Gene organization, evolution and expression of the microtubule-associated protein ASAP (MAP9)
© Venoux et al; licensee BioMed Central Ltd. 2008
Received: 20 December 2007
Accepted: 09 September 2008
Published: 09 September 2008
ASAP is a newly characterized microtubule-associated protein (MAP) essential for proper cell-cycling. We have previously shown that expression deregulation of human ASAP results in profound defects in mitotic spindle formation and mitotic progression leading to aneuploidy, cytokinesis defects and/or cell death. In the present work we analyze the structure and evolution of the ASAP gene, as well as the domain composition of the encoded protein. Mouse and Xenopus cDNAs were cloned, the tissue expression characterized and the overexpression profile analyzed.
Bona fide ASAP orthologs are found in vertebrates with more distantly related potential orthologs in invertebrates. This single-copy gene is conserved in mammals where it maps to syntenic chromosomal regions, but is also clearly identified in bird, fish and frog. The human gene is strongly expressed in brain and testis as a 2.6 Kb transcript encoding a ~110 KDa protein. The protein contains MAP, MIT-like and THY domains in the C-terminal part indicative of microtubule interaction, while the N-terminal part is more divergent. ASAP is composed of ~42% alpha helical structures, and two main coiled-coil regions have been identified. Different sequence features may suggest a role in DNA damage response. As with human ASAP, the mouse and Xenopus proteins localize to the microtubule network in interphase and to the mitotic spindle during mitosis. Overexpression of the mouse protein induces mitotic defects similar to those observed in human. In situ hybridization in testis localized ASAP to the germ cells, whereas in culture neurons ASAP localized to the cell body and growing neurites.
The conservation of ASAP indicated in our results reflects an essential function in vertebrates. We have cloned the ASAP orthologs in mouse and Xenopus, two valuable models to study the function of ASAP. Tissue expression of ASAP revealed a high expression in brain and testis, two tissues rich in microtubules. ASAP associates to the mitotic spindle and cytoplasmic microtubules, and represents a key factor of mitosis with possible involvement in other cell cycle processes. It may have a role in spermatogenesis and also represents a potential new target for antitumoral drugs. Possible involvement in neuron dynamics also highlights ASAP as a candidate target in neurodegenerative diseases.
Microtubules (MTs) are linear polymers which self-assemble from α-β tubulin heterodimers in the presence of GTP [1–3]. They participate in a number of functions within cells including chromosome movements in mitosis, vesicle and organelle motility, cell polarity, and flagellar-based motility [4–6]. During interphase, MTs are organized in astral arrays that radiate from the centrosome, and function as a scaffold to direct organelle and vesicle trafficking. During mitosis, the centrosome mediates the assembly and organization of the mitotic spindle that is required for correct chromosome segregation.
This variety of roles is made possible by the dynamic nature of the MT cytoskeleton, modulated by multiple accessory proteins [7, 8]. A delicate balance between MT stabilizing and destabilizing proteins is believed to generate the MT dynamics observed in cells [9–14]. Molecules regulating MT dynamics have been identified, and their effects on the assembly of purified tubulin into MT determined. Several spindle proteins, including XKCM1 and XMAP215 family members have been identified that either stabilize or destabilize MTs by mediating the rapid changes between polymerization and depolymerization [8, 15]. These proteins play an important role in spindle formation. Another important group of spindle proteins comprise motors of the kinesin and dynein families essential for mitotic progression [8, 15–18], and the small GTPase Ran [19–21].
MTs are especially abundant in testis and neurons. Cell-type specific MTs, such as the Sertoli cell MTs and the manchette and flagellum MTs of the spermatids, play essential roles in spermatogenesis. Others are important for flagellar-based motility. In neurons, their integrity is essential to maintaining normal neuron morphology and in the transport of materials between cell body and synaptic terminals. The MT network undergoes drastic changes in neural cells at different developmental stages. For instance, during neural proliferation, MTs assemble into the highly organized mitotic spindle on entering mitosis. In order to transmit signals, neurons stop dividing early in development and direct their efforts towards the elaboration of elongated cellular processes. Neurons are thus terminally post-mitotic cells that no longer form mitotic spindles. After the post-mitotic neurons are generated, they extend a directional process and migrate towards their destination, during which another MT network takes place. MAPs have been shown to directly regulates MT dynamics during many of these developmental processes.
We have recently characterized a novel human MAP named ASAP (AS ter-A ssociated P rotein)  that was renamed MAP9 in the nomenclature. ASAP localizes to MTs in interphase, associates with the mitotic spindle during mitosis, localizes to the central body during cytokinesis and directly binds to purified MTs by its COOH-terminal domain. Overexpression of ASAP induces profound bundling of cytoplasmic MTs in interphase cells and aberrant multipolar or monopolar spindles in mitosis. Depletion of ASAP by RNA interference results in severe mitotic defects, inducing the formation of aberrant mitotic spindle, delays in mitotic progression, defects in chromosome congression and segregation, defective cytokinesis and cell death. We have also shown that ASAP is a substrate of the oncogenic mitotic kinase Aurora-A . The phosphorylated form of ASAP localizes to centrosomes from late G2 to telophase and to the midbody during cytokinesis. Aurora-A depletion induces a proteasome-dependent degradation of ASAP. ASAP depletion provokes spindle-defects that are specifically rescued by expression of a phosphorylation-mimetic ASAP mutant indicating the need for ASAP phosphorylation by Aurora-A for bipolar spindle assembly and mitotic progression. These previous results suggest a crucial role of ASAP in the organization of the bipolar mitotic spindle, mitosis progression and cytokinesis and highlight ASAP as a putative target for cancer treatment.
Mice knock-out studies and Xenopus egg extracts have allowed detailed studies providing valuable information about the involvement of genes in regulatory pathways in the first model, and MT, mitotic spindle formation and cell cycle in the latter. In this report, we characterized the structure of the ASAP gene in human and mouse. We identified the ASAP orthologs in different species in order to perform phylogenetic and molecular evolution analyses and cloned and characterized the murine and Xenopus orthologs. We also show that ASAP is predominantly expressed in testis and brain, especially in the germ cell line and in the growing neurites, opening up new perspectives on ASAP functions.
All together, these results shed light on new putative functions of ASAP, in its role both in mitosis and post-mitotic neurons, and provide new tools for the scientific community.
Accession numbers of ASAP orthologs.
AY_690636, gi :56607126
NM_001039580, gi :88759338
Predicted XM_850247.1, gi :73978341
Predicted gene ENSCAFG00000008484
Prediction XP_855340.1, gi :73978342
Predicted protein ENSCAFP00000012464
NM_001081230, gi :124486996
XM_143366, gi :28491857
EU219608, gi :164633070
XM_689238, gi :125803791
Predicted gene ENSDARG00000037276
Predicted transcripts ENSDART00000076337
Predicted protein ENSDARP00000054224
Transcript XM_420374.2, gi:118089784
Hypothetical protein XP_4203374.2
Predicted gene ENSGALG00000020253
Predicted transcript ENSGALT00000032288
Predicted protein ENSGALP00000031652
In addition, the multiple alignment used for the phylogenetic analysis revealed two highly conserved regions (about 90% PSI between human, mouse and dog), i.e. the N- and C-terminus ends (amino acids 1–98 and amino acids 420–647, respectively), and a more divergent central region (≤50% PSI). The N-terminus region contains no known motifs or domains, whereas the C-terminus region corresponds to the MAP domain and contains three nuclear localization signal (NLS) motifs (see below) (Fig. 2C).
ASAP domains and motifs
Self-similarity regions of ASAP and MAP1A.
In addition, PSORTII software identified three NLS in the ASAP C-terminal domain corresponding to amino acids 483–501, 508–524 (bipartite NLS) and 621–624 (monopartite NLS), respectively (Fig. 4A).
Finally, as a very rich Ser/Thr protein, ASAP contains several putative phosphorylation sites, among which two are characterized and referenced on http://www.phosphosite.org: the S625 determined by ourselves and necessary for spindle formation and mitosis completion  is conserved only in mammals (Fig. 2C) and the S132 conserved in all vertebrate species as a SQ motif whose activation is typical of the ATM/ATR pathway in response to DNA damage . Other SQ sites are found across the homologs but not significantly conserved.
Overexpression of Cter and Nter ASAP deletion mutants
Mouse ASAP characterization
In order to develop the murine model as a tool, we cloned the full-length mouse ASAP (mASAP) cDNA by RT-PCR using total testis RNA and primers derived from the sequence available at the time in the database (XM_143366).
We then overexpressed the full-length EYFP-mASAP in U-2 OS (not shown) and NIH3T3 cells. As observed for human ASAP, the overexpressed mASAP induced bundles of stabilized microtubules in interphase cells and the same mitotic defects (i.e. an increased mitotic index with more than 70% of monopolar spindles)  even in the fibroblast-derived NIH3T3 cell line (Fig. 7C).
We then investigated if the protein was expressed in brain and testis extracts. As shown in Fig. 8C, ASAP protein is also strongly expressed as a 110 KDa protein in both tissues as observed in NIH3T3 and U2-OS cell line extracts. No expression was found in spleen or colon tissues, or in the colon-derived HT-29 cell line. It is noteworthy that in brain no higher molecular weight protein was detected, suggesting that the 9 Kb mRNA probably corresponded to a partially spliced ASAP mRNA.
Xenopus ASAP characterization
In a previous report, we demonstrated that ASAP/MAP9 is a novel microtubule-associated protein required for a proper cell cycle progression [22, 23]. In this paper, we detected homologs in all vertebrate species investigated and potential orthologs in different invertebrates, thus suggesting a requirement of ASAP in higher eukaryotes. The coding sequence and exon-intron structure have been conserved during vertebrate evolution, suggesting that selective constraints are exerted on this gene to maintain its function. These genes are also invariably located in regions syntenic to the human locus, demonstrating common ancestry. Invertebrate and vertebrate ASAP also likely derive from a common ancestor but which evolved independently after the separation of the two clades. This would account for the low level of homology where events such as insertion/deletion or exon shuffling shaped the ASAP gene differently.
The highly N-terminal conserved region 1–98 (Fig. 2C) corresponds to no known conserved motif and its function remains to be elucidated.
Importantly, the C-terminal MAP region is the most conserved not only within vertebrates, but also between invertebrates and vertebrates, suggesting that the conservation of the MAP function is essential. Indeed, the sequence similarity with C34D4.1 (C. elegans) or with the sea urchin protein (Table 1) is higher in the MAP region (amino-acids 428–568 and 340–680, respectively). C34D4.1 could be involved in the regulation of microtubule dynamics since it contains stathmin domains  that are known to be involved in the regulation of microtubule skeleton by acting on microtubule dynamics. In the vertebrate MAP region, other motifs such as NLS or an MIT-like domain are also conserved (Fig. 2C).
The sequence similarity of ASAP with different proteins/domains is concentrated in the C-terminal MAP domain and seems connected to microtubule binding/dynamics properties. For example, the character of ASAP as a microtubule binding protein is also found with the structural domains that ASAP shares with MAP1A. In particular there is a striking analogy with the self-similarity region SS1 that is involved in binding microtubules . Besides MAP1A, ASAP is also weakly homologous to dynein and kinesin, two motor proteins that bind to MTs, although no motor activity was found in ASAP (not shown). On the other hand, THY has been shown to be involved in the cytoskeleton organization by binding actin monomers and thus inhibiting actin polymerization. We also identified a potential MIT-like domain in the MAP domain of ASAP. This domain was first described in proteins involved in MT binding and in intracellular transport and named MIT for it being "contained within microtubule-interacting and trafficking molecules". For example the MIT domain has been identified in spastin (responsible for the dominant form of spastic paraplegia) , spartin (recessive form of spastic paraplegia) , both proteins that interact with microtubules, or VSP4  which is involved in the intracellular protein transport machinery and the regulation of membrane association of several proteins. As with spastin, ASAP overexpression causes perturbations in the MT network. The identification of related MIT domains in ASAP, spastin and spartin may therefore suggest the possible involvement of ASAP dysfunction in pathways leading to pathogenesis.
Many MAPs regulated by Aurora-A and involved in spindle assembly (TPX2, NuMA, RHAMM, TACC3) are nuclear in interphase and recruited after reorganization of the different compartments (nuclear envelope, endogenous membrane structures). They are regulated via their NLS by the small GTPase Ran, that releases and activates them from bound importins [19, 41]. The presence of these conserved NLS may suggest the regulation of ASAP by this pathway. In our fixation conditions, we observed no ASAP in the nucleus accounted for either by a very weak signal or by the fact that our antibody did not recognize the nuclear epitopes. However, when we overexpressed the Cter domain, we observed a localization of ASAP as nuclear foci, suggesting that the NLS are functional and become accessibles in this mutant. However, these nuclear foci could also suggest that ASAP, under certain conditions that need to be determined, has a nuclear function. Indeed, these foci are reminiscent of those observed after DNA damage. Although a putative BRCT-domain hit (amino acids 66–303) found in the ASAP protein sequence could not be confirmed with confidence, it may nevertheless be indicative of a putative role of ASAP in DNA damage, since BRCT domains are usually found in proteins involved in the checkpoint DNA-damage response. On the other hand, the S132, which is conserved in all vertebrates, corresponds to a SQ motif and has been identified in a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR . These data hint towards ASAP playing a role in the DNA damage response.
ASAP is very rich in Ser and Thr residues suggesting multiple putative phosphorylation sites. The S625 phosphorylated by Aurora-A is necessary for spindle assembly and completion of mitosis, but is conserved only in mammals (Fig. 2C) as the emergence of a new feature.
We have cloned the murine ortholog of ASAP and confirm an intracellular pattern similar to its human counterpart. We also showed that deregulation of this protein leads to the same mitotic defects even in the fibroblast derived NIH3T3 cell line, confirming that the phenotypes observed in U2-OS cells were not due to the transformed status of these cells. We have also cloned the Xenopus ortholog and demonstrated a similar localization when overexpressed. The identical subcellular ASAP localization within the MT network between these species suggests an evolutionary conservation of MAP function. However, the mitotic defects were not observed in the Xenopus cell line because of a lack of transfected mitosis, due either to low transfection efficiency or to the lethal issue of these transfected mitotic cells.
Tissue expression analyses have shown that ASAP is predominantly expressed in testis and brain. Such a distribution pattern has already been described for different MAPs. For example, MAP2 is neural-specific but is also expressed as a lower molecular weight isoform in the testis . The testis is one of the most abundant sources of MT networks. These include mitotic and meiotic spindles, the spermatid manchettes and axonemes, and the Sertoli cell cytoskeleton. Some MAPs, such as E-MAP-115, are required for spermatogenesis. Since ASAP is also expressed in the ovary and is involved in the mitotic spindle formation of cultured cells , its strong expression in this proliferative tissue is not surprising and a role in the meiotic spindle could be possible. We have indeed confirmed in a preliminary experiment that ASAP is specifically expressed in the germ cell line during spermatogenesis, and its expression may be stage-specific. One might expect an expression in spermatogonia which undergo rapid successive divisions or in spermatocytes where meiosis takes place. Spermatogenesis is an intricately regulated morphogenetic process during which many structural changes are necessary to produce mature spermatozoa. Differentiation and polarization of the round spermatid are associated with new microtubular configuration, that resembles that of pachytene spermatocytes . On the other hand, ASAP could be present in the perinuclear theca which is a rigid cytoskeleton that covers the entire nucleus of mammalian spermatozoa, and could coat the acrosomal vesicle of round spermatids before attachment to the anterior region of the nucleus. Different MAPs, such as Ndel1 , E-MAP-115 , MAP4 , and tau , are present in the spermatid. Together with these proteins, ASAP could be involved in nuclear shaping and the process of spermatid elongation. Deciphering the exact location of ASAP expression will require antibodies adapted for immunohistochemistry experiments.
Microtubules are also essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances. They are especially abundant in neurons, where they exhibit an extreme state of stability. They are involved in neuronal migration and positioning during cortical development. After the post-mitotic neurons are generated, they extend a directional process and migrate towards their destination, during which another MT network takes place. MAPs have been shown to be the direct regulators of MT dynamics during many of these developmental processes. In neurons, the major MAPs include tau, MAP1A, MAP1B and MAP2. These MAPs are phosphoproteins and the level of phosphorylation has been shown to regulate their activities to stimulate MT assembly. Tau has been associated with different neurodegenerative diseases such as Alzheimer's disease, Pick's disease and frontotemporal dementia associated with Parkinson's disease [48, 49]. However, several other proteins such as Ndel1, the partner of LIS1 involved in lissencephaly , ASPM involved in microcephaly , or spastin involved in hereditary spastic paraplegia , are also neuronal MAPs. The identification of numerous MAPs and the progressive elucidation of the mechanisms of MT assembly and transport are beginning to have a profound impact on the study and treatment of human genetic diseases such as neurodegenerative diseases (Huntington's disease, Alzheimer's disease) (for review see ). Here we have demonstrated the specific expression of ASAP in neurons and growing neurites, suggesting an important role in the brain. It is noteworthy that neuronal MAPs described above such as Ndel1, spastin and ASPM are also expressed at the mitotic spindles of cell cultures [36, 54, 55]. Even though neurons are quite dissimilar from typical interphase cells with regards to MT distribution and organization, several observations suggest that axonal and dendritic arrays may be established by mechanisms very similar to those used for the formation and function of the mitotic spindle .
Adult CNS neurons are considered as postmitotic but it appears that these cells must keep their cell cycle in check to avoid any reinitiation leading to an altered state. There is now growing evidence that neurons at risk of neurodegeneration are also at risk of reinitiating a cell cycle process , and several neurodegenerative disorders are related to cell cycle failures. In human, cell cycle events (loss of cell cycle control) are associated with several neurodegenerative diseases such as Alzheimer disease and ataxia telangiectasia .
ASAP is a novel MAP whose expression defects provoke aberrant mitoses leading to cell phenotypes reminiscent to those observed in cancers. ASAP is phosphorylated by the oncogenic mitotic kinase Aurora-A that plays a key role in mitotic spindle formation and the cell cycle, highlighting ASAP as a potential new target for anti-tumoral drugs [22, 23]. In this work, evolutionary and expression studies have shed light on new putative functions of ASAP both as a germ cell line and neuronal MAP that could be involved in spermatogenesis and neuronal developmental processes. Consequently, deregulation of ASAP expression in such tissues, as observed with other MAPs, may lead to spermatogenesis defects or neurodegenerative disease. Although our analysis shows an evolutionary conservation of MAP function in ASAP, it also suggests also that this protein might be involved in other cell cycle processes such as DNA damage response. Our data also validate mouse and Xenopus as models for further ASAP studies using either knock-out or MT in vitro experiments.
We performed general database searching using the BLAST program of the GCG package (University of Wisconsin) . Potential coding regions and gene structure were identified by comparison of the cDNA sequences with the genome sequences and the predicted transcripts http://www.ensembl.org, and examination by eye. We performed sequence comparisons using the Clustalw v. 1.8 software package  and PipMaker http://bio.cse.psu.edu/. Clustalw was used for the phylogenetic analysis and the tree was constructed using the neighbor joining method , based on the number of amino acid substitutions. The numbers on internal branches represent the frequency of occurrence among 1000 trees (bootstrap method with the Clustalw package). Clustal alignment was visualized using Jalview and clustal X colour codes were used as defined in the Jalview options . PipMaker was used for determining local alignments of human and mouse genes. Gap-free segments are displayed in a PIP (percent identity plot) and the corresponding dot-plot. We searched CpG islands using the cpgplot software from EMBOSS http://bioweb.pasteur.fr/seqanal/interfaces/cpgplot.html. Transcription factor binding sites were searched using TFSCAN from EMBOSS http://bioweb.pasteur.fr/seqanal/interfaces/tfscan.html and promoter regions were searched using the neural network promoter prediction software http://www.fruitfly.org/cgi-bin/seq_tools/promoter.pl.
Protein domains and motifs were searched using the following programs and databases: phi- and psi-blast programs of the NCBI platform, SMART [32, 33]http://smart.embl-heidelberg.de/, prosite http://www.expasy.ch/prosite/, pfam http://pfam.sanger.ac.uk/, MotifScan http://myhits.isb-sib.ch/cgi-bin/motif_scan, PSORTII http://www.psort.org/, all softwares being available in the Expasy package http://expasy.org.
Cter and Nter ASAP deletion mutants sub- cloning
The truncation mutants EYFP-ASAP-Cter (420–647) and EYFP-ASAP-Nter (1–420, that corresponds to the EYFP-ΔCter described in ) were constructed in pEYFP-C1 (Clontech, EYFP in N-ter) and pEAK-EGFP ((EGFP in C-ter). Since the same patterns were obtained in overexpression, only the EYFP constructions are presented in the results section.
Mouse and Xenopus cDNA cloning
BLAST analyses revealed a full-length mouse cDNA clone in the databases. We obtained the mouse ASAP cDNA (mASAP) by RT-PCR using total testis RNA and primers derived from the mouse sequence (mASAP-1F: 5'-ATGTCCGATGAAATCTTCAGCAC-3' and mASAP-1R: 5'-AAATACTTTTGAGGGCGCAGTTC). The resulting cDNA was cloned into the TA cloning vector (Invitrogen). Mouse cDNA was subcloned into EYFP-C1 (Clontech) and pGEX-4T-2 (Amersham) vectors.
At that time, BLAST analyses did not reveal any available partial or full-length Xenopus cDNA clone (X-ASAP). We PCR-screened primary and secondary DNA pools of X. laevis tadpole stage 24 (Library N° 725, RZPD, Germany) using primers derived from the AW 764609 and AW 764964 sequences that contained a partial cDNA X-ASAP sequence (X2F: 5'-AAAGCAGCATTTGAGGCATGG-3' and X1R: 5'-TGATAGCGGTTATAGTATCGTTC-3'). We isolated a single partial ORF clone (XL1) that also lacked the 3' end. However, this clone was overlapping with XL402j02 http://xenopus.nibb.ac.jp and was kindly provided by the NIBB (NBRP Xenopus ANE library) . A full cDNA clone was reconstructed by PCR, cloned into a TOPO-TA cloning vector and fully sequenced. Xenopus cDNA was subcloned into EYFP-C1.
Generation and affinity-purification of polyclonal mouse ASAP antibodies
Polyclonal rabbit sera to the full-length mouse protein were raised against the corresponding GST fusion protein purified from bacteria E. coli BL21RP+. The antibodies were affinity purified on a GST column followed by a GST-mASAP fusion protein affinity column. Antibodies were routinely used at 1:500 for immunofluorescence and 1:5000 for western blots.
Two human tissue northern blots (MTN I and MTN II, Clontech) were probed with a random-primed [32P]dCTP-labelled ASAP cDNA. Hybridizations were performed at 42°C and the blots were washed at high stringency according to the manufacturer's instructions, and then exposed in a PhosphorImager cassette for one week.
To assay mRNA transcripts expression in mouse, total cellular RNA was extracted from normal adult mouse tissues using the GenElute Total Mammalian Total RNA kit (Sigma-Aldrich) following the manufacturer's instructions. Total RNA were treated with DNAse (DNA-free kit from Ambion) as indicated by the manufacturer. cDNA synthesis and PCR amplification were performed with Superscript one-step RT-PCR (Invitrogen), using 200 ng of total RNA. Each element of a primer pair was chosen in different exons to discriminate with possible contaminations by genomic DNA (mFIS2F: 5'-AAGTGAAGACAGAAACACGAAG-3'; mFIS1R: 5'-CTGTGCATTTCATGTAAATACAC-3'), and amplification was performed for 30 cycles during which the exponential phase of PCR amplification was maintained. GAPDH cDNA was amplified for 24 cycles with the primers GAPDH1: 5'-GACCACAGTCCATGCCATCACT-3' and GAPDH2: 5'-TCCACCACCCTGTTGCTGTAG-3'. Ten microliters of PCR products were analyzed on a 1% ethidium bromide-stained agarose gel.
To assay mASAP protein expression in mouse, 1 to 2 mg of each tissue were directly crushed and lysed 2 h in (50 mM Tris-HCl, 2% SDS, 50% glycerol, 1% β-mercapto-ethanol) buffer supplemented with protease and phosphatase inhibitors). After 15 min centrifugation at 13000 rpm, supernatant was collected and proteins were analyzed by western-blot as described below.
Cell culture and transfections
All cell culture media and additives were obtained from Sigma. Human U-2 OS and Mouse NIH3T3 cells were routinely grown at 37°C in a 5% CO2 atmosphere in DMEM (Sigma) supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin. Where indicated, cells were synchronized by a thymidine-block (2 mM) 24 h, released and analyzed at S, G2 or M phases. Xenopus XL2 cells were grown in L-15 medium as described . U-2 OS and NIH3T3 cells were transfected using JetPei (Polyplus), while XL2- cells were transfected using the Amaxa-nucleofector system (solution T, program T-20), following the manufacturer's instructions. Human ASAP siRNA sequence and transfection procedure are described in .
Antibodies were used at dilutions or concentrations stated by the manufacturer, for both western blotting and immunofluorescence microscopy, unless indicated otherwise. Primary antibodies used were anti-α-tubulin (DM 1A, Sigma), anti β-tubulin (TUB 2.1, Sigma), anti-γ-tubulin (GTU-88, Sigma), anti-GFP (Zymed); mouse polyclonal anti-ASAP (1/500 for immunofluorescence and 1/3000 for western blotting). Secondary antibodies used for immunofluorescence were coupled to Alexa Fluor 488-, Alexa Fluor 546- or Alexa Fluor 642-conjugated goat anti-mouse or anti-rabbit IgG (1/1000, Molecular Probes). Secondary antibodies used for the immunoblots were peroxidase-conjugated goat anti-mouse or anti-rabbit (1/5000, Zymed). Hoechst 33258 was purchased from Sigma.
Cell extracts and Western blot analysis
Cells were washed with ice-cold PBS, scraped off the plate, and resuspended in ice-cold lysis buffer (50 mM Tris pH8.0, 120 mM NaCl, 5 mM EDTA, 0.5% NP40) supplemented with 1 mM DTT and a protease inhibitor cocktail tablet (Roche) and phosphatase inhibitors. After 15 min on ice, lysed cells were centrifuged at 13000 rpm for 10 min at 4°C. Ten to 15 mg of murine brain or testis were directly lysed in 1 ml of Laemmli buffer (50 mM Tris pH8.0, 2% SDS, 10% glycerol, 1% β-Mercaptoethanol). Protein concentrations in the cleared lysate were determined using a Bradford assay, and equal amounts were loaded on SDS-PAGE gels. Separated proteins were transferred to nitrocellulose membrane (Whatman Schleicher and Schuell) and were detected by various antibodies and visualized by enhanced chemiluminescent reagents (Supersignal West-PicoPico, Pierce Chemical).
Cells grown on coverslips were fixed either by incubation in 4% paraformaldehyde in MTSB (Microtubule Stabilization buffer: 100 mM PIPES, 1 mM EGTA, 4% PEG 8000, pH 6.9) 10 minutes at room temperature followed by 0.5% Triton X-100/MTSB for 5 min (PAF/MTSB fixation) or by incubation in formaldehyde 3.6% in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl2, pH 6.9) for 10 minutes followed by methanol 1 minute at room temperature (F/PHEM/methanol fixation). Fixed cells were incubated for 1 h at 37°C with the primary antibody, and 30 min at 37°C with the secondary antibody. All antibodies were diluted in PBS/3% BSA. Coverslips were mounted using Gel-Mount (Biomedia). Images were acquired on a Leica DM6000B fluorescence or a confocal Leica TCS SP2 microscopes using CCD cameras and subsequently processed by the Metamorph or LCS LEICA confocal softwares, respectively.
Cryosection and Immunofluorescence
Testis tissues were washed twice in PBS, fixed in 3.7% (vol/vol) paraformaldehyde/PBS for 18 h at 4°C, washed twice in PBS, and incubated overnight in 30% (wt/vol) sucrose/PBS. Samples were embedded in OCT medium (Tissue-Tek) and frozen at -70°C, and serial 12 μm sections were cut with a cryostat (Leica). After drying, the sections were rehydrated in PBS for 5 min. Tissue sections processing for immunofluorescence using the polyclonal mASAP rabbit serum (used at a 1/500 dilution) were as described .
Culture of mouse cortical neural stem cells and immunocytochemistry
The protocol used was described in Milhavet et al.  with minor modifications: during proliferation cells were cultured in a medium containing DMEM-F12 (Invitrogen Life Technologies, Cergy Pontoise, France) with modified N2 supplement and 25 ng/ml of Basic fibroblast growth factor (βFGF, Abcys, France). βFGF was added daily and medium changed every two days. Undifferentiated cells cultured in presence of βFGF expressed primarily nestin, an intermediate filament protein expressed in neural stem cells and progenitors, whereas β-III tubulin, a neuronal marker and GFAP, an astrocytic marker, were not. After two passages, cells at 80% confluence were differentiated by removal of βFGF in a culture medium containing 50% of DMEM-F12 with modified N2 supplement and 50% Neurobasal with B27 supplement (Invitrogen Life Technologies, Cergy Pontoise, France). During differentiation the medium was changed every two days. Cells were fixed in 4% paraformaldehyde plus 0.15% picric acid in PBS, and standard immunocytochemical protocols followed before observation with a Zeiss Axiovert 200 M microscope. The following primary antibodies were used: ASAP rabbit antibody at 1:500, β-tubulin type III (Tuj1) monoclonal antibody at 1:1000 (Covance Research Products, Berkeley, CA, USA) for specific detection of neuronal cells, and rabbit glial fibrillary acidic protein (GFAP) at 1:1000 (Dakocytomation, Trappes, France) for specific detection of glial cells. Appropriate fluorescence-tagged secondary antibodies (AlexaFluor 488 and 555, Invitrogen Life Technologies, Cergy Pontoise, France) were used for visualization. DAPI was used for nuclear counterstaining.
We would like to thank Drs B. Boizet-Bonhoure, B. Moniot, I. Davidson, R. Catena for helpful discussions and F. Estermann for technical help in XL2 cell transfection and immunofluorescence. MV is a recipient of a MERT fellowship. This work was supported by grants from ARC, Ligue nationale contre le Cancer (comité de l'Hérault) and Fondation Jérôme Lejeune to SR.
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