Jeffrey S. Dason

The diversity of inorganic carbon acquisition mechanisms in eukaryotic microalgae

Eukaryotic microalgae have developed CO2concentrating mechanisms to maximise the concentration of CO2 at the active site of Rubisco in response to the low CO2 concentrations in the external aquatic medium. In these organisms, the modes of inorganic carbon (Ci) uptake are diverse, ranging from diffusive CO2 uptake to the active transport of HCO3 -and CO2 and many have an external carbonic anhydrase to facilitate HCO3- use. There is unequivocal evidence for the mechanisms of Ci uptake in only about 25 species of microalgae of the chlorophyte, haptophyte, rhodophyte, diatom, and eustigmatophyte groups. Most of these species take up both CO2 and HCO3-, but the rates of uptake of each of these substrates varies with the algal species. A few species take up only one of the two forms of Ci, an adaptation that is not necessarily correlated with their ecological distribution. Evidence is presented for the active uptake of HCO3- and CO2 in two marine haptophytes,Isochrysis galbana Parke and Dicrateria inornata Parke, and for active transport of CO2 but lack of HCO3- uptake in two marine dinoflagellates, Amphidinium carteraeHulburt and Heterocapsa oceanica Stein.

Frequenin/NCS-1 and the Ca2+-channel α1-subunit co-regulate synaptic transmission and nerve-terminal growth

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca2+-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca2+ entry and enhanced nerve-terminal growth. The impaired Ca2+ entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca2+ channels, or that it regulates the PI4Kβ pathway as described in other organisms. To determine whether Frq interacts with PI4Kβ with consequent effects on Ca2+ channels, we first characterized a PI4Kβ-null mutant and found that PI4Kβ was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kβ-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kβ. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the α1-subunit of voltage-gated Ca2+ channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca2+ entry through a functional interaction with the α1 voltage-gated Ca2+-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

Source of inorganic carbon for photosynthesis in two marine dinoflagellates

Inorganic carbon uptake was investigated in two marine dinoflagellates, Amphidinium carterae Hulburt and Heterocapsa oceanica Stein. Mass spectrometric and potentiometric assays indicated that both species lacked external carbonic anhydrase (CA). The presence of internal CA was demonstrated by potentiometric assay and by the inhibition of photosynthesis upon the addition of 500 μM ethoxyzolamide a membrane‐permeable inhibitor of CA. The capacity for bicarbonate transport was investigated by comparing the calculated rate of spontaneous CO2 formation at pH 8.2 and 25°C with the rate of photosynthesis after the addition of 100 μM NaHCO3. Both species appeared to have a very limited capacity for direct bicarbonate uptake. Monitoring of CO2 and O2 fluxes in both species by mass spectrometry demonstrated a rapid uptake of CO2 on illumination, to concentrations below the CO2 equilibrium concentration, indicating an effective selective uptake of CO2. This dependence of photosynthesis on free CO2 alone suggests that these species are CO2 limited in their natural environment because the CO2 concentration of seawater is very low.

Peptide-Induced Modulation of Synaptic Transmission and Escape Response in Drosophila Requires Two G-Protein-Coupled Receptors

Neuropeptides are found in both mammals and invertebrates and can modulate neural function through activation of G-protein-coupled receptors (GPCRS). The precise mechanisms by which many of these GPCRs modulate specific signaling cascades to regulate neural function are not well defined. We used Drosophila melanogaster as a model to examine both the cellular and behavioral effects of DPKQDFMRFamide, the most abundant peptide encoded by the dFMRF gene. We show that DPKQDFMRFamide enhanced synaptic transmission through activation of two G-protein-coupled receptors, Fmrf Receptor (FR) and Dromyosupressin Receptor-2 (DmsR-2). The peptide increased both the presynaptic Ca2+ response and the quantal content of released transmitter. Peptide-induced modulation of synaptic function could be abrogated by depleting intracellular Ca2+ stores or by interfering with Ca2+ release from the endoplasmic reticulum through disruption of either the ryanodine receptor or the inositol 1,4,5-trisphosphate receptor. The peptide also altered behavior. Exogenous DPKQDFMRFamide enhanced fictive locomotion; this required both the FR and DmsR-2. Likewise, both receptors were required for an escape response to intense light exposure. Thus, coincident detection of a peptide by two GPCRs modulates synaptic function through effects of Ca2+-induced Ca2+ release, and we hypothesize that these mechanisms are involved in behavioral responses to environmental stress.

Vesicular Sterols Are Essential for Synaptic Vesicle Cycling

Synaptic vesicles have a high sterol content, but the importance of vesicular sterols during vesicle recycling is unclear. We used the Drosophila temperature-sensitive dynamin mutant, shibire-ts1, to block endocytosis of recycling synaptic vesicles and to trap them reversibly at the plasma membrane where they were accessible to sterol extraction. Depletion of sterols from trapped vesicles prevented recovery of synaptic transmission after removal of the endocytic block. Measurement of vesicle recycling with synaptopHluorin, FM1-43, and FM4-64 demonstrated impaired membrane retrieval after vesicular sterol depletion. When plasma membrane sterols were extracted before vesicle trapping, no vesicle recycling defects were observed. Ultrastructural analysis indicated accumulation of endosomes and a defect in the formation of synaptic vesicles in synaptic terminals subjected to vesicular sterol depletion. Our results demonstrate the importance of a high vesicular sterol concentration for endocytosis and suggest that vesicular and membrane sterol pools do not readily intermingle during vesicle recycling.

Chronic and acute alterations in the functional levels of Frequenins 1 and 2 reveal their roles in synaptic transmission and axon terminal morphology

Frequenin (Frq) and its mammalian homologue, neuronal calcium sensor 1 (NCS‐1), are important calcium‐binding proteins which enhance neurotransmitter release and facilitation. Here, we report the discovery of a second Frq‐encoding gene (frq2) in Drosophila. The temporal and spatial expression patterns of the two genes are very similar, and the proteins they encode, Frq1 and Frq2, are 95% identical in amino acid sequence. Frq1 is more abundant than Frq2, and is most highly expressed in larva. Loss‐of‐function phenotypes were studied using dominant negative peptides to prevent Frq target binding, RNAi to reduce gene transcription, or both methods. To discriminate chronic from acute loss‐of‐function effects, we compared the effects of transgenic expression and forward‐filling the dominant‐negative peptide into presynaptic terminals. In both cases, a 70% reduction in quantal content per bouton occurred, demonstrating that this trait does not result from homeostatic adaptations of the synapse during development. The chronic treatment also produced more synaptic boutons from MNSNb/d‐Is motorneurons, but fewer active zones per bouton. By contrast, excess‐of‐function conditions yielded a 1.4‐ to 2‐fold increase in quantal content and fewer boutons in the same motorneuron. These synaptic effects resulted in behavioural changes in the Buridan locomotion assay, showing that walking speed is dependent on Frq activity in the nervous system. All the effects were identical for both Frqs, and consistent with excess‐ and loss‐of‐function genotypes. We conclude that Frqs have two distinct functions: one in neurotransmission, regulating the probability of release per synapse, and another in axonal growth and bouton formation.

Cholesterol and F-actin are required for clustering of recycling synaptic vesicle proteins in the presynaptic plasma membrane

Extraction of cholesterol from synaptic vesicles trapped on the presynaptic plasma membrane causes synaptic vesicle proteins to disperse after exocytosis. Vesicular cholesterol regulates both presynaptic phosphatidylinositol (4,5)‐bisphosphate levels and actin distribution during synaptic vesicle recycling. Inhibition of actin polymerization results in the dispersal of proteins from trapped synaptic vesicles and impairs synaptic vesicle recycling. Vesicular cholesterol and actin together confine synaptic vesicle proteins on the presynaptic plasma membrane during synaptic vesicle recycling. Alteration of membrane or synaptic vesicle lipids might therefore affect the ability of synapses to undergo sustained exocytosis and endocytosis by compromising the recycling of synaptic vesicle proteins.

The guanine-exchange factor Ric8a binds to the Ca2+ sensor NCS-1 to regulate synapse number and neurotransmitter release

ABSTRACT The conserved Ca2+-binding protein Frequenin (homolog of the mammalian NCS-1, neural calcium sensor) is involved in pathologies that result from abnormal synapse number and probability of neurotransmitter release per synapse. Both synaptic features are likely to be co-regulated but the intervening mechanisms remain poorly understood. We show here that Drosophila Ric8a (a homolog of mammalian synembryn, which is also known as Ric8a), a receptor-independent activator of G protein complexes, binds to Frq2 but not to the virtually identical homolog Frq1. Based on crystallographic data on Frq2 and site-directed mutagenesis on Frq1, the differential amino acids R94 and T138 account for this specificity. Human NCS-1 and Ric8a reproduce the binding and maintain the structural requirements at these key positions. Drosophila Ric8a and G&agr;s regulate synapse number and neurotransmitter release, and both are functionally linked to Frq2. Frq2 negatively regulates Ric8a to control synapse number. However, the regulation of neurotransmitter release by Ric8a is independent of Frq2 binding. Thus, the antagonistic regulation of these two synaptic properties shares a common pathway, Frq2–Ric8a–G&agr;s, which diverges downstream. These mechanisms expose the Frq2–Ric8a interacting surface as a potential pharmacological target for NCS-1-related diseases and provide key data towards the corresponding drug design.