RESUMEN
By engineering the point-spread function (PSF) of single molecules, different fluorophore species can be imaged simultaneously and distinguished by their unique PSF patterns. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a maximum-likelihood estimation algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. We produced superresolution images of fixed U2OS cells using X-PSFs for dSTORM imaging with simultaneous illumination of up to three fluorophore species. The species were distinguished only by the PSF pattern. We achieved â¼21-nm lateral localization precision (FWHM) and â¼17-nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF distinguished fluorescent probes with â¼80â nm separation between spectral peaks.
RESUMEN
Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.
Asunto(s)
Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Proteínas/química , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , CongelaciónRESUMEN
The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a â¼50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the µ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as µ2 adaptin.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endocitosis , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Genes de Helminto/genética , Genoma/genética , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Sistema Nervioso/metabolismo , Fenotipo , Transporte de Proteínas , Vesículas Sinápticas/ultraestructura , Sinaptotagminas/metabolismo , Proteínas de Transporte VesicularRESUMEN
The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.
Asunto(s)
Caenorhabditis elegans/metabolismo , Transmisión Sináptica/fisiología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Apoptosis/genética , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Desarrollo Embrionario/genética , Epidermis/metabolismo , Expresión Génica , Genes Letales , Masculino , Morfogénesis/genética , Mutación , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transmisión Sináptica/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
BACKGROUND: Complexin binds the SNARE complex at synapses and regulates exocytosis, but genetic studies indicate contradictory roles: in flies it predominantly inhibits synaptic vesicle fusion, whereas in mice it promotes evoked responses. RESULTS: Here we characterize the complexin mutant in the nematode Caenorhabditis elegans and reveal bipolar functions in neurotransmission: complexin inhibits spontaneous fusion of synaptic vesicles but is also essential for evoked responses. Complexin mutants exhibit a doubling of vesicle fusion in the absence of extracellular calcium. Even more profoundly, mutants exhibit an almost complete loss of evoked responses, and current amplitudes are reduced by 94%. One possible interpretation is that complexin is required for the stabilization of docked vesicles and that, in its absence, vesicles may fuse or undock from the plasma membrane. Consistent with this hypothesis, docked synaptic vesicles are reduced by 70% in complexin-1 mutants. CONCLUSION: These data suggest that the main function of complexin is to maintain the docked state both by inhibiting fusion and by promoting priming.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Exocitosis , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas SNARE/genética , Proteínas SNARE/metabolismoRESUMEN
Serotonin modulates behavioral plasticity in both vertebrates and invertebrates and in Caenorhabditis elegans regulates key behaviors, including locomotion, aversive learning and olfaction through at least four different 5-HT receptors. In the present study, we examined the serotonergic stimulation of aversive responses to dilute octanol in animals containing null alleles of these 5-HT receptors. Both ser-1 and mod-1 null animals failed to increase sensitivity to dilute octanol on food/5-HT, in contrast to wild-type, ser-4 or ser-7 null animals. 5-HT sensitivity was restored by the expression of MOD-1 and SER-1 in the AIB or potentially the AIY, and RIA interneurons of mod-1 and ser-1 null animals, respectively. Because none of these 5-HT receptors appear to be expressed in the ASH sensory neurons mediating octanol sensitivity, we identified a 5-HT(6)-like receptor, F16D3.7(SER-5), that was required for food/5-HT-dependent increases in octanol sensitivity. ser-5 null animals failed to increase octanol sensitivity in the presence of food/5-HT and sensitivity could be restored by expression of SER-5 in the ASHs. Similarly, the RNAi knockdown of ser-5 expression in the ASHs of wild-type animals also abolished 5-HT-dependent increases in octanol sensitivity, suggesting that SER-5 modulates the octanol responsiveness of the ASHs directly. Together, these results suggest that multiple amine receptors, functioning at different levels within the locomotory circuit, are each essential for the serotonergic modulation of ASH-mediated aversive responses.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Células Quimiorreceptoras/fisiología , Actividad Motora/fisiología , Red Nerviosa/fisiología , Receptores de Serotonina/fisiología , Serotonina/fisiología , 1-Octanol/farmacología , Secuencia de Aminoácidos , Animales , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Chlorocebus aethiops , Técnicas de Silenciamiento del Gen/métodos , Interneuronas/fisiología , Datos de Secuencia Molecular , Actividad Motora/genética , Receptores de Serotonina/genética , Receptores de Serotonina 5-HT2/genética , Receptores de Serotonina 5-HT2/fisiología , Serotonina/deficiencia , Serotonina/genética , Transducción de Señal/fisiologíaRESUMEN
Serotonin (5-HT) regulates key processes in both vertebrates and invertebrates. Previously, four 5-HT receptors that contributed to the 5-HT modulation of egg laying were identified in Caenorhabditis elegans. Therefore, to assess potential receptor interactions, we generated animals containing combinations of null alleles for each receptor, especially animals expressing only individual 5-HT receptors. 5-HT-stimulated egg laying and egg retention correlated well with different combinations of predicted excitatory and inhibitory serotonergic inputs. For example, 5-HT did not stimulate egg laying in ser-1, ser-7, or ser-7 ser-1 null animals, and ser-7 ser-1 animals retained more eggs than wild-type animals. In contrast, 5-HT-stimulated egg laying in ser-4;mod-1 animals was greater than in wild-type animals, and ser-4;mod-1 animals retained fewer eggs than wild-type animals. Surprisingly, ser-4;mod-1;ser-7 ser-1 animals retained the same number of eggs as wild-type animals and exhibited significant 5-HT-stimulated egg laying that was dependent on a previously uncharacterized receptor, SER-5. 5-HT-stimulated egg laying was absent in ser-5;ser-4;mod-1;ser-7 ser-1 animals, and these animals retained more eggs than either wild-type or ser-4;mod-1;ser-7 ser-1 animals. The 5-HT sensitivity of egg laying could be restored by ser-5 muscle expression. Together, these results highlight the dual excitatory/inhibitory serotonergic inputs that combine to modulate egg laying.
Asunto(s)
Caenorhabditis elegans/fisiología , Oviposición/fisiología , Serotonina/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Femenino , Locomoción/efectos de los fármacos , Modelos Biológicos , Datos de Secuencia Molecular , Músculos/efectos de los fármacos , Músculos/metabolismo , Mutación/genética , Oviposición/efectos de los fármacos , Filogenia , Receptores de Serotonina/química , Serotonina/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Músculos/metabolismo , Receptores de Serotonina 5-HT2/fisiología , Serotonina/farmacología , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Huevos , Femenino , Datos de Secuencia Molecular , Músculos/fisiología , Neuronas/metabolismo , Estructura Terciaria de Proteína , Interferencia de ARN , Receptores Acoplados a Proteínas G , Receptores de Serotonina 5-HT2/genética , Receptores de Serotonina 5-HT2/metabolismo , Homología de Secuencia de Ácido Nucleico , Transducción de Señal , Vulva/metabolismo , Vulva/fisiologíaRESUMEN
Serotonin (5-HT) stimulates both pharyngeal pumping and egg laying in Caenorhabditis elegans. Four distinct 5-HT receptors have been partially characterized, but little is known about their function in vivo. SER-7 exhibits most sequence identity to the mammalian 5-HT7 receptors and couples to a stimulation of adenyl cyclase when expressed in COS-7 cells. However, many 5-HT7-specific agonists have low affinity for SER-7. 5-HT fails to stimulate pharyngeal pumping and the firing of the MC motorneurons in animals containing the putative ser-7(tm1325) and ser-7(tm1728) null alleles. In addition, although pumping on bacteria is upregulated in ser-7(tm1325) animals, pumping is more irregular. A similar failure to maintain "fast pumping" on bacteria also was observed in ser-1(ok345) and tph-1(mg280) animals that contain putative null alleles of a 5-HT2-like receptor and tryptophan hydroxylase, respectively, suggesting that serotonergic signaling, although not essential for the upregulation of pumping on bacteria, "fine tunes" the process. 5-HT also fails to stimulate egg laying in ser-7(tm1325), ser-1(ok345), and ser-7(tm1325) ser-1(ok345) animals, but only the ser-7 ser-1 double mutants exhibit an Egl phenotype. All of the SER-7 mutant phenotypes are rescued by the expression of full-length ser-7gfp translational fusions. ser-7gfp is expressed in several pharyngeal neurons, including the MC, M2, M3, M4, and M5, and in vulval muscle. Interestingly, 5-HT inhibits egg laying and pharyngeal pumping in ser-7 null mutants and the 5-HT inhibition of egg laying, but not pumping, is abolished in ser-7(tm1325);ser-4(ok512) double mutants. Taken together, these results suggest that SER-7 is essential for the 5-HT stimulation of both egg laying and pharyngeal pumping, but that other signaling pathways can probably fulfill similar roles in vivo.
Asunto(s)
Caenorhabditis elegans/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Oviposición/fisiología , Faringe/metabolismo , Serotonina/farmacología , Adenilil Ciclasas/metabolismo , Animales , Conducta Animal , Células COS , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Chlorocebus aethiops , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Ligandos , Neuronas Motoras/metabolismo , Músculos/fisiología , Oviposición/efectos de los fármacos , Faringe/efectos de los fármacos , Fenotipo , Receptores de Serotonina 5-HT2/química , Receptores de Serotonina 5-HT2/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Transducción de Señal , Triptófano Hidroxilasa/química , Triptófano Hidroxilasa/metabolismo , Vulva/fisiologíaRESUMEN
The biogenic amines, serotonin, octopamine, tyramine and dopamine regulate many essential processes in parasitic nematodes, such as pharyngeal pumping, muscle contraction, and egg-laying, as well as more complex behaviors, such as mechanosensation and foraging, making biogenic amine receptors excellent targets for drug discovery. This review is designed to summarize our knowledge of nematode biogenic amine signaling and preliminarily identify some of the key receptors involved in the regulation of biogenic amine-dependent behaviors through an analysis of the free-living nematode, Caenorhabditis elegans.
Asunto(s)
Caenorhabditis elegans/fisiología , Nematodos/fisiología , Receptores de Amina Biogénica/fisiología , Animales , Caenorhabditis elegans/genética , Dopamina/metabolismo , Locomoción , Contracción Muscular , Octopamina/metabolismo , Faringe/fisiología , Receptores de Amina Biogénica/genética , Reproducción , Serotonina/metabolismo , Tiramina/metabolismoRESUMEN
Serotonin plays a key role in the regulation of pharyngeal pumping in nematodes. We have isolated a Caenorhabditis elegans cDNA (C09B7.1b, ser-7b) with greatest identity to the 5-HT7 receptor family. Membranes from COS-7 cells expressing SER-7b exhibit saturable [3H]-LSD binding (Kd = 45 nm) that is inhibited by serotonin (5-HT) and tryptamine, but not by other physiological biogenic amines. Expression of SER-7b in COS-7 cells results in dramatic increase in basal cAMP levels over untransfected cells that is dependent on expression level. 5-HT further elevates cAMP levels in a dose-dependent manner (pEC50 = 7.5 +/- 0.5). Mammalian 5-HT7 receptor inverse agonists reduce constitutive activity, with methiothepin the most potent (pIC50 = 7.8 +/- 0.1). Ser-7::GFP transcriptional fusions reveal that SER-7b appears to be expressed solely in the M4 pharyngeal motorneuron after hatching. This is the first report of a Galphas coupled biogenic amine receptor in nematodes and the localization of SER-7b in the M4 pharyngeal motorneuron suggests that SER-7b may play a role in the regulation of pharyngeal pumping.
Asunto(s)
Caenorhabditis elegans/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Neuronas Motoras/metabolismo , Faringe/metabolismo , Receptores de Serotonina/genética , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células COS , Proteínas de Caenorhabditis elegans/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Metiotepina/farmacocinética , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de Aminoácido , Serotonina/farmacocinética , Antagonistas de la Serotonina/farmacocinéticaRESUMEN
Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.