Lysosome exocytosis is required for mitosis in mammalian cells.

Mitosis, the accurate segregation of duplicated genetic material into what will become two new daughter cells, is accompanied by extensive membrane remodelling and membrane trafficking activities. Early in mitosis, adherent cells partially detach from the substratum, round up and their surface area decreases. This likely results from an endocytic uptake of plasma membrane material. As cells enter cytokinesis they re-adhere, flatten and exhibit an associated increase in surface area. The identity of the membrane donor for this phase of mitosis remains unclear. In this paper we demonstrate how lysosomes dynamically redistribute during mitosis and exocytose. Antagonism of lysosomal exocytosis by pharmacological and genetic approaches causes mitosis failure in a significant proportion of cells. We speculate that either lysosomal membrane or luminal content release, possibly both, are therefore required for normal mitosis progression. These findings are important as they reveal a new process required for successful cell division.

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.

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.

α-Methyl-α-phenylsuccinimide ameliorates neurodegeneration in a C. elegans model of TDP-43 proteinopathy.

The antiepileptic drug ethosuximide has recently been shown to be neuroprotective in various Caenorhabditis elegans and rodent neurodegeneration models. It is therefore a promising repurposing candidate for the treatment of multiple neurodegenerative diseases. However, high concentrations of the drug are required for its protective effects in animal models, which may impact on its translational potential and impede the identification of its molecular mechanism of action. Therefore, we set out to develop more potent neuroprotective lead compounds based on ethosuximide as a starting scaffold. Chemoinformatic approaches were used to identify compounds with structural similarity to ethosuximide and to prioritise these based on good predicated blood-brain barrier permeability and C. elegans bioaccumulation properties. Selected compounds were initially screened for anti-convulsant activity in a C. elegans pentylenetetrazol-induced seizure assay, as a rapid primary readout of bioactivity; and then assessed for neuroprotective properties in a C. elegans TDP-43 proteinopathy model based on pan-neuronal expression of human A315T mutant TDP-43. The most potent compound screened, α-methyl-α-phenylsuccinimide (MPS), ameliorated the locomotion defects and extended the shortened lifespan of TDP-43 mutant worms. MPS also directly protected against neurodegeneration by reducing the number of neuronal breaks and cell body losses in GFP-labelled GABAergic motor neurons. Importantly, optimal neuroprotection was exhibited by external application of 50 µM MPS, compared to 8 mM for ethosuximide. This greater potency of MPS was not due to bioaccumulation to higher internal levels within the worm, based on 1H-nuclear magnetic resonance analysis. Like ethosuximide, the activity of MPS was abolished by mutation of the evolutionarily conserved FOXO transcription factor, daf-16, suggesting that both compounds act via the same neuroprotective pathway(s). In conclusion, we have revealed a novel neuroprotective activity of MPS that is >100-fold more potent than ethosuximide. This increased potency will facilitate future biochemical studies to identify the direct molecular target(s) of both compounds, as we have shown here that they share a common downstream DAF-16-dependent mechanism of action. Furthermore, MPS is the active metabolite of another approved antiepileptic drug, methsuximide. Therefore, methsuximide may have repurposing potential for treatment of TDP-43 proteinopathies and possibly other human neurodegenerative diseases.

Cysteine string protein (CSP) and its role in preventing neurodegeneration.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of co-chaperones that localises to neuronal synaptic vesicles. Its name derives from the possession of a string of 12-15 cysteine residues, palmitoylation of which is required for targeting to post-Golgi membranes. The DnaJ domain of CSP enables it to bind client proteins and recruit Hsc70 chaperones, thereby contributing to the maintenance of protein folding in the presynaptic compartment. Mutation of CSP in flies, worms and mice reduces lifespan and causes synaptic dysfunction and neurodegeneration. Furthermore, recent studies have revealed that the neurodegenerative disease, adult onset neuronal ceroid lipofuscinosis, is caused by mutations in the human CSPα-encoding DNAJC5 gene. Accumulating evidence suggests that the major mechanism by which CSP prevents neurodegeneration is by maintaining the conformation of SNAP-25, thereby facilitating its entry into the membrane-fusing SNARE complex. In this review, we focus on the role of CSP in preventing neurodegeneration and discuss how recent studies of this universal neuroprotective chaperone are being translated into potential novel therapeutics for neurodegenerative diseases.

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.

Sense and specificity in neuronal calcium signalling.

Changes in the intracellular free calcium concentration ([Ca²âº]i) in neurons regulate many and varied aspects of neuronal function over time scales from microseconds to days. The mystery is how a single signalling ion can lead to such diverse and specific changes in cell function. This is partly due to aspects of the Ca²âº signal itself, including its magnitude, duration, localisation and persistent or oscillatory nature. The transduction of the Ca²âº signal requires Ca²âºbinding to various Ca²âº sensor proteins. The different properties of these sensors are important for differential signal processing and determine the physiological specificity of Ca(2+) signalling pathways. A major factor underlying the specific roles of particular Ca²âº sensor proteins is the nature of their interaction with target proteins and how this mediates unique patterns of regulation. We review here recent progress from structural analyses and from functional analyses in model organisms that have begun to reveal the rules that underlie Ca²âº sensor protein specificity for target interaction. We discuss three case studies exemplifying different aspects of Ca²âº sensor/target interaction. This article is part of a special issue titled the 13th European Symposium on Calcium.

Caenorhabditis elegans dnj-14, the orthologue of the DNAJC5 gene mutated in adult onset neuronal ceroid lipofuscinosis, provides a new platform for neuroprotective drug screening and identifies a SIR-2.1-independent action of resveratrol.

Adult onset neuronal lipofuscinosis (ANCL) is a human neurodegenerative disorder characterized by progressive neuronal dysfunction and premature death. Recently, the mutations that cause ANCL were mapped to the DNAJC5 gene, which encodes cysteine string protein alpha. We show here that mutating dnj-14, the Caenorhabditis elegans orthologue of DNAJC5, results in shortened lifespan and a small impairment of locomotion and neurotransmission. Mutant dnj-14 worms also exhibited age-dependent neurodegeneration of sensory neurons, which was preceded by severe progressive chemosensory defects. A focussed chemical screen revealed that resveratrol could ameliorate dnj-14 mutant phenotypes, an effect mimicked by the cAMP phosphodiesterase inhibitor, rolipram. In contrast to other worm neurodegeneration models, activation of the Sirtuin, SIR-2.1, was not required, as sir-2.1; dnj-14 double mutants showed full lifespan rescue by resveratrol. The Sirtuin-independent neuroprotective action of resveratrol revealed here suggests potential therapeutic applications for ANCL and possibly other human neurodegenerative diseases.

Demonstration of binding of neuronal calcium sensor-1 to the cav2.1 p/q-type calcium channel.

In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Cav2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Cav2.1 α-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Cav2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system.

Generation and characterization of a lysosomally targeted, genetically encoded Ca(2+)-sensor.

Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

PKC-2 phosphorylation of UNC-18 Ser322 in AFD neurons regulates temperature dependency of locomotion.

Diacylglycerol (DAG)/protein kinase C (PKC) signaling plays an integral role in the regulation of neuronal function. This is certainly true in Caenorhabditis elegans and in particular for thermosensory signaling and behavior. Downstream molecular targets for transduction of this signaling cascade remain, however, virtually uncharacterized. We investigated whether PKC phosphorylation of Munc18-1, an essential protein in vesicle trafficking and exocytosis, was the downstream effector for DAG regulation of thermosensory behavior. We demonstrate here that the C. elegans ortholog of Munc18-1, UNC-18, was phosphorylated in vitro at Ser322. Transgenic rescue of unc-18-null worms with Ser322 phosphomutants displayed altered thermosensitivity. C. elegans expresses three DAG-regulated PKCs, and blocking UNC-18 Ser322 phosphorylation was phenocopied only by deletion of calcium-activated PKC-2. Expression of nonphosphorylatable UNC-18 S322A, either pan-neuronally or specifically in AFD thermosensory neurons, converted wild-type worms to a pkc-2-null phenotype. These data demonstrate that an individual DAG-dependent thermosensory behavior of an organism is effected specifically by the downstream PKC-2 phosphorylation of UNC-18 on Ser322 in AFD neurons.

Solution NMR structure of the Ca2+-bound N-terminal domain of CaBP7: a regulator of golgi trafficking.

Calcium-binding protein 7 (CaBP7) is a member of the calmodulin (CaM) superfamily that harbors two high affinity EF-hand motifs and a C-terminal transmembrane domain. CaBP7 has been previously shown to interact with and modulate phosphatidylinositol 4-kinase III-ß (PI4KIIIß) activity in in vitro assays and affects vesicle transport in neurons when overexpressed. Here we show that the N-terminal domain (NTD) of CaBP7 is sufficient to mediate the interaction of CaBP7 with PI4KIIIß. CaBP7 NTD encompasses the two high affinity Ca(2+) binding sites, and structural characterization through multiangle light scattering, circular dichroism, and NMR reveals unique properties for this domain. CaBP7 NTD binds specifically to Ca(2+) but not Mg(2+) and undergoes significant conformational changes in both secondary and tertiary structure upon Ca(2+) binding. The Ca(2+)-bound form of CaBP7 NTD is monomeric and exhibits an open conformation similar to that of CaM. Ca(2+)-bound CaBP7 NTD has a solvent-exposed hydrophobic surface that is more expansive than observed in CaM or CaBP1. Within this hydrophobic pocket, there is a significant reduction in the number of methionine residues that are conserved in CaM and CaBP1 and shown to be important for target recognition. In CaBP7 NTD, these residues are replaced with isoleucine and leucine residues with branched side chains that are intrinsically more rigid than the flexible methionine side chain. We propose that these differences in surface hydrophobicity, charge, and methionine content may be important in determining highly specific interactions of CaBP7 with target proteins, such as PI4KIIIß.

Munc18-1 tuning of vesicle merger and fusion pore properties.

The release of hormones and neurotransmitters, mediated by regulated exocytosis, can be modified by regulation of the fusion pore. The fusion pore is considered stable and narrow initially, eventually leading to the complete merger of the vesicle and the plasma membranes. By using the high-resolution patch-clamp capacitance technique, we studied single vesicles and asked whether the Sec1/Munc18 proteins, interacting with the membrane fusion-mediating SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, affect fusion pore properties. Munc18-1 mutants were transfected into lactotrophs to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mints (P242S). Compared with wild-type, Munc18-1 E466K increased the frequency of the fusion event. The latter two mutants increased the fusion pore dwell-time. All the mutants stabilized narrow fusion pores and increased the amplitude of fusion events, likely via preferential fusion of larger vesicles, since overexpression of Munc18-1 R39C did not affect the average size of vesicles, as determined by stimulated emission depletion (STED) microscopy. Single-molecule atomic force microscopy experiments revealed that wild-type Munc18-1, but not Munc18-1 R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans-complexes. However, neither form of Munc18-1 affected the interaction of Syb2 with the preformed binary cis-Synt1A-SNAP25B complexes. This indicates that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and favoring vesicular tethering to the preformed binary cis-complex of Synt1A-SNAP25B. The association of Munc18-1 with the ternary SNARE complex leads to tuning of fusion pores via multiple and converging mechanisms involving Munc18-1 interactions with Synt1A, Rab3A, and Mints.

Identification of common genetic modifiers of neurodegenerative diseases from an integrative analysis of diverse genetic screens in model organisms.

BACKGROUND: An array of experimental models have been developed in the small model organisms C. elegans, S. cerevisiae and D. melanogaster for the study of various neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and expanded polyglutamine diseases as exemplified by Huntington's disease (HD) and related ataxias. Genetic approaches to determine the nature of regulators of the disease phenotypes have ranged from small scale to essentially whole genome screens. The published data covers distinct models in all three organisms and one important question is the extent to which shared genetic factors can be uncovered that affect several or all disease models. Surprisingly it has appeared that there may be relatively little overlap and that many of the regulators may be organism or disease-specific. There is, however, a need for a fully integrated analysis of the available genetic data based on careful comparison of orthologues across the species to determine the real extent of overlap. RESULTS: We carried out an integrated analysis using C. elegans as the baseline model organism since this is the most widely studied in this context. Combination of data from 28 published studies using small to large scale screens in all three small model organisms gave a total of 950 identifications of genetic regulators. Of these 624 were separate genes with orthologues in C. elegans. In addition, 34 of these genes, which all had human orthologues, were found to overlap across studies. Of the common genetic regulators some such as chaperones, ubiquitin-related enzymes (including the E3 ligase CHIP which directly links the two pathways) and histone deacetylases were involved in expected pathways whereas others such as the peroxisomal acyl CoA-oxidase suggest novel targets for neurodegenerative disease therapy CONCLUSIONS: We identified a significant number of overlapping regulators of neurodegenerative disease models. Since the diseases have, as an underlying feature, protein aggregation phenotypes it was not surprising that some of the overlapping genes encode proteins involved in protein folding and protein degradation. Interestingly, however, some of the overlapping genes encode proteins that have not previously featured in targeted studies of neurodegeneration and this information will form a useful resource to be exploited in further studies of potential drug-targets.

InsP3receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences.

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca²âº entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP3Rs (InsP3 receptors). The co-localization between Orai1 and IP3Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca²âº store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca²âº store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP3Rs and a basolateral pool that interacts with STIM1 following the Ca²âº store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai1 localization does not require IP3Rs and that IP3Rs are not necessary for the activation of SOCE. However, the InsP3-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP3Rs could have an inhibitory effect on this Ca²âº entry mechanism.

Evidence for an interaction between Golli and STIM1 in store-operated calcium entry.

SOCCs (store-operated Ca(2+) channels) are highly selective ion channels that are activated upon release of Ca(2+) from intracellular stores to regulate a multitude of diverse cellular functions. It was reported previously that Golli-BG21, a member of the MBP (myelin basic protein) family of proteins, regulates SOCE (store-operated Ca(2+) entry) in T-cells and oligodendrocyte precursor cells, but the underlying mechanism for this regulation is unknown. In the present study we have discovered that Golli can directly interact with the ER (endoplasmic reticulum) Ca(2+)-sensing protein STIM1 (stromal interaction molecule 1). Golli interacts with the C-terminal domain of STIM1 in both in vitro and in vivo binding assays and this interaction may be modulated by the intracellular Ca(2+) concentration. Golli also co-localizes with full-length STIM1 and Orai1 complexes in HeLa cells following Ca(2+) store depletion. Overexpression of Golli reduces SOCE in HeLa cells, but this inhibition is overcome by overexpressing STIM1. We therefore suggest that Golli binds to STIM1-Orai1 complexes to negatively regulate the activity of SOCCs.

Membrane trafficking: three steps to fusion.

Membrane fusion involves the action of members of the SNARE protein family as well as Sec1/Munc18 (SM) proteins, which have been found to interact with SNAREs in three distinct ways. Recent work has established that Munc18-1 directly stimulates fusion and possibly uses all three modes of SNARE interaction.

Caenorhabditis elegans: a useful tool to decipher neurodegenerative pathways.

Neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's diseases affect millions of people. These disorders are age-dependent, progressive and, at present, incurable. A practical and relevant model is needed to investigate the molecular determinants of these debilitating diseases. Mammalian models are often prohibitively expensive, time-consuming and very complex. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for the investigation of the pathophysiology of these disorders. We describe recent findings in this area and show how C. elegans is being used to broaden our knowledge of human neurodegenerative diseases.

Presynaptic targets for acute ethanol sensitivity.

Acute exposure to ethanol is known to modulate signalling within the nervous system. Physiologically these effects are both presynaptic and postsynaptic in origin; however, considerably more research has focused primarily on postsynaptic targets. Recent research using the model organism Caenorhabditis elegans has determined a role for specific proteins (Munc18-1 and Rab3) and processes (synaptic vesicle recruitment and fusion) in transducing the presynaptic effects of ethanol. In the present paper, we review these results, identifying the proteins and protein interactions involved in ethanol sensitivity and discuss their links with mammalian studies of alcohol abuse.