Interaction of ARF-1.1 and neuronal calcium sensor-1 in the control of the temperature-dependency of locomotion in Caenorhabditis elegans

Neuronal calcium sensor-1 (NCS-1) mediates changes in cellular function by regulating various target proteins. Many potential targets have been identified but the physiological significance of only a few has been established. Upon temperature elevation, Caenorhabditis elegans exhibits reversible paralysis. In the absence of NCS-1, worms show delayed onset and a shorter duration of paralysis. This phenotype can be rescued by re-expression of ncs-1 in AIY neurons. Mutants with defects in four potential NCS-1 targets (arf-1.1, pifk-1, trp-1 and trp-2) showed qualitatively similar phenotypes to ncs-1 null worms, although the effect of pifk-1 mutation on time to paralysis was considerably delayed. Inhibition of pifk-1 also resulted in a locomotion phenotype. Analysis of double mutants showed no additive effects between mutations in ncs-1 and trp-1 or trp-2. In contrast, double mutants of arf-1.1 and ncs-1 had an intermediate phenotype, consistent with NCS-1 and ARF-1.1 acting in the same pathway. Over-expression of arf-1.1 in the AIY neurons was sufficient to rescue partially the phenotype of both the arf-1.1 and the ncs-1 null worms. These findings suggest that ARF-1.1 interacts with NCS-1 in AIY neurons and potentially pifk-1 in the Ca2+ signaling pathway that leads to inhibited locomotion at an elevated temperature

Biophysical and functional characterization of hippocalcin mutants responsible for human dystonia

Dystonia is a neurological movement disorder that forces the body into twisting, repetitive movements or sometimes painful abnormal postures. With the advent of next-generation sequencing technologies, the homozygous mutations T71N and A190T in the neuronal calcium sensor (NCS) hippocalcin were identified as the genetic cause of primary isolated dystonia (DYT2 dystonia). However, the effect of these mutations on the physiological role of hippocalcin has not yet been elucidated. Using a multidisciplinary approach, we demonstrated that hippocalcin oligomerises in a calcium-dependent manner and binds to voltage-gated calcium channels. Mutations T71N and A190T in hippocalcin did not affect stability, calcium-binding affinity or translocation to cellular membranes (Ca2+/myristoyl switch). We obtained the first crystal structure of hippocalcin and alignment with other NCS proteins showed significant variability in the orientation of the C-terminal part of the molecule, the region expected to be important for target binding. We demonstrated that the disease-causing mutations did not affect the structure of the protein, however both mutants showed a defect in oligomerisation. In addition, we observed an increased calcium influx in KCl-depolarised cells expressing mutated hippocalcin, mostly driven by N-type voltage-gated calcium channels. Our data demonstrate that the dystonia-causing mutations strongly affect hippocalcin cellular functions which suggest a central role for perturbed calcium signalling in DYT2 dystonia

A centrosome-localized calcium signal is essential for mammalian cell mitosis

Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca2+) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca2+ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca2+ signals, by flash photolysis of the caged Ca2+ chelator diazo‐2‐acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca2+ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca2+ signal as a functionally essential input into mitosis.—Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome‐localized calcium signal is essential for mammalian cell mitosis

Using Single loxP Sites to Enhance Homologous Recombination: ts Mutants in Sec1 of Dictyostelium discoideum

Dictyostelium discoideum amoebae are haploid and, as they share many features with animal cells, should be an ideal creature for studying basic processes such as cell locomotion. Isolation of mutants in this amoeba has largely been limited to non-essential genes: nsfA-the gene for NEM-sensitive factor-remains the only essential gene for which conditional (ts) mutants exist. These ts mutants were generated by gene replacement using a library of mutagenised nsfA containing a selectable marker: transformants were then screened for temperature sensitivity. The success of this approach depended on the high level of homologous recombination prevailing at this locus: approximately 95% of selected clones were homologous recombinants. This is unusually high for Dictyostelium: homologous recombination at other loci is usually much less, usually between 0-30%, making the isolation of ts mutants much more tedious.In trying to make ts mutants in sec1A, homologous recombination was found to be only approximately 25%. A new approach, involving single loxP sites, was investigated. LoxP sites are 34 bp sequences recognised by Cre recombinase and between which this enzyme catalyses recombination. A Dictyostelium line containing a single loxP site adjacent to the 3' end of the sec1A gene was engineered. A sec1A replacement DNA also containing a single loxP site in a homologous position was then introduced into this cell line. In the presence of CRE recombinase, homologous recombination increased to approximately 80% at this locus, presumably largely driven by intermolecular recombination between the two single loxP sites.A route to increase the rate of homologous recombination at a specific locus, sec1A, is described which enabled the isolation of 30 ts mutants in sec1A. One of these, sec1Ats1,has been studied and found to cease moving at the restrictive temperature. The approach described here may be valuable for enhancing homologous recombination at specified loci and thus for introducing mutations into specific genes in Dictyostelium and other creatures

Differential Calcium Signaling by Cone Specific Guanylate Cyclase-Activating Proteins from the Zebrafish Retina

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1–4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca2+ around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca2+/Mg2+-dependent conformational changes and in the Ca2+-dependent monomer-dimer equilibrium. Direct Ca2+-binding revealed that all zGCAPs bound at least three Ca2+. The corresponding apparent affinity constants reflect binding of Ca2+ with high (≤100 nM), medium (0.1–5 µM) and/or low (≥5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca2+-sensor system in zebrafish rod and cone cells supporting a Ca2+-relay model of differential zGCAP operation in these cells

Structural and Functional Deficits in a Neuronal Calcium Sensor-1 Mutant Identified in a Case of Autistic Spectrum Disorder

Neuronal calcium sensor-1 (NCS-1) is a Ca2+ sensor protein that has been implicated in the regulation of various aspects of neuronal development and neurotransmission. It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca2+ or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca2+ -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain

1H, 15N, and 13C chemical shift assignments of neuronal calcium sensor-1 homolog from fission yeast

The neuronal calcium sensor (NCS) proteins regulate signal transduction processes and are highly conserved from yeast to humans. We report complete NMR chemical shift assignments of the NCS homolog from fission yeast (Schizosaccharomyces pombe), referred to in this study as Ncs1p. (BMRB no. 16446)

Real-Time Measurement of F-Actin Remodelling during Exocytosis Using Lifeact-EGFP Transgenic Animals

F-actin remodelling is essential for a wide variety of cell processes. It is important in exocytosis, where F-actin coats fusing exocytic granules. The purpose of these F-actin coats is unknown. They may be important in stabilizing the fused granules, they may play a contractile role and promote expulsion of granule content and finally may be important in endocytosis. To elucidate these functions of F-actin remodelling requires a reliable method to visualize F-actin dynamics in living cells. The recent development of Lifeact-EGFP transgenic animals offers such an opportunity. Here, we studied the characteristics of exocytosis in pancreatic acinar cells obtained from the Lifeact-EGFP transgenic mice. We show that the time-course of agonist-evoked exocytic events and the kinetics of each single exocytic event are the same for wild type and Lifeact-EGFP transgenic animals. We conclude that Lifeact-EGFP animals are a good model to study of exocytosis and reveal that F-actin coating is dependent on the de novo synthesis of F-actin and that development of actin polymerization occurs simultaneously in all regions of the granule. Our insights using the Lifeact-EGFP mice demonstrate that F-actin coating occurs after granule fusion and is a granule-wide event

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead,we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation,and suggest a prefusion rather than postfusion role for vesicular cation channels