Behavioral response of Caenorhabditis elegans to localized thermal stimuli.

BACKGROUND: Nociception evokes a rapid withdrawal behavior designed to protect the animal from potential danger. C. elegans performs a reflexive reversal or forward locomotory response when presented with noxious stimuli at the head or tail, respectively. Here, we have developed an assay with precise spatial and temporal control of an infrared laser stimulus that targets one-fifth of the worm's body and quantifies multiple aspects of the worm's escape response. RESULTS: When stimulated at the head, we found that the escape response can be elicited by changes in temperature as small as a fraction of a degree Celsius, and that aspects of the escape behavior such as the response latency and the escape direction change advantageously as the amplitude of the noxious stimulus increases. We have mapped the behavioral receptive field of thermal nociception along the entire body of the worm, and show a midbody avoidance behavior distinct from the head and tail responses. At the midbody, the worm is sensitive to a change in the stimulus location as small as 80 µm. This midbody response is probabilistic, producing either a backward, forward or pause state after the stimulus. The distribution of these states shifts from reverse-biased to forward-biased as the location of the stimulus moves from the middle towards the anterior or posterior of the worm, respectively. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. Analyses of mutants suggest the possibility that TRPV channels and glutamate are involved in facilitating the midbody noxious response. CONCLUSION: Through high resolution quantitative behavioral analysis, we have comprehensively characterized the C. elegans escape response to noxious thermal stimuli applied along its body, and found a novel midbody response. We further identified the nociceptor PVD as required to sense noxious heat at the midbody and can spatially differentiate localized thermal stimuli.

An ultramarine fluorescent protein with increased photostability and pH insensitivity.

We report a pH-insensitive and photostable ultramarine fluorescent protein, Sirius, with an emission peak at 424 nm, the shortest emission wavelength among fluorescent proteins reported to date. The pH-insensitivity of Sirius allowed prolonged visualization of biological events in an acidic environment. Two fluorescence resonance energy transfer (FRET) pairs, Sirius-mseCFP and Sapphire-DsRed, allowed dual-FRET imaging with single-wavelength excitation, enabling detection of Ca(2+) concentration and caspase-3 activation in the same apoptotic cells.

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block.

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.

A high-throughput and single-tube recombination of crude PCR products using a DNA polymerase inhibitor and type IIS restriction enzyme.

Type IIS restriction enzymes have been successfully used as "universal" restriction enzymes in DNA manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in a single step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA ligase to initiate a spontaneous and one-way recombination reaction. Highly efficient DNA recombination can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution; thereby the entire procedure takes less than 15 min. Owing to its simplicity, efficiency and rapidity, one-step FASTR can be applied to a wide range of DNA manipulations including those involving high-throughput applications where significant reduction in time and cost is expected.

Genetically encoded ratiometric fluorescent thermometer with wide range and rapid response.

Temperature is a fundamental physical parameter that plays an important role in biological reactions and events. Although thermometers developed previously have been used to investigate several important phenomena, such as heterogeneous temperature distribution in a single living cell and heat generation in mitochondria, the development of a thermometer with a sensitivity over a wide temperature range and rapid response is still desired to quantify temperature change in not only homeotherms but also poikilotherms from the cellular level to in vivo. To overcome the weaknesses of the conventional thermometers, such as a limitation of applicable species and a low temporal resolution, owing to the narrow temperature range of sensitivity and the thermometry method, respectively, we developed a genetically encoded ratiometric fluorescent temperature indicator, gTEMP, by using two fluorescent proteins with different temperature sensitivities. Our thermometric method enabled a fast tracking of the temperature change with a time resolution of 50 ms. We used this method to observe the spatiotemporal temperature change between the cytoplasm and nucleus in cells, and quantified thermogenesis from the mitochondria matrix in a single living cell after stimulation with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which was an uncoupler of oxidative phosphorylation. Moreover, exploiting the wide temperature range of sensitivity from 5°C to 50°C of gTEMP, we monitored the temperature in a living medaka embryo for 15 hours and showed the feasibility of in vivo thermometry in various living species.

Pan-neuronal screening in Caenorhabditis elegans reveals asymmetric dynamics of AWC neurons is critical for thermal avoidance behavior.

Understanding neural functions inevitably involves arguments traversing multiple levels of hierarchy in biological systems. However, finding new components or mechanisms of such systems is extremely time-consuming due to the low efficiency of currently available functional screening techniques. To overcome such obstacles, we utilize pan-neuronal calcium imaging to broadly screen the activity of the C. elegans nervous system in response to thermal stimuli. A single pass of the screening procedure can identify much of the previously reported thermosensory circuitry as well as identify several unreported thermosensory neurons. Among the newly discovered neural functions, we investigated in detail the role of the AWCOFF neuron in thermal nociception. Combining functional calcium imaging and behavioral assays, we show that AWCOFF is essential for avoidance behavior following noxious heat stimulation by modifying the forward-to-reversal behavioral transition rate. We also show that the AWCOFF signals adapt to repeated noxious thermal stimuli and quantify the corresponding behavioral adaptation.

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators.

While genetically encoded Ca(2+) indicators (GECIs) allow Ca(2+) imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca(2+) imaging, it had a, diminished Ca(2+) response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca(2+) response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca(2+) signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.

Reversible dimerization of Aequorea victoria fluorescent proteins increases the dynamic range of FRET-based indicators.

Fluorescent protein (FP)-based Forster resonance energy transfer (FRET) technology is useful for development of functional indicators to visualize second messenger molecules and activation of signaling components in living cells. However, the design and construction of the functional indicators require careful optimization of their structure at the atomic level. Therefore, routine procedures for constructing FRET-based indicators currently include the adjustment of the linker length between the FPs and the sensor domain and relative dipole orientation of the FP chromophore. Here we report that, in addition to these techniques, optimization of the dimerization interface of Aequorea FPs is essential to achieve the highest possible dynamic range of signal change by FRET-based indicators. We performed spectroscopic analyses of various indicators (cameleon, TN-XL, and ATeam) and their variants. We chose variants containing mutant FPs with different dimerization properties, i.e., no, weak, or enhanced dimerization of the donor or acceptor FP. Our findings revealed that the FPs that dimerized weakly yielded high-performance FRET-based indicators with the greatest dynamic range.

In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase.

A cell proliferation marker protein, pKi-67, distributes to the chromosome periphery during mitosis and nucleolar heterochromatin in the interphase. We report here on the structural domains of pKi-67 that are required for its correct distribution. While both the LR domain and the conserved domain were involved in localization to the nucleolar heterochromatin, both the LR domain and the Ki-67 repeat domain were required for its distribution to the mitotic chromosome periphery. Using in vivo time-lapse microscopy, GFP-pKi-67 was dynamically tracked from the mitotic chromosome periphery to reforming nucleoli via prenucleolar bodies (PNBs). The signals in PNBs then moved towards and fused into the reforming nucleoli with a thin string-like fluorescence during early G1 phase. An analysis of the in vivo kinetics of pKi-67 using photobleaching indicated that the association of pKi-67 with chromatin was progressively altered from "loose" to "tight" after the onset of anaphase. These findings indicate that pKi-67 dynamically alters the nature of the interaction with chromatin structure during the cell cycle, which is closely related to the reformation process of the interphase nucleolar chromatin.

Importin alpha transports CaMKIV to the nucleus without utilizing importin beta.

Ca(2+)/calmodulin-dependent protein kinase type IV (CaMKIV) plays an essential role in the transcriptional activation of cAMP response element-binding protein-mediated signaling pathways. Although CaMKIV is localized predominantly in the nucleus, the molecular mechanism of the nuclear import of CaMKIV has not been elucidated. We report here that importin alpha is able to carry CaMKIV into the nucleus without the need for importin beta or any other soluble proteins in digitonin-permeabilized cells. An importin beta binding-deficient mutant (DeltaIBB) of importin alpha also carried CaMKIV into the nucleus, which strongly suggests that CaMKIV is transported in an importin beta-independent manner. While CaMKIV directly interacted with the C-terminal region of importin alpha, the CaMKIV/importin alpha complex did not form a ternary complex with importin beta, which explains the nonrequirement of importin beta for the nuclear transport of CaMKIV. The cytoplasmic microinjection of importin alpha-DeltaIBB enhanced the rate of nuclear translocation of CaMKIV in vivo. This is the first report to demonstrate definitely that mammalian importin alpha solely carries a cargo protein into the nucleus without utilizing the classical importin beta-dependent transport system.

Importin alpha/beta-mediated nuclear protein import is regulated in a cell cycle-dependent manner.

Functional nuclear proteins are selectively imported into the nucleus by transport factors such as importins alpha and beta. The relationship between the efficiency of nuclear protein import and the cell cycle was measured using specific import substrates for the importin alpha/beta-mediated pathway. After the microinjection of SV40 T antigen nuclear localization signal (NLS)-containing substrates into the cytoplasm of synchronized culture cells at a certain phase of the cell cycle, the nuclear import of the substrates was measured kinetically. Cell cycle-dependent change in import efficiency, but not capacity, was found. That is, import efficiency was found low in the early S, G2/M, and M/G1 phases compared with other phases. In addition, we found that the extent of co-imunoprecipitation of importin alpha with importin beta from cell extracts was strongly associated with import efficiency. These results indicate that the importin alpha/beta-mediated nuclear import machinery is regulated in a cell cycle-dependent manner through the modulation of interaction modes between importins alpha and beta.