Your account will be automatically transferred for no fee or charge to you to Folio as noted above. All shares including fractional shares and cash remaining in accounts eligible for transfer as of the close of business on May 20, will be transferred to Folio Investing. Your shares and cash will be in your Folio Investing account S on May 21, A: If your account does not have the necessary cash to cover any charges due, Motif will sell a portion of your equity position S as necessary to pay any debit balance prior to the transfer.

Q: Will I have access to the same securities and types of services and fees that I had at Motif? A: Folio is a leading self-directed online brokerage, and offers many securities and portfolio investing features, some of which were not available to you at Motif. Please see the Pricing and Sweep Disclosures below, or visit www. Q: Where can I obtain additional information about Folio Investing? A: Please visit www. Q: How do the brokerage fees on Folio Investing compare with my Motif account?

Depending on the services you were using at Motif and the services you use at Folio Investing, your fees at Folio Investing may be higher or lower than you have been paying at Motif. Q: Do I have to move my account to Folio Investing? A: No, you are free to liquidate your account or move your account to another broker-dealer of your choosing. However, if you have not completed a transfer to another firm by May 12, , your account will be transferred to the Folio Investing platform and you will be responsible for any Folio charges you incur there, including their monthly fees and fees to transfer to another firm.

If you wish to liquidate and close your account instead of transferring it, you may incur a taxable event when selling your securities holdings. Q: Where do I get my tax forms? A: Pershing will issue you by mail a copy of your tax forms for activity in your Motif account to your address of record. If you would like to access historical tax records, please download them from the Motif site by the end of day, May 20, , when the site will no longer be available.

Q: Can I still get statements and trade confirmations from the Motif site? A: Pershing will issue you by mail copy of your May account statement for activity in your Motif Account. If you would like to access historical documents generated while your Motif account as open, please download them from the Motif site by the end of day, May 20, , when the site will no longer be available.

Q: Will my dividends be reinvested? A: As part of this conversion, Folio will setup all accounts for dividend reinvestment. You can change this setting on the Folio site at any time. A: As part of this conversion, Folio will setup all accounts to automatically select tax lots for sale that minimize gains. Q: Will my bank link and recurring deposits or withdrawals carry over to Folio? A: If your account is transferred to Folio Investing, then your electronic funds transfer EFT bank link will be carried over and setup at Folio but you will need to setup your automated recurring deposits and or withdrawals, as well as automatic recurring purchases and sales on the Folio site.

Additional disclosure: I wrote this article myself, and it expresses my own opinions. I am not receiving compensation for it other than from Seeking Alpha. I have no business relationship with any company whose stock is mentioned in this article.

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