Luiza Ghila andMarie Gomez
Department of Zoology and Animal Biology and NCCR Frontiers in Genetics, University of Geneva, Sciences III, 30 quai Ernest-Ansermet, 1211 Gene`ve 4, Switzerland
Keywords:C. elegans, localization, neurotransmission, synaptic vesicle, transport
Abstract
The control of vesicle-mediated transport in nerve cells is of great importance in the function, development and maintenance of synapse. In this paper, we characterize the newCaenorhabditis elegansgene,lnp-1. Thelnp-1gene is broadly distributed in many neuronal structures and its localization is dependent of the UNC-104⁄kinesin protein. Deletion mutations inlnp-1result in increased resistance to aldicarb, an acetylcholinesterase inhibitor, and in locomotor defects. However, sensitivity to levamisole, a nicotinic agonist which, unlike aldicarb, only affects postsynaptic function, was similar to that of wild-type animals, suggesting a presynaptic function for LNP-1 in neurotransmission. The mislocalization of presynaptic proteins, such as synaptobrevin-1 or RAB-3, in lnp-1 mutants further supports this hypothesis. In summary, our studies suggest that LNP-1 plays a role in synaptogenesis by regulating vesicular transport or localization.
Introduction
Synapses are essential structures that mediate signal transmission by neurons, allowing the establishment of interneuronal as well as sensory and motor functions. The control of synaptic vesicle transport is crucial for synapse formation and regulation of neuronal networks, as well as learning and memory (Malenka & Nicoll, 1999).
The nematode Caenorhabditis elegans (C. elegans) has been extensively used as a model system for studying neural development and function, due to its simple nervous system, its well-described neuronal circuitry and the high degree of evolutionary conservation of neuronal genes. These attributes, allied with the relative ease of genetic manipulation, have allowed the elucidation of complex molecular pathways regulating neural development. Classical genetic screens for mutants displaying specific behavioural defects, e.g.
locomotion, have resulted in the discovery of a large number of neuronal genes (Brenner, 1974). However, mutants with more subtle phenotypes are not efficiently identified in these screens and require the use of alternative approaches (Hutter, 2000).
We have opted for a candidate gene approach that investigates the role of specific genes based on previous observations and we have focused on the C. elegans lnp-1. The mouse lnp is part of the lnp-evx2-hoxd gene cluster (Spitz et al., 2003). It is expressed in neuronal structures such as the cerebellum and the neural tube and its neuronal expression coincides with that of evx genes (Spitz et al., 2003). Data assembled from whole-genome expression profiling studies have permitted the identification of groups of coregulated genes that may be part of common functional circuit (Hugheset al., 2000; Kimet al., 2001). In C. elegans, lnp-1 is part of a group of coregulated genes, named mount 1, which includes many known neuronal and muscle-specific members (Kimet al., 2001) that tend to
encode receptors or receptor-associated proteins, such as unc-16, klc-2, unc-5orsax-3.Unc-16andklc-2genes encode for proteins that regulate synaptic vesicular transport (Byrd et al., 2001), and the protein products ofunc-5andsax-3genes are molecules involved in axonal guidance (reviewed in Hobert & Bulow, 2003).
Lnp-1 has been conserved through evolution, from yeast to mammals, although the overall function of LNP remains unknown.
In this manuscript, we show that the neuronal expression pattern of lnp-1 described in mice is conserved in C. elegans. LNP-1 is expressed in different neuronal structures including cell bodies, neuritic processes and commissures, and this localization requires the motor protein kinesin UNC-104. Loss-of-function deletions inlnp-1 result in the mislocalization of the synaptic vesicle cargos synaptob-revin-1 (SNB-1) and RAB-3. The lnp-1 mutants display increased resistance to aldicarb, an inhibitor of acetylcholinesterase, and have subtle locomotion defects. We propose that LNP-1 is involved in synaptic vesicle transport or localization and plays a role in the regulation of synapse function.
Materials and methods Strains
The strains used in this study were maintained as described (Brenner, 1974). lnp-1-Deleted strains lnp-1(tm1247) and lnp-1(tm733) were obtained from The National Bioresource Project for the Experimental Animal NematodeC. elegans (NBRP; Japan) and outcrossed three times with N2 animals. NW1229 (evIs111 (F25B3.3::GFP) from O. Hobert, Columbia University, New York, USA). NM306 (jsIs1 (pSB120[psnb-1::SNB::1::GFP];pRF4[rol-6]) from M. L. Nonet, Washington University, St Louis, USA). KP3931 nuIs168 (p unc-129::RAB-3::Venus from L. Dreier, UCLA, Los Angeles, USA).
Correspondence: Dr M. Gomez, as above.
E-mail: marie.gomez@zoo.unige.ch
European Journal of Neuroscience, Vol. 27, pp. 621–630, 2008 doi:10.1111/j.1460-9568.2008.06049.x
nuIs168. Some nematode strains used in this work were provided by theCaenorhabditisGenetics Center (CGC; University of Minnesota, Minneapolis, USA).
Constructplnp-1::LNP-1: consists of a 4158-bp genomic fragment, including a 2746-bp genomic region upstream of thelnp-1ATG start (plnp-1) and the genomiclnp-1coding sequence (LNP-1). All these fragments were generated by PCR and subcloned into either the pPD95.75 green fluorescent protein (GFP) vector (from A. Fire, Stanford University School of Medicine, Stanford, CA, USA) or pPD95.75 RFP (from O. Hobert, Columbia University, New York, USA).
Rescued plasmid: plnp-1::LNP-1 subcloned in pPD95.75 vector where GFP was deleted by enzyme digestion.
GST::LNP-1 and GST::DLNP-1: C. elegans lnp-1 cDNA (full-length coding region) was generated from total RNA as described by the supplier (cDNA synthesis system, Promega). Total RNA was isolated as described by the manufacturer (RNeasy kit, Qiagen).lnp-1 cDNA and Dlnp-1 cDNA were amplified by PCR. The resulting fragments were subcloned into the pGEX4T1 vector (GE⁄Healthcare).
All constructs were checked for correct orientation and sequence.
For oligo sequences and DNA templates used in PCR reactions and resulting fragments see Supplementary material, Table S2.
Anti-LNP-1 antibody production
C. elegansGST::DLNP-1 protein was produced in BL21 E. colias described (Guan & Dixon, 1991). The protein was expressed at 37!C and purified using glutathione–sepharose beads (GE⁄Healthcare).
For antibody production, DLNP-1 was recovered from the super-natant by thrombin digestion (GE⁄Healthcare). Rabbits were immu-nized with DLNP-1 according to the manufacturer’s instructions (Eurogentec, Belgium). The resulting antisera were affinity-immuno-purified onDLNP-CNBR-activated sepharose beads (GE⁄Healthcare).
Subcellular fractionation
All procedures were performed at 4!C as previously described (Rao et al., 2005). Briefly, synchronized worms were washed three times in phosphate-buffered saline (PBS) and resuspended in cold buffer (TrisHCl, pH 7.5, 20 mm; EDTA, 1 mm; dithiothreitol (DTT), 1 mm;
and sucrose, 250 mm) at a ratio of 1 : 2 (w⁄v), then frozen in liquid nitrogen, thawed and homogenized in a glass douncer. The freezing–
homogenization cycle was performed three times. The homogenates were centrifuged twice at 1000g for 10 min to remove cuticle and large debris, then ultracentrifugated to 20 psi (100 000g) for 2 h to obtain a pellet fraction rich in organelles and membranes, and a supernatant fraction rich in cytosol. The pellet was resuspended in 50lL of the previous buffer and protein concentration was deter-mined by the Bradford assay. For immunoblotting, lysates were heated to 100!C for 10 min in the presence of SDS sample buffer containing DTT. Proteins were separated by SDS-PAGE, transferred to nitrocel-lulose and detected by the Western Lightning Chemiluminescence kit (PerkinElmer) using anti-LNP-1 and antirabbit horseradish peroxidase (HRP) secondary antibodies (Jackson).
Transgenic animals and rescue experiments
Transgenic worms were generated as previously described (Mello et al., 1991). Transgenes were microinjected at 20 ng⁄lL.Rol-6was used as injection marker and injected at 50 ng⁄lL. In rescue
pttx-3::RFP or the intestinal markerelt-2::GFP were used as markers and injected at 50 ng⁄lL. At least three independent transgenics were analysed.
Behavioural assays Sensitivity to aldicarb
Radial migration assays were performed on agar plates containing 0.5 mmaldicarb (Wicom, Germany) and seeded with OP50 bacteria at the outer edge of the plate such that after overnight growth a ring-shaped lawn was formed as described (Salciniet al., 2001). Between 50 and 200 synchronous worms were washed off from bacteria and transferred to the centre of a new plate. After 4 h at 18!C, the worms that had reached the bacteria were counted and their number was reported as a fraction of the total number of tested worms.
Acute sensitivity to aldicarb was determined by an assay of the full time course of the onset of paralysis following acute exposure of a population of animals to aldicarb as previously described (Sieburth et al., 2005). For each strain, 25–30 worms were placed on agar plates plus 0.5 mmaldicarb and prodded every 30 min over a 6-h period to determine whether they retained the ability to move; worms that did not respond to repeated prodding were classified as paralysed. Each experiment was performed three times.
Thrashing behaviour
This assay was performed as previously described (Milleret al., 1996).
Young adult hermaphrodites were placed in glass chambers containing 100lL of M9 buffer (Brenner, 1974). After a 5-min recovery period, thrashes were photographed for 1 min using Improvision OpenLab 5.0.1 and a Leica DFC320 camera connected to a Leica MZ125 binocular microscope, and counted manually. A thrash was considered a change in the direction of bending at the mid-body.
Serotonin-induced egg-laying assay
Young adult hermaphrodites were placed individually in microtitre wells containing 100lL of 7.5 mmserotonin (Sigma) in M9 solution as described (Trentet al., 1983). After 90 min the number of eggs in each well was counted.
Immunocytochemistry On whole worm
L4 larvae and young adults were washed in PBS and fixed for 4 h in Lavdowsky fixative (50% ethanol, 10% acetic acid and 4% formal-dehyde in water). Worms were then washed in PBS and incubated overnight in 2% Triton X-100 in PBS. The larvae and eggs were permeabilized by microwave for 7 min at 750 W and incubated for 30 min in 0.1% Na-citrate and 0.5% Triton X-100 in PBS. They were then washed in 0.5% TritonX-100 in PBS (PBST) and treated with 3%
H2O2 in PBS (blocking the endogenous peroxidase), blocked in 2%
bovine serum albumin in PBST and incubated overnight at 4!C with the appropriate first antibody (anti-LNP-1, 1 : 500; anti-GFP, 1 : 200;
Roche). Animals were washed in PBST and incubated for 3 h at room temperature with the corresponding secondary antibody diluted 1 : 100 (antirabbit HRP, Jackson; antimouse HRP, Sigma) as described in the TSA Kit no. 2, 15 or 40 kit protocols (Molecular Probes) and with a detection time of 12 min. The larvae and young adults were washed in PBS and water, mounted in Mowiol and examined on an Axioplan2 Zeiss microscope equipped with a Plan-622 L. Ghila and M. Gomez
DFC480 camera using the Openlab System Software (Improvision).
Confocal imaging was performed on a Leica TCS⁄SP2⁄AOBS microscope. The images were processed using PhotoshopCS software (Adobe Systems).
For all colocalization imaging,Z-stacks were acquired and average or maximum projection was recorded.
Fluorescence analysis and quantification
Measurements of SNB-1::GFP and RAB-3::Venus puncta were performed on confocal images as described previously (Ackley et al., 2005; Daiet al., 2006). Synchronized worms were imaged in the same day, in the region between the nerve ring and the anterior gonad bend (50lm was selected in this area). Images with minimal fluorescence saturation were obtained using identical laser settings.
For each worm a 3.5-lmZ-stack containing 20Z-planes was acquired.
Confocal images were projected into a single plane using maximum projection and exported as a tiff file with a scale bar. Using ImageJ 1.34s (NIH, USA), the files were converted to a binary image and the fluorescence profile was analysed using the plot profile command.
This command displays a two-dimensional graph of the intensities of pixels along a line within the image (in our case ventral cord or dorsal cord). Thex-axis represents distance along the line and they-axis is the pixel intensity. The listed pixel intensity table was generated in one-pixel increments. The total and average pixel intensities were calculated. The threshold was set by using the threshold command so that the binary images resembled the original image. The ‘analyse particle’ command was used with a minimum size of 4 pixels and a maximum size of 10 000 pixels, and the ‘outline particles’, ‘exclude on edges’, ‘include interior holes’ and ‘reset counter options’ were
selected. Scaling was set by measuring the scale bar (GFP images:
12 pixels¼1lm). The resulting measurement were exported to Microsoft Excel for statistical analysis.
Results
LNP-1: evolutionary aspects
Like all the lnp orthologs (e.g. Drosophila, D. rerio, mouse, etc.) examined so far (Spitzet al., 2003), a single gene located on the X chromosome codes forlnpinC. elegans: C05E11.1. This gene, which we renamed lnp-1 (accepted and registered by the Caenorhabditis Genetics Center), encodes a 342-amino-acid protein that contains the domains conserved in all orthologs, including two predicted trans-membrane domains in the N-terminal part of the protein and an atypical zinc finger domain (C2HC2), which may represent a novel class of zinc-finger motif (Fig. 1A).
Neuronal expression oflnp-1inC. elegans
To elucidate a possible role forlnp-1in the nervous system, we first determined its expression pattern. We generated worm lines expressing the plnp-1::LNP-1::GFP transgene (Figs 1B and 2A). Broad, and possibly ubiquitous, expression was first visible during gastrulation (data not shown). In larvae and adult stages, the LNP-1::GFP fusion protein was expressed in numerous cell bodies along the ventral cord, around the pharynx and the tail. To confirm this expression pattern, we made transgenes expressing GFP or red fluorescent protein (RFP) under the control of the 2748-bp region upstream of the first ATG codon (p lnp-1::GFP or RFP; Figs 1B and 2B and C). These constructs were injected
Fig. 1. (A) Structure of the LNP-1 protein and alignments of the transmembrane (TM) and zinc finger domains of mouse,Drosophila, zebrafish andC. elegans LNP-1. Identities and similarities are highlighted in dark grey and light grey, respectively. The main conserved domains (boxed) are the two predicted transmembrane domains in the N-terminal part of the protein, an atypical zinc finger and the consensus sequence LNPARK. (B) Genomic organization oflnp-1inC. elegansand LNP-1 and synaptic vesicle trafficking 623
either into wild-type animals or into animals expressing GFP under the control of a pan-neuronal promoter (evIs111strain; Fig. 2D). As shown, motor neurons in the ventral nerve cord, and sensory- and interneurons in the nerve ring and in the tail, were labelled. Expression was also observed in muscles and hypodermal cells. The overall expression patterns of the two transgenes were quite similar; however, the expression of the LNP-1::GFP fusion protein appeared more localized than the GFP reporter protein alone. To further investigate the distribution of LNP-1 into the cell, subcellular fractionation experiments and Western blot analyses were performed. LNP-1 was highly concentrated in the membrane and organelle fraction, suggesting that LNP-1 is a membrane protein (supplementary Fig. S1, B).
The transgene expression pattern was further confirmed by immuno-histochemistry in whole worms using an antibody that we generated against theC. elegansLNP-1 (Fig. 2E). The specificity of the antibody was tested by Western blot on total protein extracts and by whole-mount immunohistochemistry of wild-type andlnp-1mutant animals (Fig. 2J and supplementary Fig. S1, A). Within neurons, endogenous LNP-1 was not present in the nucleus but was localized in cell bodies. The processes of motor neurons of the ventral nerve cord (Fig. 2F), the dendrites of sensory neurons of the head (Fig. 2G), the dorsal and sublateral nerve cords and commissures were all labelled (Fig. 2H and I).
Subcellular localization of LNP-1 required the neuronal UNC-104⁄kinesin
The neuronal expression pattern of LNP-1 resembles that of proteins involved in neuronal transport, such as the motor protein
UNC-from cell bodies to synapses through the microtubule network (Hall &
Hedgecock, 1991). LNP-1 and UNC-104 are both broadly expressed in neuronal structures, including cell bodies and neuronal processes (Zhouet al., 2001). To address whether LNP-1 may be associated with UNC-104⁄kinesin synaptic vesicles, the localization of LNP-1 in unc-104(e1265) mutants was examined. Whereas in wild-type worms LNP-1 was broadly distributed in many neuronal structures (Fig. 2K), in theunc-104(e1265)mutant background LNP-1 remained confined to cell bodies of the ventral cord (Fig. 2L), suggesting that LNP-1 localization is dependent on UNC-104.
Synaptobrevin-1 and RAB-3 were mislocalized in lnp-1 mutants The distributions of many presynaptic vesicle proteins, such as SNB-1 or RAB-3, are also altered in theunc-104(e1265)mutant background, where they are not transported to presynaptic structures (Nonetet al., 1993, 1998) (Figs 3C–D¢and 4C–D¢). To address whether LNP-1 may function to regulate transport or localization of UNC-104⁄synaptic vesicle cargos, we looked at the distribution of SNB-1::GFP inlnp-1 mutants. Twolnp-1mutants strains were tested:lnp-1(tm1247), which lacks part of exon 2 to exon 5, and lnp-1(tm733), in which part of exons 3 and 4 have been deleted (Fig. 5). Both of these mutations result in out-of-frame deletion alleles with premature stop codons, resulting in putative truncated forms of LNP-1 in which only the N-terminal part of the protein, including the two predicted transmem-brane domains, remain. LNP-1 was not detected in total protein extracts fromlnp-1(tm733)orlnp-1(tm1247)animals (supplementary Fig. S1, A), presumably because these two mutants either express a Fig. 2. Expression pattern and intracellular localization oflnp-1. (A) Expression ofplnp-1::LNP-1::GFP in different cell types including neurons in the pharyngeal region (arrow), the ventral cord (asterisk) and the tail. (B) plnp-1::GFP transgenic animals show diffuse GFP expression, filling the entire cell bodies (arrows), nerve processes (asterisk) and commissures (arrowhead) of motor neurons in the ventral cord. (C and D)plnp-1::RFP construct injected into theevIs111strain, which expresses GFP in all neurons. Neurons coexpressing both (C) RFP and (D) GFP were found in the ventral nerve cord (asterisks in C and D) and in the nerve ring (arrows in C and D). Sensory neurons in the nerve ring coexpressing both reporters are demonstrated by the staining of the dendritic processes that extend to the tip of the head (arrowheads in C and D). (E–I) Endogenous LNP-1 visualized by anti-LNP-1 immunostaining (E). Signal detected in ventral (F, arrows) and dorsal (H, arrow) nerve cords. LNP-1 localized in cell bodies (arrowheads in F and G), dendrites and commissures (respectively G and I, arrows). (J) Note that no signal was detected inlnp-1mutants. (K and L) LNP-1 localization was dependent of the UNC-104⁄kinesin motor protein. Localization of endogenous LNP-1 in wild-type animals (K, arrow and arrowhead indicate ventral and dorsal nerve cords, respectively). (L) LNP-1 trapped in neuronal cell bodies but absent from other structures such as dorsal cords (arrow and arrowheads, respectively) in theunc-104(e1265)⁄kinesin mutant. Scale bars, 10lm(A, B, E–L), 50lm(C and D).
624 L. Ghila and M. Gomez
LNP-1 antibody or express an unstable form of LNP-1. These two homozygous strains are viable and do not show any obvious behavioural or developmental defects.
ThejsIs1strain expresses at low level the SNB-1::GFP transgene, driven by its endogenous promoter, representing the overall expression of endogenous SNB-1 (Nonet, 1999). As previously described (Nonet, 1999), SNB-1::GFP is concentrated in presynaptic elements of the three major neuronal structures, the nerve ring (data not shown) and the ventral and dorsal nerve cords (Fig. 3A–B¢). No staining was detected in commissures, consistent with the fact these structures are devoid of synapses. A faint signal is detected in neuronal cell bodies, resulting from overexpression of the transgene. Inlnp-1mutants, the distribution of SNB-1::GFP was significantly altered. GFP fluores-cence was no longer exclusively restricted to presynaptic sites of dorsal and ventral nerve cords but was also detected in cell bodies and
(Fig. 3E–I). To explain the increase in ventral cord fluorescence intensity oflnp-1mutants, the fluorescence profile of SNB-1::GFP in this cord was determined (Ackleyet al., 2005; Daiet al., 2006). The main difference in SNB-1::GFP fluorescence profiles between wild-type andlnp-1mutants was the almost complete lack of distinguish-able individual puncta in mutant animals, displaying a ‘linear’ signal along the ventral cord (supplementary Fig. S2, A). Therefore accurate quantification of the signal distribution (e.g. puncta area, puncta density, etc.) could not be performed. However the maximal value of the fluorescence signal inlnp-1mutants was similar to that of wild-type animals but the period and amplitude of the graph were smaller in lnp-1mutants, reflecting an increase in the total fluorescence in the ventral cord of these animals.
To determine whether the SNB-1::GFP protein level was altered in mutant backgrounds, we performed Western blot analysis with equal Fig. 3. SNB-1::GFP was mislocalized inlnp-1mutants. (A and B) Characteristic punctate localization of SNB-1::GFP (jsIs1strain) in presynaptic structures of the ventral and dorsal nerve cords in wild-type animals. (C and D) Mislocalization of SNB-1::GFP inunc-104mutants. Note that signal was detected in cell bodies (arrow in C and C¢) but only weakly in the dorsal cord (D and D¢). (E and G) Mislocalization of SNB-1::GFP inlnp-1mutants in cell bodies (arrow) and in commissures (arrowheads).
(F and H) SNB-1::GFP distribution in dorsal cord oflnp-1mutant. (A¢–H¢) Images magnified 3·of the boxed regions indicated in A–H. Ventral cords are indicated by asterisks (*) in A, C, E and G, and cell bodies and commissures by arrows and arrowheads, respectively, in A¢, C¢, E¢and G¢. Note that the phenotype of SNB-1::GFP misdistribution inlnp-1mutants is different from that in theunc-104(e1265)mutant. Compare panels E–H with panels C and D. (J and K) Double immunostaining using
(F and H) SNB-1::GFP distribution in dorsal cord oflnp-1mutant. (A¢–H¢) Images magnified 3·of the boxed regions indicated in A–H. Ventral cords are indicated by asterisks (*) in A, C, E and G, and cell bodies and commissures by arrows and arrowheads, respectively, in A¢, C¢, E¢and G¢. Note that the phenotype of SNB-1::GFP misdistribution inlnp-1mutants is different from that in theunc-104(e1265)mutant. Compare panels E–H with panels C and D. (J and K) Double immunostaining using