Diane Henry

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophylla/c-containing protein from the chrysophyteIsochrysis galbana: implications for evolution of thecab gene family

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyteIsochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophylla/c-binding light-harvesting protein (FCP) ofI. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chla/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity betweenI. galbana FCP and othercab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related tocab genes encoding LHC I, CP 29, and CP 24 of higher plants than tocab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chla/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.

Reduced expression of Nav1.6 sodium channels and compensation by Nav1.2 channels in mice heterozygous for a null mutation in Scn8a

The voltage-gated sodium channel alpha subunit Na(v)1.6, encoded by the Scn8a gene, accumulates at high density at mature nodes of Ranvier of myelinated axons, replacing the Na(v)1.2 channels found at nodes earlier in development. To investigate this preferential expression of Na(v)1.6 at adult nodes, we examined isoform-specific expression of sodium channels in mice heterozygous for a null mutation in Scn8a. Immunoblots from these +/- mice had 50% of the wild-type level of Na(v)1.6 protein, and their optic-nerve nodes of Ranvier had correspondingly less anti-Na(v)1.6 immunofluorescence. Protein level and nodal immunofluorescence of the Na(v)1.2 alpha subunit increased in Scn8a(+/-) mice, keeping total sodium channel expression approximately constant despite partial loss of Na(v)1.6 channels. The results are consistent with a model in which Na(v)1.6 and Na(v)1.2 compete for binding partners at sites of high channel density, such as nodes of Ranvier. We suggest that Na(v)1.6 channels normally occupy most of the molecular machinery responsible for channel clustering because they have higher binding affinity, and not because they are exclusively recognized by mechanisms for transport and insertion of sodium channels in myelinated axons. The reduced amount of Na(v)1.6 protein in Scn8a(+/-) mice is apparently insufficient to saturate the nodal binding sites, allowing Na(v)1.2 channels to compete more successfully.

Stabilization of Spontaneous Neurotransmitter Release at Ribbon Synapses by Ribbon-Specific Subtypes of Complexin

Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific complexins in transmitter release, we combined presynaptic voltage clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses.

Functional Roles of Complexin in Neurotransmitter Release at Ribbon Synapses of Mouse Retinal Bipolar Neurons

Ribbon synapses of photoreceptor cells and bipolar neurons in the retina signal graded changes in light intensity via sustained release of neurotransmitter. One molecular specialization of retinal ribbon synapses is the expression of complexin protein subtypes Cplx3 and Cplx4, whereas conventional synapses express Cplx1 and Cplx2. Because complexins bind to the molecular machinery for synaptic vesicle fusion (the SNARE complex) and modulate transmitter release at conventional synapses, we examined the roles of ribbon-specific complexin in regulating release at ribbon synapses of ON bipolar neurons from mouse retina. To interfere acutely with the interaction of native complexins with the SNARE complex, a peptide consisting of the highly conserved SNARE-binding domain of Cplx3 was introduced via a whole-cell patch pipette placed directly on the synaptic terminal, and vesicle fusion was monitored using capacitance measurements and FM-dye destaining. The inhibitory peptide, but not control peptides, increased spontaneous synaptic vesicle fusion, partially depleted reserve synaptic vesicles, and reduced fusion triggered by opening voltage-gated calcium channels under voltage clamp, without affecting the number of synaptic vesicles associated with ribbons, as revealed by electron microscopy of recorded terminals. The results are consistent with a dual role for ribbon-specific complexin, acting as a brake on the SNARE complex to prevent spontaneous fusion in the absence of calcium influx, while at the same time facilitating release evoked by depolarization.

A novel cyclic nucleotide-gated ion channel enriched in synaptic terminals of isotocin neurons in zebrafish brain and pituitary

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels opened by binding of intracellular cyclic GMP or cyclic AMP. CNG channels mediate sensory transduction in the rods and cones of the retina and in olfactory sensory neurons, but in addition, CNG channels are also expressed elsewhere in the CNS, where their physiological roles have not yet been well defined. Besides the CNG channel subtypes that mediate vision and olfaction, zebrafish has an additional subtype, CNGA5, which is expressed almost exclusively in the brain. We have generated CNGA5-specific monoclonal antibodies, which we use here to show that immunoreactivity for CNGA5 channels is highly enriched in synaptic terminals of a discrete set of neurons that project to a subregion of the pituitary, as well as diffusely in the brain and spinal cord. Double labeling with a variety of antibodies against pituitary hormones revealed that CNGA5 is located in the terminals of neuroendocrine cells that secrete the nonapeptide hormone/transmitter isotocin in the neurohypophysis, brain, and spinal cord. Furthermore, we show that CNGA5 channels expressed in Xenopus oocytes are highly permeable to Ca(2+), which suggests that the channels are capable of modulating isotocin release in the zebrafish brain and pituitary. Isotocin is the teleost homolog of the mammalian hormone oxytocin, and like oxytocin, it regulates reproductive and social behavior. Therefore, the high calcium permeability of CNGA5 channels and their strategic location in isotocin-secreting synaptic terminals suggest that activation of CNGA5 channels in response to cyclic nucleotide signaling may have wide-ranging neuroendocrine and behavioral effects.

Nanoscale dynamics of synaptic vesicle trafficking and fusion at the presynaptic active zone

The cytomatrix at the active zone (CAZ) is a macromolecular complex that facilitates the supply of release-ready synaptic vesicles to support neurotransmitter release at synapses. To reveal the dynamics of this supply process in living synapses, we used super-resolution imaging to track single vesicles at voltage-clamped presynaptic terminals of retinal bipolar neurons, whose CAZ contains a specialized structure—the synaptic ribbon—that supports both fast, transient and slow, sustained modes of transmission. We find that the synaptic ribbon serves a dual function as a conduit for diffusion of synaptic vesicles and a platform for vesicles to fuse distal to the plasma membrane itself, via compound fusion. The combination of these functions allows the ribbon-type CAZ to achieve the continuous transmitter release required by synapses of neurons that carry tonic, graded visual signals in the retina. DOI: http://dx.doi.org/10.7554/eLife.13245.001