Novel imaging strategies reveal a conformational shift in a receptor tyrosine kinase domain that controls ligand shedding by an ADAM metalloprotease. trans. Fluorescence lifetime imaging microscopy and electron tomography reveal that activation extends the Eph receptor tyrosine kinase intracellular domain away from the cell membrane into a conformation that facilitates productive association with ADAM10. Accordingly, EphA3 mutants with constitutively-released kinase domains efficiently support shedding, even when their kinase is disabled. Our data suggest that this phosphorylation-activated conformational switch of EphA3 directly controls ADAM-mediated shedding.Release of cell surface-bound ligands by A-Disintegrin-And-Metalloprotease (ADAM) transmembrane metalloproteases is essential for signalling by cytokine, cell adhesion, and tyrosine kinase receptors. For Eph receptor ligands, it provides the switch between cell-cell adhesion and repulsion. Ligand shedding is tightly controlled by intrinsic tyrosine kinase activity, which for Eph receptors relies on the release of an inhibitory interaction of the cytoplasmic juxtamembrane segment with the kinase domain. However, a mechanism linking kinase and sheddase activities had remained elusive. We demonstrate that it is a membrane-proximal localisation of the latent kinase domain that prevents ephrin ligand shedding in  The Eph transmembrane receptors are part of the receptor tyrosine kinase family and play important roles in communication between neighbouring cells. An Eph receptor binds to its ligand, membrane-tethered ephrin, on a neighbouring cell so as to form a stable complex and activate downstream signalling events. One such event is regulation of ADAM10, a transmembrane protease of the ADAM metalloprotease family, which provides a feedback mechanism to Eph signalling. ADAM10 is located on Eph-expressing cells and cleaves ephrin from its membrane tether on the opposite cell , thereby separating the cell-cell connection and allowing the signalling complex to internalise. In other biological contexts, activity of the ADAM metalloprotease family underlies signalling mechanisms such as oncogenic EGF-receptor transactivation, adhesion molecule shedding and cytokine/chemokine release. In general, ADAM function is enhanced when receptor tyrosine signalling is active and repressed when tyrosine kinase signalling is inhibited. However, the mechanism through which receptor tyrosine kinase signalling regulates ADAM10, have remained elusive. By combining fluorescence lifetime imaging microscopy (FLIM) and electron microscopic tomography of EphA3, we have demonstrated in live cells at molecular resolution that tyrosine phosphorylation of activated EphA3 triggers a measurable movement of the kinase domain away from the plasma membrane. Only this conformation of the EphA3 kinase domain away from the plasma membrane permits ADAM10 to come close enough to EphA3 so that it can reach its tightly EphA3-bound substrate, ephrin-A5. Our findings delineate a new regulatory concept in cell-cell communication, whereby control over proteolytic sheddase activity is provided by an activation-induced switch in the conformation of the cytoplasmic domain of a receptor tyrosine kinase, rather than by a cytosolic signalling pathway. The ADAM  transmembrane proteases fulfil essential functions during normal and pathological tissue- and organ-development Ephs and their membrane bound ligands (ephrins) control cell positioning during normal and oncogenic development by modulating cell-cell adhesion and cell-cell segregation or repulsion ADAM proteins are produced as inactive precursors that become catalytically active upon removal of the prodomain during secretion. Interestingly, there is little evidence for a substrate cleavage sequence motif, and the regulation of proteolytic specificity is achieved by interaction of substrates with the non-catalytic disintegrin and cysteine-rich extracellular ADAM domains. For ephrin cleavage, a substrate recognition module within the cysteine-rich domain of ADAM10 specifically binds only the high-affinity ligand-receptor complex to ensure that only Eph receptor-bound ephrins are cleaved n addition to this direct control of ADAM10-facilitated shedding, substantial evidence documents intracellular regulation of ADAM proteases To elaborate the intracellular regulation of ADAMs by RTK signalling we investigated ADAM10 catalysed ephrin-A5 cleavage that is mediated by EphA3-expressing cells, as previous studies demonstrated that this sheddase activity depends on ephrins binding and activating the EphA3 RTK 653M]. Immunoblot analysis confirmed that the release of ephrin-A5 from the Fc complexes was dependent on pre-clustering and indeed was greatly reduced in cultures of cells expressing the kinase-inactive EphA3 mutant  segment from an inhibitory interaction with the kinase domain We hypothesized that the inactive/constrained JM segment positions the kinase domain close to the membrane, a configuration that imparts a steric obstruction to the productive ADAM10/EphA3 interaction and thereby controls ephrin shedding. To test this hypothesis we examined whether forced approximation of the EphA3 kinase domain to the plasma membrane affects ADAM10 association and function. For these experiments we designed a series of EphA3 mutants  includinImportantly, all EphA3 mutants retained the capacity to associate and co-immunoprecipitate with ADAM10 , in agre594ephrin-A5-Fc was used as substrate  cleavage to occur.In addition to controlling RTK function, ADAMs are key modulators of cell–matrix interactions To test the hypothesis that a bulky protein domain at the JM position would impair a productive ADAM/EphA3 alignment, the EphA3 cytoplasmic domain was replaced with that of L-selectin. We surmised that CaM-loaded EphA3/L-selectin could not effectively promote ephrin cleavage, while conversely inhibition of CaM binding to this chimeric receptor using CaM inhibitors should favour ADAM10 association and ephrin-A5 cleavage. Control experiments confirmed inhibition of CaM binding  and incrtkRasRFP (red-fluorescent protein)  imaging approach protein)  [33].tkRasRFP (acceptor) will not only depend on their distance but also on the local acceptor density that may vary as a function of time and space in a FLIM time-lapse series. To account for this, the FRET rate (Tk) per acceptor density (Tk/acceptor) that does not depend on the concentration of acceptors in the plasma membrane −1)–acceptor intensity (Iaccep) 2D-histograms of the confocal images at selected time points  of cell-surface EphA3-GFP increased (showing reduced FRET) 10–20 min after stimulation , indicate points . Ephrin-r values , indicatresidues  [16],-GFP mutant exhibited significantly (p = 7×10−14) lower FRET efficiencies  is proportional to the fourth power of the distance between the donor chromophore and acceptor plane Tk/acceptor of EphA3[3YF]-GFP and EphA3[2YE]-GFP  To examine at increased resolution the change in the span between the plasma membrane and the carboxy-terminus of the latent and activated receptor, we used electron microscopy (EM) to image the EphA3 C-terminus that had been labelled with streptavidin-conjugated Qdots SA-Qdots . We achi2-terminal AP-tagged and biotinylated EphA3 (APN-EphA3). Confocal microscopy revealed that SA-Qdot staining of APN-EphA3 cells correlated with anti-EphA3 staining and shifted to a cytoplasmic compartment upon ephrin-stimulation . Microinjection of SA-Qdots into APC-EphA3-expressing cells and sectioning of non-permeabilised cells allowed EM of EphA3-bound Qdots at the intact plasma membrane  receptor JM domain with a maximal theoretical (β-sheet) span of ∼20 nm. While it would seem formally possible that extension of the activated EphA3 ICD reflects an unfolding of the linker connecting the kinase with the C-terminal SAM domain, recently elucidated crystal structures of the EphA3 ICD argue against this possibility Furthermore, marking the EphA3 C-terminus with fluorescent (GFP) and electron-dense (Qdots) tags allowed for the first time to demonstrate in live cells at molecular resolution that receptor activation and tyrosine phosphorylation triggers a measurable shift of the kinase domain away from the plasma membrane. The increased span of activated EphA3 that we estimated by FLIM and EM tomography is consistent with partial extension of the 64-residue  were inserted, with a terminal stop codon. For EphA3-APN the AP-tag 20), and for EphA3-APC the AP-tag was inserted into a XmaI site engineered into the EphA3 C-terminus .Inactive EphA3 was made by substitution of residue K653 to M in EphA3-GFP Cleaved ephrin-A5 was extracted from pooled Protein-A Sepharose-pre-cleared lysates and culture supernatants of cells that had been treated with pre-clustered or non-clustered ephrin-A5-Fc by using EphA3-Fc coupled to Protein-A-Sepharose. Pull-downs were analysed by anti-ephrin-A5 immunoblot. Cleavage of cell-surface ephrin-A5 was assayed in 1-h co-cultures of ephrin-A5-expressing HEK293T cells and EphA3/L-selectin transfected cells by extracting ephrin-A5 from cell lysates with EphA3-Fc coated Protein-A Sepharose. Where indicated, cells were treated prior to ephrin-A5-stimulation with CaM inhibitors trifluoperazine, calmidazolium , or W7, or metalloprotase inhibitors TAPI1 or GM6001 . For CaM-co-precipitation EphA3/L-selectin and Wt EphA3 tagged with a biotin AP were biotinylated with BirA 488-ephrin labelling relative to the expression level of the Alexa647IIIA4 anti-EphA3 antibody-stained receptor 3-channel confocal microscopy was performed by sequential scanning on Olympus FV1000 or Leica SP5 confocal microscopes. Quantitation of internalised ephrin-A5-associated fluorescence was achieved using ImageJ or Metamorph image analysis software by selecting regions of cells to exclude bead-associated fluorescence. Microscopic evaluation of ephrin cleavage by EphA3/L-selectin expressing cells, where ephrin-A5 was not internalised but remained complexed at the plasma membrane, was done by estimating the level of Alexa8 photons. Images of the donor fluorescence decays were processed using the SymPhoTime software package  and the calculated average fluorescence lifetime (τ) images are presented in pseudo-colour. The average fluorescence lifetime τ(xy) images were calculated from the parameters  of a double exponential fit of the fluorescence decay curves [F] in each pixel:−1-acceptor intensity (Ia) 2D-histograms were computed from the confocal FLIM images as described below for wide-field frequency-domain FLIM except that a bin size of 100 counts was used for the acceptor intensity.Time-domain confocal FLIM was performed in transiently transfected Cos7 cells grown on coverslips or glass bottom dishes (MatTek Corp.). FLIM images were obtained using an Olympus Fluoview 1000 microscope, equipped with a Picoharp 300 photon counting setup . GFP was excited with a 470 nm diode . Images of 512×512 pixels were acquired detecting approximately 10For wide-field frequency-domain FLIM  we used http://www.python.org) with the Numarray extension for numerical computing (http://www.stsci.edu/resources/software_hardware/numarray) further augmented with low-level routines written in C a) was generated from the multiple fluorescence phase-lifetime images (≥16 images) and corresponding acceptor intensity images using a bin size of 320 intensity units (arbitrary units). The standard error in the fluorescence lifetimes for each bin was calculated from the averages of all the images. The donor to acceptor energy transfer rate kT normalized to acceptor density (kT/acceptor) was obtained from the slope of a linear fit to the τ−1−Ia (acceptor intensity) 2D-histograms. The τ−1−Ia 2D-histograms were fitted to a linear equation:d = 1.96 ns) was used to constrain the intercept, kd, to 0.51. The slope kT/acceptor is proportional to the energy transfer rate per acceptor yielding 1.9+/−0.16 for EphA3[3YF]-GFP, 0.56+/−0.08 for EphA3[2YE]-GFP and 1.27+/−0.16 for EphA3[2YE-KM]-GFP. The relative distance increase from GFP to the plasma membrane, comparing both conformations, (R2YE/R3YF) can be calculated from:Raw FLIM data were processed in IPLab  to generate a binary mask for the intensity threshold operation data and a mask for the ROI used in background correction of the raw data. Using the raw FLIM data and mask, phase- and modulation lifetime images were generated using scripts written in Python programming language (605 (Invitrogen). Labelled cells were washed in PBS, fixed in 2.5% Glutaraldehyde, 2% sucrose for 40 min, and prepared on ice for EM by “epon” embedding: cells were rinsed in CaCo buffer (30 min), post-fixed in 2% osmium tetroxide (40 min), washed (water), and stained with 0.5% uranyl acetate (30 min). Fixed, washed cells were dehydrated in graded ethanol solutions, embedded in epon 812 (Serva), and hardened (48 h) at 60°C. Epoxy-embedded blocks were cut into 50 or 250 nm sections (Leica Ultracut S microtome) and mounted on Formvar coated grids. Grids were post-stained with led-citrate (1 min) at room temperature, rinsed with water, and air dried. APC-EphA3 expressing cells, stably co-expressing APc-EphA3 together with a cytoplasmic form of BirA for efficient biotinylation of the AP-tagged EphA3 C-terminus, were either microinjected with Qdots prior to fixation (where indicated) or were fixed in 4% PFA, 0.5% Glutaraldehyde, 2% sucrose (30 min), permeabilised with 0.1% Triton ×100, and incubated with SA-Qdots605 for 1 h. Washed samples were then fixed in 2.5% Glutaraldehyde, 2% sucrose, and prepared for EM as described above. This approach partially solubilises the plasma membrane and required computer-assisted assignment of the exact plasma membrane/cytoplasm boundaries.For EM we biotinylated AP-tagged EphA3 receptors in intact cells using either exogenous or co-transfected biotin ligase (BirA) www.amiravis.com). We de-noised three-dimensional images with nonlinear anisotropic diffusion and semi-automatically segmented those using erosion and dilation operations after roughly segmenting regions of the reconstructions manually. Plasma membranes localisation in the electronic images was semi-automated, with their boundaries determined using dilation and erosion operations. Qdot detection was fully automated according to their size and contrast using thresholding techniques We collected at room temperature single-axis tilt series of chemically fixed cells at 1–2° angular increment between −67° to +67° using CM200 and Tecnai 30 microscopes  and the Tietz tomography interface  for data acquisition. Serial EM images were recorded on 2 k×2 k and 4 k×4 k pixel CCD cameras at a defocus level of −2 µm, with a pixel size at the specimen level of 0.7 nm. We aligned the projection images of the samples using cross-correlation techniques. The merit figure of the aligned tilt-series had a value of approximately 1 nm, indicating no significant shrinkage of the sample. Reconstructions were performed Figure S1Tyrosine phosphorylation and ADAM10 association of EphA3 mutants. (A) Phosphorylation of Wt and mutant EphA3 after incubation with clustered ephrinA5 Fc. HEK293T cell clones stably expressing either Wt or kinase inactive EphA3[K653M], or parental HEK293T cells, were incubated with vehicle control (−), with non-clustered (NC) or clustered (C) ephrinA5-Fc for 15 min prior to lysis. EphA3 immuno-precipitates were analysed by Western blot with anti-phosphotyrosine (α-PY) and lysates with anti-EphA3 antibodies as indicated. (B) The EphA3/ADAM10 association does not require their ICDs. α-HA immunoprecipitates from cells expressing HA-ADAM10, and/or Wt EphA3 or EphA3[ΔICD], were immunoblotted for EphA3 (top) or ADAM10 (bottom); total lysates were probed for EphA3 (right). (U), unprocessed; (P), processed ADAM10. Single exposures of blots are shown with non-relevant lanes removed.(0.64 MB TIF)Click here for additional data file.Figure S2Inhibition of EphA3ΔICD-dependent ephrin cleavage by metalloprotease inhibitors. Cells transiently expressing EphA3[ΔICD] were incubated 1 h with the metalloprotease inhibitor GM6001 (10 and 20 µM) or the ADAM-specific inhibitor TAPI1 (50 µM) prior to incubation with Alexa594-ephrin-A5-coated beads. After 40 min the cells were placed on ice, stained with anti-EphA3 (IIIA4)-Alexa647, fixed and imaged by confocal microscopy.(4.45 MB TIF)Click here for additional data file.Figure S3Cell surface expression and ephrin binding capacity of EphA3 mutants. HEK293T cells were transfected with Wt or mutant EphA3-GFP constructs as indicated and analysed for cell surface EphA3 expression by labelling with Alexa647-conjugated IIIA4 anti-EphA3 antibody specific for the native EphA3 conformation 594-conjugated ephrinA5-Fc (ephrinA5-Alexa594). Flow cytometric analysis shows cell surface receptor expression (α-EphA3-Alexa647) relative to overall expression level (GFP) and to the ability of cells to bind ephrin-A5-Alexa594. The fraction of GFP-tagged EphA3 protein on the cell surface was estimated as fraction of GFP-tagged receptor recognised by the anti-EphA3 antibody: all EphA3 ICD mutants are expressed at the plasma membrane and bind the IIIA4 antibody and ephrin-A5 at levels similar to the Wt receptor.(0.96 MB TIF)Click here for additional data file.Figure S4Phosphotyrosine profile and ADAM10 binding capacity of EphA3 JM mutants. (A) Phosphotyrosine profile in cells transfected transiently to express Wt EphA3 and JM mutants. HEK293T cells were transfected with expression constructs for Wt EphA3-GFP or derived mutants, as indicated, and cells treated with non-clustered or pre-clustered ephrin-A5 Fc for 10 min. Anti-phosphotyrosine (PY) antibody (4G10) immuno-precipitates from whole cell lysates were probed with anti-PY, and total lysates with anti-EphA3 antibodies, as indicated. Positions on the Western blot corresponding to molecular weights of GFP-EphA3 and IgG (heavy and light chains) are indicated on the left. Phosphorylated protein bands at the GFP-EphA3 position in the left panel are likely due to auto-phosphorylation due to high transient over-expression of the EphA3 constructs in these samples. (B) ADAM10 association with Wt and mutant EphA3. ADAM10 immunoprecipitates and total cell lysates from Wt or mutant (as indicated) EphA3-transfected cells (ephrin-A5-treated) were analysed for EphA3 and ADAM10 by immunoblot. Single exposures of blots are shown with non-relevant lanes removed.(1.02 MB TIF)Click here for additional data file.Figure S5The extended (active) EphA3 ICD conformation is sufficient for ephrin cleavage, while internalisation requires an intact kinase. (A) Cells expressing Wt EphA3-GFP, EphA3[2YE]-GFP, or kinase-inactive EphA3[2YE KM]-GFP were incubated with Alexa594ephrinA5-coated beads or (B) pre-clustered, soluble Alexa594ephrinA5. EphA3-GFP (green) and Alexa594ephrin (red) fluorescence in fixed cells was imaged by confocal microscopy. Individual micrographs from fluorescent channels, the merged images, and phase-contrast images are shown. Yellow arrow heads denote areas of sustained interactions between cell surface EphA3 and ephrin-A5 beads. White arrows mark cell-membrane areas with bound- but not internalised Alexa594ephrin.(2.71 MB TIF)Click here for additional data file.Figure S6Removal of the ADAM10 ICD reconstitutes ephrin shedding in cells expressing EphA3 JX mutants. (A) Confirmation that ADAM10−/− MEFs do not contain detectable ADAM10. Lysates of HEK293Ts, ADAM10−/−, or Wt MEFs were immunoprecipitated with 1, protein A beads alone; or with 2, anti-human specific ADAM10 monoclonal antibodies (RND); or 3, with anti-ADAM10 polyclonal antibodies (Abcam). Immunoprecipitates were immuno-blotted with polyclonal anti-ADAM10 antibodies. U, unprocessed; P, processed ADAM10. (B) ADAM10−/− MEFs transfected with combinations of GFP-tagged EphA3  and HA-tagged ADAM10  were incubated with Alexa594ephrinA5-coated beads. After 40 min the cells were fixed, permeabilised, and stained with anti-HA and Alexa647-labelled secondary antibodies. Images show single-section confocal micrographs, together with the merged images .(4.57 MB TIF)Click here for additional data file.Figure S7CaM inhibitors regulate association of EphA3-L-selectin with CaM and with ADAM10 and trigger ephrinA5 shedding by EphA3-L-selectin expressing cells. (A) CaM inhibitors block association of CaM with EphA3-L-selectin. Cells expressing AP-tagged EphA3/L-selectin or Wt AP-EphA3 were treated with CaM inhibitors trifluoperazine (TFP) or Calm or vehicle control, as indicated. Following biotinylation of AP-tagged receptors, EphA3 complexes were recovered by SA pulldown and analysed by Western blot with anti-CaM and anti-EphA3 antibodies. The positions of Wt EphA3 and of the EphA3/L-selectin fusion protein are indicated. (B) CaM inhibitors modulate the association of EphA3/L-selectin with ADAM10. HEK293T cells expressing EphA3/L-selectin (left panels) or Wt EphA3 (right panels) were pre-treated (30 min) with CaM inhibitors TFP (20 µM), Calm (2 µM), N-6-Aminohexyl0-5-chloro-1-naphthalenesulfonamide , or vehicle control before lysis. ADAM10 immunoprecipitates were analysed by Western blot with anti-EphA3 or anti-ADAM10 antibodies, and total lysates with anti-EphA3 antibodies, as indicated. The graph shows amounts of EphA3/L-selectin (left panels) or EphA3 (right panels) in ADAM10 immunoprecipitates relative to control lanes as determined by densitometry. (C) Mutation of the CaM-binding site in EphA3-L-selectin reduces its ability to support ephrinA5 cleavage. L358E and K359E substitutions, reported to affect CaM binding to the L selectin cytoplasmic domain, were introduced into the EphA3/L selectin chimera to produce EphLsel EE. HEK293T cells, transfected with Wt EphA3-L-selectin or with EphLsel EE were incubated with Alexa594-labelled ephrinA5 beads; the capacity to promote ephrin cleavage was monitored by measuring ephrin labelling of the cell membrane. Ephrin labelling relative to receptor expression was determined in 50 regions from five individual micrographs for each sample. The mean+/−SEM are shown in the graph.(1.48 MB TIF)Click here for additional data file.Figure S8CaM-binding to chimeric EphA3/L-selectin regulates ephrin cleavage from cells. (A) Microscopic analysis of cleavage of GFP-ephrinA5 from cells. EphA3/L-selectin transfected HEK293T cells were pre-treated (15 min) with CaM inhibitors trifluoperazine , Calm (2 µM), W7 (50 µM), or vehicle control before incubation (1 h) with cells expressing GFP-ephrinA5. Cell surface EphA3/L-selectin  and GFP-ephrinA5 (green) were imaged in fixed cells by confocal microscopy, micrographs from individual green and red fluorescence channels, and merged images are shown. The outline of GFP-ephrin-A5 expressing cells is indicated (….) for clarity. The open arrow head points at the interface between untreated, EphA3/L-selectin, and GFP-ephrinA5 cells. Yellow arrowheads indicate areas on CaM-inhibitor-treated EphA3/L-selectin cells that are not in direct contact with GFP-ephrin-A5 expressing cells but reveal obvious ephrin staining. (B) Biochemical analysis of GFP-ephrinA5 cleavage from cells. EphA3/L-selectin transfected HEK293T cells were pre-treated as in (A) with TFP, Calm, or vehicle control, then incubated for 1 h with stably transfected ephrinA5/HEK293T cells. Ephrin-A5 was recovered from cell lysates by pulldown with EphA3-Fc beads and detected on Western blot with α-ephrinA5 antibodies. Full-length and cleaved ephrin are indicated. Total lysates were also probed for EphA3/L-selectin expression with α-EphA3 antibodies (bottom). Cleaved ephrin-A5 was quantitated by densitometry.(2.57 MB TIF)Click here for additional data file.Figure S9FLIM analysis of the EphA3-ICD reveals extension of the activated EphA3 transmembrane domain. (A) Wide field frequency domain FLIM time-series of EphA3-GFP (green) and tkRasRFP (red) co-transfected COS7 cells at indicated times (min) after ephrin-A5 stimulation. Upper row: EphA3-GFP fluorescence intensity images. Lower row: fluorescence phase lifetime (τϕ) images of EphA3-GFP colour bar inset indicates the fluorescence lifetime range in ns. Lower image: tkRasRFP fluorescence intensity image. (B) Histograms of GFP phase lifetimes τΦ calculated on a pixel-by-pixel basis for the cells displayed in (A). (C) Example of GFP phase (τΦ) and modulation (τM) fluorescence lifetime images obtained by FLIM to generate 1τ-acceptor 2D-histograms  EphA3 GFP fluorescence was blacked out to exclude areas where the detector was saturated. Cumulative (2D) phase (τΦ) and modulation (τM) fluorescence lifetime histograms of cell populations (right panels) indicate significant fluorescence lifetime differences between EphA3-GFP-[2YE] and EphA3-GFP-[3YF]. (D) The Kolmogorov-Smirnov (KS) test was performed to assess if fluorescence lifetimes of either EphA3-GFP [3YF] or EphA3-GFP [2YE] measured in a large population of tkRasRFP-Cos7 cells are distinct. A highly significant (p<0.001) difference between the two datasets suggests a measurable change in the donor (EphA3-GFP)/acceptor (RFP-labelled membrane) distance. The two datasets were entered for the KS test at http://www.physics.csbsju.edu/stats/KS-test.html. (E) Acceptor photobleaching confirms the reliability of fluorescence lifetime imaging to detect FRET. tkRasRFP-transfected (red) Cos7 cells co-expressing EphA3-GFP [3YF] (green) were imaged by FLIM. The images were taken from the same cell before (bottom row) and following (top row) RFP (acceptor) photobleaching. Areas of the cell where the fluorescent acceptor was photobleached show an increase in the GFP lifetime of the donor compared to the nonbleached sample, confirming the fidelity of lifetime imaging to detect FRET.stograms  of tkRas(2.47 MB TIF)Click here for additional data file.Figure S10EM of Qdot-labelled EphA3. EM images of fixed APC-EphA3-expressing HEK293T cells without pretreatment (A) or following ephrin stimulation (B), showing plasma membrane disruption and endocytosis, respectively. Cells were fixed, permeabilized, and stained with SA-Qdots 605 prior to EM plastic embedding. Inserts show an enlarged section of the boxed (red) areas, using computer-assisted assignment to delineate the exact plasma membrane/cytoplasm boundary. Arrowheads mark EphA3-tethered Qdots on the inner cell membrane.(1.01 MB TIF)Click here for additional data file.Video S1EM tomographic reconstruction of Qdot-labelled EphA3-bearing cell membrane. EM tomography of a protrusion of APC-EphA3 [3YF]-transfected cells, where Qdots label the EphA3 COOH-terminus. The movie shows an iso-surface visualization of the 3D tomographic reconstruction displayed in (0.47 MB AVI)Click here for additional data file.


Caenorhabditis elegans CYB-3 has essential and distinct functions from cyclin B1 and B2 in the early embryo. CYB-3 is required for the timely execution of a number of cell cycle events including completion of the MII meiotic division of the oocyte nucleus, pronuclear migration, centrosome maturation, mitotic chromosome condensation and congression, and, most strikingly, progression through the metaphase-to-anaphase transition. Our experiments reveal that the extended metaphase delay in CYB-3–depleted embryos is dependent on an intact spindle assembly checkpoint (SAC) and results in salient defects in the architecture of holocentric metaphase chromosomes. Furthermore, genetically increasing or decreasing dynein activity results in the respective suppression or enhancement of CYB-3–dependent defects in cell cycle progression. Altogether, these data reveal that CYB-3 plays a unique, essential role in the cell cycle including promoting mitotic dynein functionality and alleviation of a SAC–dependent block in anaphase chromosome segregation.The master regulators of the cell cycle are cyclin-dependent kinases (Cdks), which influence the function of a myriad of proteins via phosphorylation. Mitotic Cdk1 is activated by A-type, as well as B1- and B2-type, cyclins. However, the role of a third, conserved cyclin B family member, cyclin B3, is less well defined. Here, we show that  Every time a cell divides in two, the genetic material, DNA, is copied; each copied chromosome is referred to as a pair of sister chromatids. Each chromatid must be cleanly separated from its sister so that each daughter cell inherits the same DNA complement as the starting cell. The mitotic spindle is a cellular machine that physically separates the sister chromatids from one another. The chromatids are attached to the spindle at kinetochores, which are structures built at specific sites (centromeres) on each chromatid. The cell monitors the attachment of each chromatid and blocks their separation until they are all properly attached. This process is called the spindle assembly checkpoint (SAC). Here we report that loss of an evolutionarily conserved cell cycle regulator, Cyclin B3/CYB-3, results in an unusual and strikingly persistent SAC–dependent delay in sister chromatid separation. Furthermore, CYB-3 promotes the activity of a cellular motor, dynein, in this and other mitotic processes. Altogether, our results indicate that Cyclin B3 genetically interacts with mitotic dynein and is absolutely required to satisfy a SAC–dependent inhibition in sister chromatid separation. The eukaryotic cell cycle is driven by the temporally controlled activation of cyclin-dependent kinases (CDKs) in association with their requisite cofactors, the cyclins C. elegans, have holocentric chromosomes with kinetochores along their entire length As cells prepare to enter mitosis, cyclin B/Cdk1 complexes phosphorylate a host of substrates leading to chromosome condensation, centrosome maturation, and nuclear envelope breakdown raison d'être for mitosis is the equal partitioning of replicated genetic material to each daughter cell. Hence, progression through mitosis is inextricably linked to the state of kinetochore-microtubule attachment. To be properly segregated, each pair of sister chromatids must be attached to the mitotic spindle in a bipolar fashion The i.e., all chromosomes are attached and under tension) It has become increasingly clear that once a cell engages a checkpoint such as the SAC, the checkpoint must be shut-off or silenced once the checkpoint is satisfied  were fed bacteria expressing cyb-3 dsRNA. This RNAi treatment resulted in efficient CYB-3 depletion (cyb-3(RNAi) oocytes underwent an apparently normal first meiotic division followed by extrusion of the first polar body at the anterior end of the embryo (cyb-3(RNAi) embryos, anaphase II did not occur. Sister chromatids failed to separate from one another and a second polar body was not extruded  embryos  from the anterior end   embryos , S5, S6. embryos , S6.C. elegans oocytes are devoid of centrioles and centrosomes cyb-3(RNAi) embryos were much smaller compared to controls. At the time of PNM, cyb-3(RNAi) centrosomes were approximately two-fold smaller than control centrosomes (cyb-3(RNAi) embryos; condensation of the paternal pronucleus was significantly delayed with respect to the maternal pronucleus .Asynchrony of pronuclear condensation is a feature of mutants that fail to undergo pronuclear migration control or cyb-3(RNAi) using specific mitotic landmarks as follows: Prophase: interval between pronuclear meeting  and nuclear envelope breakdown ; Prometaphase: interval between NEB and chromosome congression to the metaphase plate; Metaphase: interval between complete (or nearly complete) congression and the initiation of anaphase chromosome segregation; Anaphase: interval between the initiation of chromosome segregation and the beginning of chromosome decondensation; Telophase/mitotic exit: interval between the initiation of chromosome decondensation and centrosome breakdown.We quantified the duration of the first mitotic division in OD57 embryos treated with cyb-3(RNAi) embryos compared to control, and chromosome congression was often incomplete (cyb-3(RNAi) embryos had at least one chromosome that initially congressed to the metaphase plate but subsequently underwent movement towards the centrosome, followed by re-alignment in the majority of embryos (cyb-3(RNAi) mitotic spindles also had an abnormal appearance, with microtubule bundles appearing to be pinched at the centrosomes rather than the more spread out, straight microtubules of control spindles  (cyb-3(RNAi) embryos was defined as the continued alignment of chromosomes at the metaphase plate until centrosome breakdown (cyb-3(RNAi) embryos were disassembled only to reform, separate, and nucleate microtubules in the presence of aligned chromosomes  . The pinssembly) . Since areakdown . In thesreakdown . In addiomosomes . These rcyb-3(RNAi) embryos with two extruded polar bodies, which is indicative of complete MI and MII divisions. All of these embryos (n = 7) displayed metaphase delays comparable to cyb-3(RNAi) embryos with single polar bodies  embryos (data not shown). However, in sharp contrast to cyb-3(RNAi), cyb-1&2(RNAi) mitotic chromosomes did not align to a metaphase plate but still underwent anaphase  = 11; cyb-1&2(RNAi): avg.  = 124±20 seconds, n = 7), suggesting that there were no appreciable delays in prometaphase or the metaphase-to-anaphase transition. In conclusion, embryos depleted of CYB-3 exhibit a phenotype distinct from that caused by co-depletion of CYB-1 and CYB-2.CYB-3 is one of four B-type cyclins in le dsRNA . As receanaphase . Surpriscyb-3(RNAi) embryos was dependent on a functional SAC. Hence, OD57 embryos co-depleted of CeMad1/MDF-1 cyb-3 and mdf-1 dsRNA-expressing bacteria were diluted with control bacteria (see mdf-1+control(RNAi) did not result in any apparent defects in the timing or execution of mitosis as compared to control(RNAi) embryos (cyb-3+control(RNAi) embryos were indistinguishable from undiluted cyb-3(RNAi) (cyb-3(RNAi) embryos, since the duration of prometaphase in cyb-3+mdf-1(RNAi) embryos was shortened compared to cyb-3+control(RNAi); however, this interval remained lengthened as compared to control(RNAi) (cyb-3+mdf-1(RNAi) embryos entered anaphase after a brief metaphase delay, suggesting that the SAC is required for the prolonged metaphase in cyb-3(RNAi) embryos (Since the spindle assembly checkpoint (SAC) delays the metaphase-to-anaphase transition in the presence of unattached kinetochores or defective microtubule attachments, we asked whether the prolonged metaphase delay in eria see . Consist embryos  [48]. Th-3(RNAi) , S8. MDFol(RNAi) , S8, S10 embryos , S10. In embryos  also res embryos , S12.unc-46(e177); mdf-1(gk2) L4 hermaphrodite offspring (F1) from unc-46(e177);mdf-1(gk2) heterozygous mothers were fed control or cyb-3 dsRNA-expressing bacteria. unc-46(e177) is a recessive linked visible marker for homozygous mdf-1(gk2) animals gk2 is a strong loss-of-function deletion allele of mdf-1mdf-1(gk2) homozygotes are viable but display a low level of sterility (23%), while the majority of F2 mdf-1(gk2) progeny arrest as embryos or larvae unc-46(e177);mdf-1(gk2) or unc-46(e177) hermaphrodites were fixed, immunostained with kinetochore (CeBub1/BUB-1)cyb-3(RNAi);unc-46(e177) one-cell embryos were in mitotic metaphase and none in anaphase, while 50% of cyb-3(RNAi);unc-46(e177)mdf-1(gk2) one-cell embryos were in mitotic metaphase and 36% were in anaphase (cyb-3(RNAi) embryos and permits anaphase onset.To confirm these results, homozygous anaphase . These dsmc-4(RNAi) embryos, although highly defective with respect to chromosome condensation, do not display significant delays at any mitotic stage (cyb-3(RNAi) embryos is not a secondary consequence of chromosome condensation defects.Given that CYB-3-depleted embryos display chromosome condensation defects , mitoticic stage . These rC. elegans chromosomes are holocentric, providing a large centromere advantageous for studying changes in kinetochore structure and centromere resolution cyb-3(RNAi) embryos, we tested whether kinetochore architecture was also changed. Hence, control and cyb-3(RNAi) embryos were fixed and co-stained with antibodies recognizing two kinetochore proteins, CeCENP-F/HCP-1 cyb-3(RNAi) embryos had no clear BUB-1 or HCP-1 kinetochore stripes, and no BUB-1 or HCP-1 localization to metaphase K-Mts  Ndc80 (strain OD11) transgenic embryos se K-Mts . Rather,se K-Mts . ImmunosC. elegans kinetochore is built on centromeric chromatin containing the histone variant CENP-A (CeHCP-3) As in other organisms, the cyb-3(RNAi) embryos is dependent on the spindle assembly checkpoint, we determined whether depletion of SAC components affected kinetochore geometry. As above, the majority of one-cell embryos treated with cyb-3+control(RNAi) displayed “twisted” kinetochores  embryos in control and mdf-1+control(RNAi) embryos, no K-Mt BUB-1 localization was apparent in cyb-3(RNAi) or cyb-3+mdf-1(RNAi) embryos.Since the prolonged metaphase delay in tochores . The twiC. elegans is directly reflected by the timing and rate of spindle pole separation. C. elegans chromosomes do not undergo anaphase A movements cyb-3(RNAi) embryos was significantly greater than control embryos prior to NEB (cyb-3(RNAi) embryos is stabilized within 60 seconds after NEB at the same length (14.5 µm±0.73 (SEM); n = 6) as the metaphase-to-anaphase transition spindle in control embryos (15.7 µm±0.56 (SEM); n = 6; p = 0.2)(180 seconds post-NEB)(cyb-3(RNAi) spindles until the centrosomes of the former separated coincident with anaphase chromosome segregation (approximately 240 seconds post-NEB) (cyb-3(RNAi) spindles then stabilize at the same length as control metaphase spindles (180 seconds post-NEB), indicating that kinetochore-microtubule interactions reach levels that balance cortical pulling forces similarly to control spindles. This balance could also be achieved if kinetochore-microtubule interactions were compromised coincident with a diminution of cortical pulling forces. However, spindle pole separation and sister chromatid segregation in cyb-3+mdf-1(RNAi) embryos are not consistent with this latter model. While we cannot rule out the presence of underlying spindle abnormalities or assembly defects, these data reveal that CYB-3-depleted embryos are capable of generating at least grossly functional kinetochore-microtubule attachments.The quality of kinetochore-microtubule attachments in r to NEB , indicatr to NEB . Spindlepost-NEB). Spindleost-NEB) . Mitoticost-NEB) . Hence, ost-NEB) . cyb-3 treated with control or cyb-3(RNAi). As previously reported, GFP::MDF-2 localizes to prophase and prometaphase nuclei but is not apparent on metaphase kinetochores in control embryos (cyb-3(RNAi) embryos, GFP::MDF-2 accumulated on chromosomes beginning in prophase and remained on chromosomes throughout the prolonged metaphase in these cells  treated embryos (cyb-3(RNAi) embryos, consistent with persistent SAC signaling.A second hallmark of an engaged SAC is the accumulation of phospho-specific epitopes recognized by the 3F3/2 antibody e.g., attach all chromosomes) but also actively silence these checkpoints once the damage or defects have been repaired cyb-3(RNAi) embryos; hence, we examined dynein behavior in C. elegans embryos harboring a GFP-tagged dynein heavy chain (GFP::DHC-1) transgene (strain OD203)smc-4(RNAi) or mdf-1+control(RNAi) (cyb-3(RNAi) embryos, GFP::DHC-1 localized to the nuclear periphery and centrosomes during prophase and also accumulated at kinetochores during prometaphase and metaphase as in controls  , S25. Incontrols , S27. Stcontrols , S29. Imcontrols .cyb-3(RNAi) embryos could reflect a global defect in microtubule-associated proteins (MAPs) binding to K-Mts. However, the CeBimC/BMK-1 kinesin control and cyb-3(RNAi) embryos  embryos, leading to timely anaphase entry. A recent study in C. elegans revealed that specific dynein light chains negatively regulate the activity of the dynein heavy chain (DHC-1) dhc-1 allele cyb-3 dsRNA bacteria 20-fold with control or control+dylt-1 dsRNA bacteria and fed these mixtures to young adult OD57 hermaphrodites. Embryos treated with diluted cyb-3(RNAi)(20x dilution with control bacteria) experienced significant metaphase delays, but ultimately underwent anaphase chromosome segregation approximately 90 seconds after control embryos , this phenotype was rescued by concomitant loss of DYLT-1 , both before and after NEB  phenotypes. Hence we utilized a dhc-1(ts) allele to test this model. Embryos from dhc-1(ts) hermaphrodites reared at semi-permissive temperatures (22°C and 24°C) were fed cyb-3 dsRNA bacteria diluted 20x with control bacteria. Embryos were fixed after 24 hours on dsRNA bacteria and the number of one-cell embryos at different stages of mitosis was counted (dhc-1(ts) embryos treated with control(RNAi) or wild-type embryos treated with diluted cyb-3(RNAi). However, in dhc-1(ts) embryos treated with diluted cyb-3(RNAi), there was a significant increase in the number of prometaphase embryos and a concomitant decrease in the number of anaphase embryos (dhc-1(ts) embryos treated with control(RNAi) also showed a significant increase in the number of prometaphase embryos and a decrease in anaphase embryos  embryos and rescue of this phenotype by modulation of dynein activity suggest that centrosome attachment to the nuclear envelope is compromised in CYB-3-deficient embryos. Consequently, the shortening of the post-NEB centrosome-centrosome distance in cyb-3;dylt-1(RNAi) compared to cyb-3(RNAi) embryos may be a secondary consequence of stronger centrosome-nuclear envelope attachments and thus abrogation of pre-NEB separation. Alternatively, it may reflect the more timely formation of, or more robust, kinetochore-microtubule attachments than in CYB-3-depleted embryos. Unfortunately, our data neither allow us to distinguish between these two possibilities nor address whether modulating dynein activity also affected CYB-3-dependent prophase events since diluted cyb-3(RNAi) embryos did not display consistent defects in early mitosis. Experiments to titrate the amount of CYB-3 and dynein activity required for different mitotic events are underway.Given that other cyclins have a variety of targets, it is not surprising that CYB-3 affects a number of different cellular events. A commonality of many of these processes is that they are dynein-dependent. The significantly slower migration rate of the female pronucleus and the failure of the male pronucleus to move from the embryo posterior in CYB-3-depleted embryos are strikingly similar to the pronuclear defects of C. elegans embryo. Several groups have reported that the C. elegans SAC is relatively weak and can only mount, at most, a two-to-three fold delay in the metaphase-to-anaphase transition under all conditions tested, including severe spindle damage after nocodazole exposure Although loss of CYB-3 affects a number of processes, the most striking abnormality is the persistent SAC-dependent delay in the initiation of anaphase chromosome segregation. To our knowledge, this is an unprecedented phenotype in the early cyb-3(RNAi) embryos reaches and is maintained at the same length as control spindles at the metaphase-to-anaphase transition. Furthermore, when the metaphase delay is abrogated by loss of the SAC or DYLT-1, twisting is not apparent and sister chromatid separation readily occurs, suggesting that kinetochore-microtubule attachments are made and are at least partially functional. Hence, although CYB-3 loss may very well lead to spindle assembly defects that engage the SAC, the unusual persistence of SAC signaling in this circumstance and not in embryos with qualitatively more severe spindle and chromosome segregation defects is not easily reconciled.Loss of CYB-3 leads to gross defects in kinetochore and kinetochore-microtubule architecture. We posit that the twisted centromeres and kinetochores are the result of multiple cycles of microtubule attachment and detachment occurring during the metaphase delay. Microtubule attachment appears to play a role since prophase kinetochores are not affected by CYB-3 depletion and the twisting correlates with increased AIR-2/Aurora B activity , which is congruent with increased kinetochore-microtubule turnover. This twisting is phenotypically distinct from that seen in embryos depleted of the condensin proteins SMC-4 and HCP-6 C. elegans, and Drosophilavis-à-vis SAC silencing. Furthermore, a role for CYB-3 in SAC silencing is more easily reconciled with the rare and unusually persistent metaphase delay in CYB-3-depleted embryos. While a plethora of proteins are required for spindle assembly C. elegans embryo, many fewer would be necessary to turn off the SAC and allow cell cycle progression. It will be very interesting to determine whether proteins implicated in SAC silencing in other systems, such as the phosphatase PP1 C. elegans embryo.A second potential mechanism, and one we favor, is that among other essential mitotic functions, CYB-3 is required to inactivate or silence the SAC. Our data are consistent with a model whereby CYB-3 participates in SAC silencing by either directly or indirectly affecting the ability of dynein to strip SAC components from kinetochores. The dynein motor has been implicated in SAC silencing in mammals, cyb-3 loss-of-function phenotype is that despite a complete inhibition of sister chromatid separation and chromosome decondensation, centrosomes breakdown and duplicate with the same timing as mitotic centrosomes in wild-type cells . Engagement of the SAC should inhibit all aspects of cell cycle progression. Strikingly, an uncoupling of the nuclear and centrosome cell cycle occurred upon depletion of Drosophila mitotic cyclins One puzzling aspect of the C. elegans CYB-3 associates with CDK-1 in vitro, and CYB-3 complexes display H1 kinase activity; H1 is commonly used as a Cdk1 substrate C. elegans ortholog, DLI-1. Furthermore, unlike CYB-3 and DHC-1, DLI-1 does not appear to be required for the MII division of the oocyte nucleus C. elegans are directly phosphorylated by CYB-3/CDK-1 and the functional consequence of these phosphorylation events on mitotic progression are important investigations for the future.Altogether, our data demonstrate that CYB-3 plays a distinct, non-redundant role in mitosis by influencing dynein-dependent mitotic processes. That CYB-3 depletion does not mirror all dynein/DHC-1 loss-of-function phenotypes may reflect a requirement for CYB-3 in some dynein-related processes but not others, or may indicate that different processes require varying doses of dynein activity. This hypothesis is supported by the isolation of hypomorphic dynein alleles that display a range and severity of defects C. elegans strains were maintained at 15°C–25°C C. elegans wild type, DR subclone of CB original (Tc1 pattern I)) unc-119(ed3); ltIs37 [pAA64: pie-1p::mCherry::his-58+ unc-119 (+)]; ltIs25 ) unc-46(e177) V) unc-46(e177)mdf-1(gk2) V/nT1[let-X] IV;V) unc-119(ed3) III; ltIs52 ; ltIs37  IV)unc-119(ed3) III; orls17 ; ltIs37  IV)unc-119(ed3) III; ltIs7 /+)unc-119(ed3) III; ruIs32  III; ddIs6 V) dhc-1(or195) I) pie-1/GFP::hcp-3 and pie-1/mCherry::his-58 transgenes were isolated.cyb-3, mdf-1, san-1, bub-1, smc-4, knl-1, and dylt-1 were obtained from the Geneservice Ltd. C. elegans feeding library control). To deplete CYB-3 alone, a three ml LB + 100 µg/µl ampicillin liquid culture was seeded with a single colony of HT115 bacteria transformed with the cyb-3(RNAi) L4440 plasmid and shaken overnight (O/N) at 37°C. The next day, the O/N culture was expanded to 50 ml with the same media and grown until the OD600 of the culture was between 0.6–0.8 (∼ two hours). IPTG was added to a final concentration of 1 mM and the culture was grown an additional three hours at 37°C to induce cyb-3 dsRNA expression. The culture was then centrifuged at 5000 rpm for 10 minutes, the pellet was resuspended in 800 µl LB, and 200 µl of the suspension plated on nematode growth (NG) media containing 100 µg/µl ampicillin and three mM ITPG (NG/AMP/IPTG). Plates were incubated at 37°C O/N and then seeded with L4 larvae. Seeded plates were incubated at 25°C O/N and embryos from the young adult worms (L4+24 hours) were utilized for experiments.RNAi plasmids for i.e., cyb-3 and mdf-1 dsRNA-expressing bacteria) were thoroughly mixed and transferred to NG/AMP/IPTG plates, incubated at 37°C O/N, and then seeded with L4 larvae. To generate highly dilute cyb-3(RNAi) conditions for dylt-1 and dhc-1(ts) experiments, control and cyb-3 dsRNA expressing bacteria were induced, pelleted, and resuspended as above. 10 µl cyb-3(RNAi) bacteria were thoroughly mixed with 190 µl control or dylt-1(RNAi) bacteria in a 15 ml conical tube and briefly centrifuged at low speed. The pellet was resuspended in the supernatant and plated as above.To co-deplete CYB-3 and MDF-1, SAN-1, or BUB-1, the induction conditions were as described above. However, after resuspension of the pellets in 800 µl LB, 200 µl of each suspension (cyb-1&2(RNAi) experiments, sense and anti-sense mRNAs corresponding to ZC168.4 (CYB-1) were transcribed from linearized templates using a T7 in vitro transcription kit . Complementary RNAs were mixed, heated at 90°C for five minutes, and annealed at room temperature (RT). cyb-3 dsRNA was also generated in this manner for direct comparison of injected animals. dsRNAs were injected into the gonads of OD57 L4 larvae and the injected animals incubated at 25°C O/N.For Embryos from adult hermaphrodites were fixed and stained as previously described Immunofluorescent images were acquired on a Nikon 2000U inverted microscope equipped with a Photometrics Coolsnap HQ camera. Metamorph software was used for image acquisition. Z-sections were acquired at 0.2 µm steps using a 60X/1.49 NA objective. Z-stacks were projected and deconvolved for 10 iterations using Autodeblur . Images were processed for figures using Adobe Photoshop.http://rsbweb.nih.gov/ij) was used to measure centrosome size, centrosome-centrosome distance, and pronuclear migration rates.For live imaging, embryos cut from RNAi-treated hermaphrodites (24 hours post-RNAi exposure) were mounted on 2% agarose pads and imaged using a spinning disk confocal  attached to a Nikon TE2000U inverted microscope. Images were acquired using an ORCA-ER digital camera  and a 60×1.45 NA Plan Apo VC lens. Ultraview software (Perkin Elmer) was used to control the confocal, microscope, and camera. Images were captured at 30 second intervals; Z-sections were 1 µm. For condensation assays, condensation of male pronucleui in TH32 RNAi-treated embryos were imaged and the condensation parameter calculated as previously described Figure S1cyb-3(RNAi) efficiency and an alignment of C. elegans B-type cyclins. A) Protein extracts from control and cyb-3(RNAi) embryos were immunoprecipitated with a CYB-3 antibody and subjected to western analysis with the same antibody. α-tubulin was used as a loading control. Asterisk: non-specific protein band. B) A Clustal-W alignment of approximately 1000 nucleotides from the N-terminal protein coding region of cyb-1, cyb-2.1, cyb-2.2, and cyb-3 cDNAs. The percent identity among the four C. elegans B-type cyclins is listed in the table below.(0.87 MB TIF)Click here for additional data file.Figure S2Mitotic chromosome condensation is modestly delayed in CYB-3 depleted embryosSelected images of the male pronucleus from TH32 embryos treated with the indicated RNAi are shown. Time 0:00 =  NEB, intervals are 30 seconds. Images are flattened from five 1 µm optical slices.(0.73 MB TIF)Click here for additional data file.Figure S3C. elegans Cyclin B proteins leads to distinct mitotic defects. A) Embryos from OD57 hermaphrodites microinjected with control, cyb-3, and cyb-1&2 double-stranded RNA were subjected to live imaging. Time 0:00 corresponds to NEB. Frames to the right of 0:00 depict mitotic progression in minutes after NEB. PNM: Pronuclear meeting; NEB: nuclear envelope breakdown. Scale bar: 10 µm. B) Mitotic progression in control and cyb-3(RNAi) treated OD57 embryos undergoing the first mitotic division. Error bars: SEM, n = 4 embryos for each condition. Metaphase*: metaphase in cyb-3(RNAi) embryos was defined as the interval between near complete chromosome alignment and centrosome breakdown.Depletion of different (1.21 MB TIF)Click here for additional data file.Figure S4cyb-3(RNAi) embryosCompletion of meiosis does not affect the metaphase delay in control or cyb-3(RNAi) were subjected to live imaging. 0:00 =  NEB. Arrows point to two extruded polar bodies indicating that the MI and MII divisions were complete. Scale bar: 10 µm.OD57 embryos treated with (1.21 MB TIF)Click here for additional data file.Figure S5C. elegans chromosomes is increased upon taxol exposure3F3/2 immunostaining of Wild-type embryos treated with vehicle or taxol were fixed and stained as described in (0.45 MB TIF)Click here for additional data file.Figure S6DNC-1/p150(glued) is sequestered at chromosomes in CYB-3 depleted cellsControl and cyb-3(RNAi) embryos were fixed and stained with DAPI (blue) and antibodies recognizing α-tubulin (green) and DNC-1 (red). Arrows: DNC-1 centrosome staining in control embryos that is decreased upon CYB-3 depletion. Arrowhead: centrosome breakdown. Scale bar: 10 µm.(3.28 MB TIF)Click here for additional data file.Figure S7cyb-3(RNAi) K-Mts are accessible to microtubule-associated proteinsControl and cyb-3(RNAi) embryos were fixed and stained with DAPI (blue) and antibodies recognizing α-tubulin (green) and BMK-1 (red). Scale bar: 10 µm.(1.38 MB TIF)Click here for additional data file.Text S1Supplemental (0.04 MB DOC)Click here for additional data file.Video S1control(RNAi) oocyte. A fertilized oocyte from a control(RNAi)-treated OD57 hermaphrodite .Meiotic divisions in a fertilized (1.29 MB MOV)Click here for additional data file.Video S2cyb-3(RNAi) oocyte. A fertilized oocyte from a cyb-3(RNAi)-treated OD57 hermaphrodite .Meiotic divisions in a fertilized (2.10 MB MOV)Click here for additional data file.Video S3control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated TH32 hermaphrodite .Pronuclear migration in a (1.83 MB MOV)Click here for additional data file.Video S4cyb-3(RNAi) embryo. A one-cell embryo from a cyb-3(RNAi)-treated TH32 hermaphrodite .Pronuclear migration in a (1.31 MB MOV)Click here for additional data file.Video S5control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.75 MB MOV)Click here for additional data file.Video S6cyb-3(RNAi) embryo. A one-cell embryo from a cyb-3(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.56 MB MOV)Click here for additional data file.Video S7cyb-1&2(RNAi) embryo. A one-cell embryo from a cyb-1&2(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.48 MB MOV)Click here for additional data file.Video S8cyb-3+control(RNAi) embryo. A one-cell embryo from a cyb-3+control(RNAi)-treated OD57 hermaphrodite .First mitosis in a (1.08 MB MOV)Click here for additional data file.Video S9mdf-1+control(RNAi) embryo. A one-cell embryo from a mdf-1+control(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.47 MB MOV)Click here for additional data file.Video S10cyb-3+mdf-1(RNAi) embryo. A one-cell embryo from a cyb-3+mdf-1(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.56 MB MOV)Click here for additional data file.Video S11cyb-3+san-1(RNAi) embryo. A one-cell embryo from a cyb-3+san-1(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.84 MB MOV)Click here for additional data file.Video S12cyb-3+bub-1(RNAi) embryo. A one-cell embryo from a cyb-3+bub-1(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.24 MB MOV)Click here for additional data file.Video S13smc-4(RNAi) embryo. A one-cell embryo from a smc-4(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.43 MB MOV)Click here for additional data file.Video S14control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated OD110 hermaphrodite. Only the GFP channel is shown. .GFP::MDF-2 dynamics in a (0.21 MB MOV)Click here for additional data file.Video S15control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated OD110 hermaphrodite. Both the GFP and mCherry signals are shown .GFP::MDF-2 and chromosome dynamics in a (0.20 MB MOV)Click here for additional data file.Video S16cyb-3(RNAi) embryo. A one-cell embryo from a cyb-3(RNAi)-treated OD110 hermaphrodite. Only the GFP channel is shown. .GFP::MDF-2 dynamics in a (0.18 MB MOV)Click here for additional data file.Video S17cyb-3(RNAi) embryo. A one-cell embryo from a cyb-3(RNAi)-treated OD110 hermaphrodite. Both the GFP and mCherry signals are shown .GFP::MDF-2 and chromosome dynamics in a (0.17 MB MOV)Click here for additional data file.Video S18smc-4(RNAi) embryo. A one-cell embryo from a smc-4(RNAi)-treated OD110 hermaphrodite. Only the GFP channel is shown. .GFP::MDF-2 dynamics in a (0.25 MB MOV)Click here for additional data file.Video S19smc-4(RNAi) embryo. A one-cell embryo from a smc-4(RNAi)-treated OD110 hermaphrodite. Both the GFP and mCherry signals are shown .GFP::MDF-2 and chromosome dynamics in a (0.25 MB MOV)Click here for additional data file.Video S20control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 dynamics in a (0.46 MB MOV)Click here for additional data file.Video S21control(RNAi) embryo. A one-cell embryo from a control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 and chromosome dynamics in a (0.45 MB MOV)Click here for additional data file.Video S22smc-4(RNAi) embryo. A one-cell embryo from a smc-4(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 dynamics in a (0.49 MB MOV)Click here for additional data file.Video S23smc-4(RNAi) embryo. A one-cell embryo from a smc-4(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 and chromosome dynamics in a (0.51 MB MOV)Click here for additional data file.Video S24mdf-1+control(RNAi) embryo. A one-cell embryo from a mdf-1+control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 dynamics in a (0.16 MB MOV)Click here for additional data file.Video S25mdf-1+control(RNAi) embryo. A one-cell embryo from a mdf-1+control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 and chromosome dynamics in a (0.17 MB MOV)Click here for additional data file.Video S26cyb-3+control(RNAi) embryo. A one-cell embryo from a cyb-3+control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 dynamics in a (0.51 MB MOV)Click here for additional data file.Video S27cyb-3+control(RNAi) embryo. A one-cell embryo from a cyb-3+control(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 and chromosome dynamics in a (0.49 MB MOV)Click here for additional data file.Video S28cyb-3+mdf-1(RNAi) embryo. A one-cell embryo from a cyb-3+mdf-1(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 dynamics in a (0.86 MB MOV)Click here for additional data file.Video S29cyb-3+mdf-1(RNAi) embryo. A one-cell embryo from a cyb-3+mdf-1(RNAi)-treated OD203 hermaphrodite .GFP::DHC-1 and chromosome dynamics in a (0.84 MB MOV)Click here for additional data file.Video S30dilute cyb-3(RNAi) embryo. A one-cell embryo from a dilute cyb-3(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.32 MB MOV)Click here for additional data file.Video S31dilute cyb-3+dylt-1(RNAi) embryo. A one-cell embryo from a dilute cyb-3+dylt-1(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.30 MB MOV)Click here for additional data file.Video S32dylt-1(RNAi) embryo. A one-cell embryo from a dylt-1(RNAi)-treated OD57 hermaphrodite .First mitosis in a (0.44 MB MOV)Click here for additional data file.


Caenorhabditis elegans is inherently attracted to specific odors that are sensed by the paired AWC olfactory sensory neurons. The attraction diminishes if the animal experiences these odors for a prolonged period of time in the absence of food. The AWC neuron responds acutely to odor-exposure by closing calcium channels. While odortaxis requires a Gα subunit protein, cGMP-gated channels, and guanylyl cyclases, adaptation to prolonged odor exposure requires nuclear entry of the cGMP-dependent protein kinase, EGL-4. We asked which candidate members of the olfactory signal transduction pathway promote nuclear entry of EGL-4 and which molecules might induce long-term adaptation downstream of EGL-4 nuclear entry. We found that initiation of long-term adaptation, as assessed by nuclear entry of EGL-4, is dependent on G-protein mediated signaling but is independent of fluxes in calcium levels. We show that long-term adaptation requires polyunsaturated fatty acids (PUFAs) that may act on the transient receptor potential (TRP) channel type V OSM-9 downstream of EGL-4 nuclear entry. We also present evidence that high diacylglycerol (DAG) levels block long-term adaptation without affecting EGL-4 nuclear entry. Our analysis provides a model for the process of long-term adaptation that occurs within the AWC neuron of C. elegans: G-protein signaling initiates long-lasting olfactory adaptation by promoting the nuclear entry of EGL-4, and once EGL-4 has entered the nucleus, processes such as PUFA activation of the TRP channel OSM-9 may dampen the output of the AWC neuron.While most sensory neurons will adapt to prolonged stimulation by down-regulating their responsiveness to the signal, it is not clear which events initiate long-lasting sensory adaptation. Likewise, we are just beginning to understand how the physiology of the adapted cell is altered.  Caenorhabditis elegans is capable of sensing a variety of attractive volatile compounds. These odors are the worm's “best guesses” as to how to track down food. Employing calculated approximations underlies a foraging strategy that is open to failure. When C. elegans track an odor which proves unrewarding, they must modify their behavior based on this experience. They also need to prevent over-stimulating their neurons. To accomplish this, C. elegans olfactory sensory neurons adapt to odors after a sustained exposure to odor in the absence of food. Within the pair of primary odor-sensory neurons, termed the AWCs, adaptation requires the cGMP-dependent protein kinase G (PKG), EGL-4. Exposing animals to AWC–sensed odors for approximately 60 minutes results in a long-lasting (∼3 hour) adaptation that requires the nuclear translocation of EGL-4. To understand how sensory transduction and desensitization machinery converge to achieve olfactory adaptation, we asked whether odor-induced EGL-4 nuclear accumulation was affected by gene mutations that abrogate either odor sensation of or adaptation to AWC–sensed odors. We find that G-protein signaling represents the integration point where primary odor sensation and odor adaptation pathways diverge. PUFA signaling, calcium, and decreased diacylglycerol all dampen the response of the AWC neuron to odor downstream of this divergence. C. elegans. Since the sources of many odors that are inherently attractive to C. elegans do not, in fact, provide a source of nutrition, the worm may have to discriminate between rewarding and unrewarding stimuli by adapting to nutritionally profitless odors. This modification in olfactory behavior, as a function of experience, represents a finely regulated signaling circuit Olfactory adaptation may subserve a food-seeking strategy in The AWC cilia contain olfactory receptors that enable the nematode to sense inherently attractive volatile odors C. elegans by Colbert and Bargmann adp-1(ky20), which fails to adapt to the AWC sensed odors benzaldehyde and butanone, and the TRPV channel mutant osm-9(ky10), which is defective in adaptation to the AWC sensed odors butanone and isoamyl alcohol. The Bargmann group and other laboratories have since identified additional mutants that are unable to adapt to AWC-sensed odors; these mutants include: the arrestin arr-1tbx-2tax-6, which actually down regulates adaptation oα/Gqα DAG signaling mutants The genetics of olfactory adaptation were first investigated in Examination of the temporal sequence of events leading up to long-term adaptation revealed that it occurs in at least two phases: an initial “early” adaptation within the first 10 minutes of exposure To determine which step along the olfactory signal transduction pathway is integrated over time to evoke long-term adaptation, we examined olfactory behavior as well as nuclear accumulation of a GFP-tagged form of EGL-4 (GFP::EGL-4)  in genetSignal transduction in the primary sensory neuron AWC begins at the most distal portion of the dendrite, in the sensory cilia (arrowhead in che-2(e1033) and che-11(e1810)Chlamydomonas IFT complex A protein C. eleganse1033 allele of che-2 is a predicted null p)odr-1::RFP, which expresses soluble RFP throughout AWC (che-2(e1033) and che-11(e1810) mutants displayed AWC cilia defects in nearly all animals examined (che-2(e1033) or naïve che-11(e1810) mutant animals  – a regulator of G protein signaling protein tax-4(p678) – a cyclic nucleotide gated channel alpha subunit tax-2(p671) – a cyclic nucleotide gated channel beta subunit che-2(e1033) and che-11(e1810) mutants (che-2(e1033), che-11(e1810), rgs-3(vs19), tax-2(p671) or tax-4(p678) mutant worms. We found a strong correlation  between the penetrance of the cilia defects and the incidence of nuclear GFP::EGL-4, regardless of the genotype  allele - n2150 allele we found it exhibited wildtype subcellular localization of GFP::EGL-4 in naive animals  and the che-2(e1033), che-11(e1810), rgs-3(vs19), tax-2(p678) and tax-4(p671) strains we examined (odr-3(n2150), however, were intact and the cilia resembled the more condensed fork-shaped cilia of AWB (odr-1(n1936) mutant strain has visually intact AWC cilia odr-1 mutant worms are non-functional, indeed, Mukhopadhyay et al. odr-1(n1936) mutant worms were not like those of their well-fed wildtype counterparts; they more closely resembled the cilia of starved animals. This raises the possibility that the AWC cilia are also defective in odr-1 mutants, perhaps in a way that specifically affects EGL-4 cytoplsamic localization. On the whole, the data suggest that intact cilia/dendrite morphology and perhaps function is required to keep EGL-4 in the AWC cytoplasm of the naïve animal.There are certain exceptions to these findings. The Gα signaling  animals . This maexamined . We obsea of AWB . Indeed,To better understand how odor-exposure adapts AWC-mediated chemotaxis, we asked first how the initial events of sensory signaling might affect odor-adaptation and GFP::EGL-4 nuclear entry. Since AWC-mediated chemotaxis represents the behavioral output from a G-protein coupled receptor (GPCR) initiated signaling pathway  we firstgrk). This phosphorylation leads to recruitment of cytosolic arrestin, which then leads to desensitization of the GPCR by disallowing further Gα binding odr-3(n2150)gpb-2(sa603) and gpb-2(pk751)gpc-1(pk298)rgs-3(vs19)arr-1(ok401)grk-2(rt97)odr-3(n2150) and rgs-3(vs19) are shown in Stimulation of a GPCR causes the Gα subunit to dissociate from the β and γ subunits to initiate second messenger cascades often by activating an adenylyl cyclase to increase or a phosphodiesterase to decrease second messenger levels odr-3(n2150) mutant strain. 80 minute exposure to benzaldehyde failed to induce nuclear accumulation of EGL-4 (odr-3 mutant (P = 0.09) or naïve wildtype worms  mutant strain is only mildly defective in chemotaxis to butanone and so could be examined for its ability to adapt to this odor. We found that this odr-3 mutant strain was unable to adapt to butanone  and two known genes encoding the Gγ subunits (gpc-1 and gpc-2) in the C. elegans genome. Both gpc-2 and gpb-1 mutants are lethal and could not be assayed. The GPB-2 β subunit was previously shown to be required for adaptation to the odor benzaldehyde and we confirmed this result using another predicted null allele, gpb-2(sa603) (gpb-2(sa603) animals with nuclear GFP::EGL-4 over that of the naïve population . Though this is significantly different from the percent of wildtype animals that exhibited nuclear GFP::EGL-4 , the fact that we saw a significant increase with odor exposure encouraged us to examine the deletion allele of gpb-2, pk751gpb-2(pk751) did not affect odor-induced nuclear accumulation of GFP::EGL-4 (gpc-1 γ subunit mutant was neither defective for this long-term adaptation nor defective for odor-induced nuclear accumulation of GFP::EGL-4 (We next examined the contribution of β and γ subunits of the G-protein trimer to odor-induced GFP::EGL-4 nuclear accumulation. There are two known genes encoding the Gβ subunits (2(sa603) . When weP::EGL-4 . This isC. elegans arrestin, ARR-1 rgs-3(vs19) mutants also exhibited reduced chemotaxis to either benzaldehyde or butanone (rgs-3(vs19) mutant animals exhibited nuclear GFP::EGL-4 . Prolonged odor-exposure was able to further increase the percentage of rgs-3 animals that exhibited nuclear GFP::EGL-4  mutant strain was unable to adapt to either butanone or benzaldehyde in our hands as well (data not shown), GFP::EGL-4 accumulated within the nuclei of the same percentage of an arr-1 mutant population of animals as in a wildtype population  or isoamyl alcohol (1∶1000 dilution) . Likewise, after prolonged exposure to isoamyl alcohol dispersed in air, 75% of grk-2 and 92% of wildtype animals exhibited nuclear GFP::EGL-4 (p = 0.012). Thus, grk-2 animals would seem to have a lower sensitivity than wildtype animals to prolonged  . These milution) . We did,e strain . That isrolonged , as wellrolonged , exposurThe next step in the signal transduction pathway is regulation of the second messenger, which, in AWC, is thought to be cGMP. Since the calcium current is likely to be mediated by opening of the cGMP-gated TAX-2/4 channel Another approach to interfere with the flux of cGMP is to overexpress a cGMP-producing guanylyl cyclase. At least two guanylyl cyclases, DAF-11 and ODR-1, are known to be expressed in AWC and loss of either cyclase renders the animal unable to seek AWC-sensed odors tax-2/4 mutant animals was correlated with ciliopathies in the same way that was observed for rgs-3 . This suggests that odor was still able to induce nuclear accumulation of GFP::EGL-4 even in the absence of TAX-2/TAX-4 mediated calcium fluctuations. To ask whether the nuclear accumulation of EGL-4 might be a direct consequence of decreased calcium, we incubated GFP::EGL-4 expressing animals with odor and the calcium chelator, ethylene glycol tetraacetic acid (EGTA). We found that though EGTA was effective enough to blunt adaptation of the chemotaxis response in wildtype animals (p = 0.101 for buffer versus buffer plus odor pre-exposure after EGTA treatment), it was unable to induce nuclear accumulation of EGL-4 in odor-naïve animals or perturb the nuclear accumulation of EGL-4 in odor-exposed animals (p<0.005). Taken together, our analysis of mutants defective in calcium signaling  mutant background, we found that though these animals failed to adapt to the odor butanone  mutants are so severely defective for adaptation (osm-9(ky10);egl-4(n479) strain. Therefore, we examined the role of the possible regulators of TRP channel function, the Polyunsaturated Fatty Acids (PUFAs).Though the TRPV channel OSM-9 was shown previously to be required for adaptation to butanone , its rolbutanone , they diaptation  that onefat-3 mutant accumulates the 18 carbon linoleic acid fat-4(wa14) fat-1(wa9) double mutant animals and fat-3(wa22) single mutant animals displayed defects in adaptation of AWC-mediated responses (Since PUFAs were shown to function through OSM-9 in nociceptive neurons esponses .(p)ceh-36::FAT-3::GFP see fat-3(wa22) mutant background. This transgenic line was able to adapt to benzaldehyde odor exposure significantly better than the parental fat-3(wa22) mutant strain . This indicates that FAT-3 is likely to act within the AWC neurons to promote adaptation.To investigate whether PUFA signaling is required in the AWC neuron itself, we expressed FAT-3 tagged with GFP under the control of an AWC-specific promoter  and fat-4(wa14) fat-1(wa9) mutant animals with the 20-carbon PUFA eicosapentaenoic acid (EPA) was able to restore chemosensory behavior. To ask whether the adaptation defects of the desaturase defective mutants could be restored by such dietary supplementation either during their development or in adulthood immediately prior to odor-exposure, we prepared Nematode growth media (NGM) plates containing 160ìM EPA, seeded with OP50 E. coli, then grew C. elegans on these plates prior to collection for assays. By supplementing their diet with EPA throughout development, we were able to rescue the adaptation behavioral defects of fat-3 and fat-4 fat-1 animals . Though EPA treatment did not rescue the mutant strain's adapted C.I. back to that of wildtype , the EPA treatment was as effective in the adult as if the animals had been exposed to EPA for their entire lives . These results indicate that the PUFA EPA is required dynamically in adult animals for adaptation of the AWC-mediated olfactory response. Consistent with this finding, expression from the GPCR, STR-2 promoter which is down stream of EGL-4 activity fat-3 or fat-4 fat-1 mutants  and fat-4(wa14) fat-1(wa9) mutant animals displayed normal nuclear localization of EGL-4 after prolonged odor exposure  mutant strain. When we supplemented the diets of fat-3(wa22), fat-4(wa14) fat-1(wa9) and osm-9(ky10) mutants animals with EPA 24 hours prior to assaying adaptation responses, we found that EPA supplementation could rescue the butanone adaptation defect of fat-3 and fat-4 fat-1 mutant animals but could not rescue the adaptation defect of osm-9 mutant animals (p = 0.667 for odor-exposed osm-9 populations with and without EPA treatment). This result suggests that PUFAs might signal through the TRPV channel OSM-9 to promote butanone adaptation.To determine if PUFA signaling requires the TRPV channel OSM-9 to promote adaptation, we asked whether dietary supplementation with EPA could rescue the adaptation defects of an egl-4 null animals would relieve their adaptation defects. Khan-Kirby et al., egl-4 null strain  animals as a transgene. Strains expressing the NLS::GFP::EGL-4 transgene displayed GFP::EGL-4 in the AWC nuclei of both naïve and odor-exposed animals (egl-4(n479) genetic background (crh-1(tz2)crh-1 = 0.09, p = 0.137). When we examined the behavior of fat-3 mutant animals that expressed this transgene, however, we found that they showed significantly higher chemotaxis indexes than their wild-type siblings that expressed NLS::GFP::EGL-4 (fat-3(wa22) was 0.40, p = 0.005). In each case, the transgenic array was introduced by mating into the mutant background and wildtype siblings were used to derive the wildtype control lines. Thus, PUFAs are likely to function downstream of EGL-4's nuclear accumulation after prolonged odor exposure.Another way to assess whether PUFA signaling might be downstream of EGL-4 nuclear entry is to ask whether loss of PUFA signaling can suppress the gain-of-function phenotype of a constitutively nuclear form of EGL-4. In Lee  animals . The NLSckground . One exposm-9(ky10) null mutation did suppress the chemotaxis defects of the NLS::GFP::EGL-4 expressing transgenic animals (osm-9(ky10) was 0.72, p = 0.014). The extent of the rescue was greater than that of the fat-3 loss-of-function in the same context. That is, osm-9 loss rescued the C.I. back to 100% of the wildtype and osm-9 values , while fat-3 loss increased the C.I. to up to 65% of the fat-3 alone .Finally, as evidence points to the TRP channel OSM-9 acting downstream of EGL-4 nuclear entry  qα protein, EGL-30 and decreased by the Goα GOA-1 egl-30(js126) stimulates the phospholipase C, EGL-8, to increase DAG levels egl-8(n488) and egl-8(ok934)mutants, which lack the phospholipase C beta homolog and, therefore, the ability to make DAG egl-30(n686) that would decrease DAG levels and egl-30(js126) gain-of-function mutants that would increase DAG levels; the GOA-1 specific RGS, egl-10 mutants which should have more active GOA-1 and thus lower DAG; and in the pkc-1(ok563) mutants which should have lower dense core vesicle release. We found that GFP::EGL-4 nuclear accumulation was not affected by any of these mutations  could affect EGL-4 nuclear accumulation. As was previously described (Matsuki egl-4 (egl-30(js126) mutant background that is proposed to have high DAG levels within the AWC neuron egl-30(js126) mutation did indeed suppress the chemotaxis defects of NLS::GFP::EGL-4 animals (egl-30(js126) = 0.6, p = 0.003). This implies that high DAG may act downstream of EGL-4's nuclear entry to block adaptation and thus, the ability of the cell, perhaps via EGL-4, to regulate DAG levels is important for appropriate down-regulation of chemotaxis in response to odor stimulation. It is also possible that the high DAG levels produced in the egl-30 gain-of-function block adaptation by another process that is independent of, rather than down stream of nuclear EGL-4.To test the hypothesis that DAG acts downstream of nuclear EGL-4, we asked whether the chemotaxis defects of the constitutively nuclear allele of egl-4  could beThe mechanisms of AWC olfaction and vertebrate phototransduction appear to be quite similar. In the absence of light, photoreceptor cells exhibit an inward “dark current” of calcium through cGMP-gated channels. Upon absorbing a photon, the receptor rhodopsin becomes enzymatically active and catalyzes the activation of the G-protein, transducin, which activates a phosphodiesterase (PDE). The PDE then hydrolyzes cGMP causing the closure of the cGMP-gated channels resulting in a decrease in calcium influx The rapid adaptation of the vertebrate visual response is primarily regulated by the dampening of the Gα by the regulator of G protein signaling RGS9 After 30 minutes of exposure to odor, the attractiveness of the odor, as measured by the chemotaxis index, (CI) falls to about 75% of the initial value and, as the exposure time is lengthened, the odor's attractiveness is further diminished so that by 80 minutes of exposure, the odor may be completely ignored (the CI is close to 0). The short-term adapted state is labile in that it can be reversed after only 30 minutes of recovery in the absence of odor Using odor-induced nuclear accumulation as a cell-specific biological tool to probe this one part in the process of olfactory adaptation, we examined players in the pathways that are required for generating the odor signal such as: the G-protein and its modulators; the guanylyl cyclase; the calcium channels and calcium based modulators; the PUFA pathway and finally the diacylglycerol pathway that may be involved in synaptic transmission. From this analysis, the Gα protein ODR-3 emerged as the only signal transduction molecule that was required to integrate odor signaling over time to allow adaptation of AWC-mediated olfactory responses . This isC. elegansfat-3 and fat-1 fat-4 mutants are defective for their ability to adapt to benzaldehyde while the osm-9 mutant is able to adapt to benzaldehyde like wildtype. Loss of either PUFA signaling or OSM-9 was able to suppress the gain-of-function EGL-4 allele (NLS::GFP::EGL-4). This indicates that PUFA signaling via OSM-9 might act directly downstream of EGL-4 nuclear entry.Studies by Kahn-Kirby et al. into the nociceptive neuron pair, the ASH, have provided much insight into TRP channel function within sensory neurons of C. elegans, unlike humans, are capable of synthesizing long chain PUFAs by desaturating and elongating saturated fats obtained from its bacterial food source Humans require essential PUFAs in their diet C. elegans have shown that PUFA signaling is required for efficient synaptic transmission et al.fat-3 mutant animals have a reduction in the number of synaptic vesicles at release sites and exhibit a concomitant reduction in neurotransmitter release. Furthermore, Marza et al.fat-3(wa22) mutant animals exhibit mislocalization of the presynaptic proteins synaptojanin and synaptobrevin. Synaptojanin is a phosphoinositide phosphatase that promotes the uncoating of presynaptic endocytic vesicles C. elegans synaptojanin mutants (unc-26) are defective in synaptic vesicle recycling and neurotransmitter release due to a reduction in synaptic vesicle numbers fat-3 mutant animals phenocopy these defects demonstrating that PUFAs are required to promote endocytosis at synapses by regulating synaptojanin localization at sites of release Our findings place PUFAs downstream of EGL-4's odor-induced nuclear entry. This observation implies a potential role for PUFAs at the presynaptic site of AWC. Previous studies in Synaptic vesicles are produced in cell bodies and transported along the axon to the nerve terminal. After fusion at the pre-synaptic plasma membrane, the synaptic vesicle proteins and lipids are rapidly endocytosed via clathrin mediated mechanisms and are reused to form new synaptic vesicles. To facilitate olfactory plasticity, perhaps PUFAs control adaptation responses in AWC by regulating synaptic vesicle recycling downstream of EGL-4's nuclear entry. The fact that the TRP channel OSM-9 is required for adaptation to two of the three odors the PUFAs are required for might indicate an odor-specific role for PUFA regulated processes perhaps in the vicinity of the synapse.oα/Gqα mediated DAG signaling has been shown to increase neurotransmitter release in motor neurons gain-of-function egl-30 allele blocks adaptation, it is likely that synaptic transmission needs to be down regulated (or at least tightly regulated) in order for AWC to adapt to prolonged odor exposure. Once in the nucleus, EGL-4 may down-regulate EGL-30 or another factor responsible for high DAG levels since the gain-of-function egl-30 suppresses the constitutively nuclear EGL-4's chemotaxis defects. Indeed, odor-induced dampening of synaptic transmission would be a simple way to silence AWC's output. This interpretation, however, must be tempered by the fact that such a gain-of-function could ectopically or non-specifically suppress this phenotype.DAG may act downstream of EGL-4 nuclear entry since increasing DAG levels blocks adaptation without affecting EGL-4 nuclear accumulation . Goα/GqαWe have reproduced the finding that calcium is required for adaptation but we also show that this requirement is actually downstream of or in parallel to EGL-4 nuclear accumulation. Thus, calcium dependent processes must be at work in promoting adaptation but what they are remains obscure. NCS-1 was an obvious candidate rgs-3 and tax-2, tax-4 butanone and benzaldehyde exposed bars versus unexposed bars; p = 0.005 for rgs-3 and p = 0.05 for tax-2 and tax-4), this suggests that the mutations that cause severely deformed cilia and distal region of dendrites do not affect the dynamic translocation of EGL-4 to the nucleus.Odor-naive wildtype and most mutant animals we examined in our candidate screen displayed diffusely cytoplasmic GFP::EGL-4 . Furtheret al.et al. demonstrated that continual signaling by molecules including TAX-2 and TAX-4 is required to maintain proper AWB cilia architecture. In tax-2 and tax-4 mutants Mukhopadhyay et al., et al., perhaps baseline and continuous sensory signaling in AWC is required to both maintain cilia structure and also to keep EGL-4 in the cytosol. Thus, a certain level of continuous sensory signaling may be responsible for the localization of EGL-4 in a naive animal.There are several explanations for the constitutively nuclear EGL-4 phenotype of severely AWC cilia/dendrite defective mutants. One explanation is that EGL-4 may be tethered in the cytosol of the cilia/dendrite in the AWC by association with other proteins. These tethering proteins may be sensitive to cilia morphology and be unable to correctly retain EGL-4 in the cytosol when this region of the cell is deformed. This tether model suggests that EGL-4 may have a propensity to enter the nucleus and that dissociation of the link between certain tethering proteins and EGL-4 may be a key step in the translocation of EGL-4. Indeed, as Mukhopadhyay odr-3(n2150) mutant strain might be because there is just enough signaling in these animals to preserve both cilia structure and normal localization of GFP::EGL-4 (see odr-3(n2150) allele that we examined was not significantly different from the wildtype strain in its ability to respond to butanone odr-3(1605) allele that was significantly defective for chemotaxis to this odor and showed very severe cilia defects in AWC odr-3(n2150) showed a decrease in cilia size, as determined both by electron microscopic analysis rgs-3, tax-2 or tax-4 mutant backgrounds. Thus, we were able to dissociate the role for the Gα ODR-3 in promoting intact cilia from its role in integrating prolonged odor signaling by using the less severe odr-3(n2150) allele.The reason that we failed to see aberrant nuclear localization of GFP::EGL-4 in the signaling-impaired naive GL-4 see . In factWe have also examined the localization of EGL-4 in other neurons and the AWCs are the only cells that we could identify in which EGL-4 is not constitutively nuclear (unpublished observations by Jeff Eastham-Anderson and N.D.L). Furthermore, EGL-4's ability to accumulate within the AWC nucleus seems to be specific for EGL-4 as other GFP-tagged proteins that are both cytosolic and nuclear do not translocate to the nucleus after odor exposure (unpublished observations by Bi-Tzen Juang and N.D.L).One interesting aspect of odor adaptation is its specificity. In the starved animal, adaptation to the AWC-sensed odor benzaldehyde does not affect isoamyl alcohol chemotaxis and vice versa Since we can force adaptation to occur even in the absence of odor by expressing a constitutively nuclear form of EGL-4 , it suggosm-9 renders animals defective for adaptation to butanone but leaves their ability to adapt to benzaldehyde intact Our evidence is that there are parts of the adaptation machinery that act down stream of EGL-4's nuclear entry that are required for adaptation to specific odors, (OSM-9) and that there are parts that are required for all odors we have tested (PUFAs). Since loss of odr-3(n2150), egl-4(n479), gpb-2(pk751), gpb-2(sa603), gpc-1(pk298), fat-3(wa22), fat-4(wa14) fat-1(wa9), rgs-3(vs19), egl-30(js126), crh-1(tz2), tax-2(p671), tax-4(p678), osm-9(ky10) strain JZ1032 (made by out-crossing CX10 one extra time prior to assaying), pyIs500 him-5, gpb-2(sa603);him-5 pyIs500, gpb-2(pk751);him-5 pyIs500, gpc-1(pk298);him-5 pyIs500, unc-18(md299);him-5 pyIs500, mbk-1(pk1389);him-5 pyIs500, daf-2(e1370);him-5 pyIs500, daf-3(e1376);him-5 pyIs500, let-60(n1046);him-5 pyIs500, let-23(sa62);him-5 pyIs500, gpa-2(pk16);Ex[pyIs500], arr-1(ok401);him-5 pyIs500, rgs-3(vs19);him-5 pyIs500, gcy-31(ok296);him-5 pyIs500, gcy-36(db42);him-5 pyIs500, grk-2(rt97);him-5 pyIs500, tax-4(pr678);him-5 pyIs500, tax-2(p671);him-5 pyIs500, cng-1(jh111);him-5 pyIs500, cng-3(jh113);him-5 pyIs500, ncs-1(qa406);him-5 pyIs500, unc-2(e55);him-5 pyIs500, tax-6(jh107);him-5 pyIs500, tax-6(p675);him-5 pyIs500, ttx-3(ks5);him-5 pyIs500, pkc-1(ok563);him-5 pyIs500, egl-30(n686);him-5 pyIs500, itr-1(sa73);him-5 pyIs500, egl-8(n488);him-5 pyIs500, egl-8(ok934);him-5 pyIs500, egl-10(md176);him-5 pyIs500, egl-30(js126);him-5 pyIs500, fat-3(wa22);him-5 pyIs500, osm-9(ky10);him-5 pyIs500, fat-4(wa14) fat-1(wa9);Ex[pyIs500], sel-12(ar131);him-5 pyIs500, che-2(e1033);him-5 pyIs500, che-11(e1810);him-5 pyIs500, pyIs500 him-5; Ex, odr-3(n2150);Ex[pyIs500], adp-1(ky20) is a dominant mutation ; him-5 pyIs500/+++, N2;Ex, fat-4(wa14) fat-1(wa9);Ex, fat-3(wa22);Ex, osm-9(ky10);Ex, adp-1(ky20);Ex, fat-3(wa22); Ex, N2; Ex[(p)odr-3::NLS::GFP::EGL-4], crh-1(tz2); Ex[(p)odr-3::NLS::GFP::EGL-4], osm-9(ky10); Ex[(p)odr-3::NLS::GFP::EGL-4], fat-3(wa22); Ex[(p)odr-3::NLS::GFP::EGL-4], egl-30(js126); Ex[(p)odr-3::NLS::GFP::EGL-4]. NGM plates were seeded with E. coli strain OP50 and maintained according to standard protocol pyIs500 males and F1 cross progeny picked by identifying transgene-containing (green) worms. F2 animals were picked and genotyped by behavioral analysis, PCR, visual phenotypes or sequencing and then homozygosed for the mutation of interest and the pyIs500 transgene.Bristol N2, osm-9(ky10)) (egl-30(js126) animals  phenotypes, modified adaptation assays were performed )  and egl- animals . For theerformed  by placi animals . For all(p)odr-3::GFP::EGL-4;(p)ofm-1::GFP;(p)odr-1::RFP (strain name pyIs500) to odor in S-Basal (pre-exposed) or S-Basal (un-exposed) for 80 minutes and then scoring the number of worms exhibiting GFP in one AWC nucleus after butanone exposure or in both AWC nuclei after benzaldehyde or isoamyl alcohol exposure under 40× magnification. Wildtype pyIs500 worms were included for every translocation assay as a positive control using ≥0.75 percentile as the baseline for successful control assays and treatment assay inclusion. Between twenty and fifty animals were scored for each translocation assay and repeated on separate days three to five times.Four to five L4 animals were picked onto a 9cm OP50 seeded plate and incubated at 25°C. Animals were washed from these plates and accumulation assays were performed by exposing animals containing an integrated copy of pyIs500 animals were washed three times with ddH2O and soaked in buffer plus benzaldehyde  with 0.5µg/ml PMA (Sigma) for 80 minutes. After this pre-exposure, animals were washed three times with ddH2O and tested for chemotaxis and the subcellular localization of GFP::EGL-4 in AWC. For EGTA treatment, wildtype pyIs500 animals were pre-incubated in 50mM EGTA (pH 7.0 in water) for 2 hours before standard benzaldehyde exposure in S-Basal. Benzaldehyde exposed animals were then assayed for chemotaxis and examined for their subcellular localization of GFP::EGL-4 in AWC, as described. For the preparation of 8-br-cGMP (Sigma) containing plates, 5mM final concentration of 8-br-cGMP was prepared as described by van der Linden et al. E. coli OP50 24 hrs later. Four to five L4 animals were picked to these plates and their progeny assayed. Naïve and odor exposed worms were examined for the localization of GFP::EGL-4 (Wildtype (p)odr-3::GFP::EGL-4 was constructed from (p)egl-4::GFP::EGL-4 (a generous gift from M. Fujiwara) by placing GFP sequences and the first few coding sequences of EGL-4 downstream of (p)odr-3. (p)odr-3::GFP::EGL-4 was co-injected (50 ng/µl) with the AWC marker (p)odr-1::RFP (25 ng/µl) and coelomocyte marker (p)ofm-1::GFP (25 ng/µl) into N2 animals, and the subsequent transgenic line was integrated by TMP method to form the integrated strain pyIs500. The egl-4 gain-of-function allele was made by inserting an extra NLS coding sequence to the 5′ end of GFP coding sequences in the (p)odr-3::GFP::EGL-4 construct. This was accomplished by site directed mutagenesis. The resulting construct was called (p)odr-3::NLS::GFP::EGL-4. ODR-3 overexpression lines were generated using (p)odr-3::ODR-3 (a kind gift from Denise Ferkey). ODR-1 overexpression lines were generated using (p)odr-1::ODR-1 (injected at 1µg/µl). The (p)AWC::FAT-3::GFP rescue construct was made by sub-cloning cDNA into an AWC specific promoter construct, (p)ceh-36::GFP (a kind gift from John F. Etchberger and Oliver Hobert) at the N terminus of GFP by using KpnI and MscI sites. The ceh-36 promoter was truncated by John F. Etchberger and Oliver Hobert until AWC-specific expression was achieved.20 immediately prior to making plates as described by Watts et al.et al.E. coli.Eicosapentaenoic acid (EPA) stocks were prepared by diluting EPA fatty acid salt  to 100 mM in ddHFigure S1Mutant animals for a variety of signaling pathways were assayed for the ability of EGL-4 to translocate to the nucleus after prolonged odor exposure. Error bars represent S.E.M.(0.90 MB TIF)Click here for additional data file.Figure S2Overexpression of ODR-1 causes developmental and adaptation defects in AWC ((0.25 MB TIF)Click here for additional data file.Figure S3The authors have previously demonstrated that appending an extra nuclear localization sequence (NLS) onto the N terminus of EGL-4 to make a (p)odr-3::NLS::GFP::EGL-4 expressing line was sufficient to constitutively force EGL-4 into the nucleus of AWC (0.77 MB TIF)Click here for additional data file.Figure S4odr-3(n2150) mutant animal. Structural defects of odr-3(n2150) are juxtaposed with representative images of wild-type and rgs-3(vs19) for comparison. The white dotted box highlights the defect. The n2150 mutant fails to form wild-type fan shaped cilia.Fluorescent confocal image of the AWC neuron of an (0.45 MB TIF)Click here for additional data file.Figure S5fat-4(wa14)fat-1(wa9) and fat-3(wa22), which all exhibited uncoordinated (unc) phenotypes, were assayed using modified adaptation assays by placing worms at the center of the assay plate. By modifying the origin point, shorter distances were traveled to overcome the unc phenotype problem with assaying these animals. Modified assays were also used to assay fat-3(wa22); Ex [(p)odr-3::NLS::GFP::EGL-4] animals and fat-3(wa22); Ex[(p)AWC::FAT-3] animals.Representation of standard and modified assay plates. 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A pilot study of continuous infusional 5-fluorouracil 200 mg m-2 per 24 h by ambulatory pump and Hickman line for the entire treatment cycle with mitomycin C 8 mg m-2 i.v. on day 1 and cisplatin 75 mg m-2 i.v. on day 1, both repeated every 28 days, was carried out in 31 previously untreated patients with advanced non-small-cell lung cancer (NSCLC). Of 31 patients assessable for response, one attained a complete remission and eight a partial remission, an overall response rate of 29%. Haematological toxicity was minimal, with only 3% of patients developing WHO grade III/IV neutropenia and 13% grade III/IV thrombocytopenia. Significant side-effects included moderate to severe emesis (41%), mucositis (34%), diarrhoea (31%) and palmar-plantar syndrome (14%). Seven patients (23%) had Hickman line complications requiring line removal. Continuous infusional chemotherapy with this regimen is active in advanced non-small-cell lung cancer, but its complexity and associated treatment toxicity offer little advantage over equally active but simpler and less toxic cisplatin-based regimens.