GFP-SLAIN1 and -SLAIN2 expression constructs and their deletion mutants were generated using the mouse cDNA IMAGE (integrated molecular analysis of genomes and their expression) clone 6811096 and the human cDNA KIAA1458 (a gift from Kazusa DNA Research Institute) in pEGFP-C1 by PCR-based strategies. In BioGFP fusions, a linker encoding the sequence MASGLNDIFEAQKIEWHEGGG, which is the substrate of biotin ligase BirA is inserted into the NheI and AgeI sites in front of the GFP (pBioGFP-C1). The BirA ligase expression construct, which contains the Escherichia coli
BirA open reading frame fused to the triple HA tag and cloned into the cytomegalovirus enhancer and promoter-containing vector pSCT (Driegen et al., 2005
) was a gift from D. Meijer (Erasmus MC, Rotterdam, Netherlands). mCherry-SLAIN1/2 were made by recloning SLAIN1/2 into MluI–EcoRI sites of a modified pEGFP vector in which the GFP open reading frame was substituted for that of mCherry (a gift from R. Tsien, University of California, San Diego, San Diego, CA; Shaner et al., 2004
). GFP–ch-TOG, based on a human ch-TOG cDNA in pEGFP-C–based vector, was a gift from L. Wordeman (University of Washington, Seattle, WA); this construct was used for generating ch-TOG deletion fragments in pBioGFP-C1 by a PCR-based strategy. We also used the following previously described pEGFP-C1–based expression constructs: GFP–α-tubulin, encoding human α-tubulin (Takara Bio Inc.), GFP–CLIP-115, which encodes full-length rat CLIP-115 (De Zeeuw et al., 1997
), GFP–CLIP-170, which encodes full-length rat CLIP-170 (Hoogenraad et al., 2000
), GFP–CLIP-170-N, which encodes amino acids 4–309 of the rat CLIP-170 (Komarova et al., 2002
) and GFP–CLASP2-ΔM, which encodes amino acids 36–340 fused in frame to amino acids 581–1,294 of CLASP2-γ (Mimori-Kiyosue et al., 2005
). mCherry–α-tubulin contained a human α-tubulin coding sequence N-terminally fused to mCherry (Shaner et al., 2004
). EB3-GFP, based on the human cDNA, was generated in pEGFP-N1 (Stepanova et al., 2003
). Point mutations in the GFP–SLAIN2-C3 fragment were introduced by overlapping PCR. pSuper-based small hairpin RNA (shRNA) vectors (Brummelkamp et al., 2002
) were directed against the following target sequences: mouse/rat ch-TOG, 5′-AGAGTCCAGAATGGTCCAA-3′; and mouse/rat/human SLAIN2, 5′-CTCTATAGATAGTGAGTTA-3′.
Cell culture, stable cell lines, and transfection of DNA constructs
HeLa, Swiss 3T3, CHO, and HEK293 were cultured in medium which consisted of 45% DME, 45% Ham’s F10, and 10% fetal calf serum supplemented with penicillin and streptomycin (Akhmanova et al., 2001
). HeLa cell lines stably expressing GFP–α-tubulin, EB3-GFP (Mimori-Kiyosue et al., 2005
), mCherry–α-tubulin (Splinter et al., 2010
), GFP-SLAIN2, and the GFP-SLAIN2 #3 rescue construct and the 3T3 cell line stably expressing EB3-GFP were selected using FACS and cultured in the presence of 0.4 mg/ml G418 (Roche). PolyFect (QIAGEN), FuGENE 6 (Roche), or Lipofectamine 2000 (Invitrogen) reagents were used for plasmid transfection.
siRNAs were synthesized by Invitrogen or Thermo Fisher Scientific. They were directed against the following target sequences: SLAIN1 #1, 5′-GACAUGUAGUGAACAAGAA-3′; SLAIN1 #2, 5′-GUAACAUGCCUUUAUCAAA-3′; SLAIN1 #3, 5′-GCAGCAACAGUAUUAUUC-3′; SLAIN2 #1, 5′-GCGCAGUUCUGGUUCAUCU-3′; SLAIN2 #2, 5′-CUCUAUAGAUAGUGAGUUA-3′; SLAIN2 #3, 5′-GGAACUUGAUGCACAAAGU-3′; control, 5′-GCACUCAUUAUGACUCCAU-3′ (Mimori-Kiyosue et al., 2005
); human ch-TOG, 5′-GAGCCCAGAGUGGUCCAAA-3′ (Cassimeris and Morabito, 2004
); mouse ch-TOG, ON-TARGETplus SMARTpool L-0470; EB1, 5′-AUUCCAAGCUAAGCUAGAA-3′ (Watson and Stephens, 2006
); EB3, 5′-CUAUGAUGGAAAGGAUUAC-3′ (Komarova et al., 2005
); CLIP-170, 5′-GGAGAAGCAGCAGCACAUU-3′ (Lansbergen et al., 2004
); CLASP1, 5′-GCCAUUAUGCCAACUAUCU-3′; and CLASP2, 5′-GUUCAGAAAGCCCUUGAUG-3′ (Mimori-Kiyosue et al., 2005
). Synthetic oligos were transfected using a transfection reagent (HiPerFect; QIAGEN) at a concentration of 5 nM. Cells were analyzed 72 h after transfection.
In the case of siRNA-mediated ch-TOG and SLAIN2 knockdown, 3T3 and HeLa cells were blocked in interphase 1 d after transfection by adding 2 mM thymidine (Sigma-Aldrich) to culture medium for 2 d. HeLa cells were blocked in mitosis by a 16 h treatment with 0.1 µM nocodazole (Sigma-Aldrich) or with 7.5 µM S-trityl-l-cysteine (STLC; Eg5 inhibitor; Sigma-Aldrich). Cells were released from the mitotic block for 60 min in the presence of the following inhibitors: 10 µM RO-3306 (EMD), 100 nM BI-2536 (Selleck), 20 mM LiCl, 10 µM flavopiridol (Sigma-Aldrich), and 20 µM MG132 (Sigma-Aldrich). Nocodazole washout experiments were performed by applying 10 µM nocodazole for 2 h followed by washout of the drug for 5–20 min.
Total RNA was isolated from HeLa cells using RNA-Bee (Tel-Test, Inc.) according to the manufacturer’s protocol. cDNA was generated using a reverse transcription system (First-Strand cDNA synthesis SuperScript II RT; Invitrogen). Human brain cDNA was a gift from E. Mientjes (Erasmus MC, Rotterdam, Netherlands). Primers used for amplification of SLAIN2 were as follows: forward, 5′-TAAGTGCTTCAGAATTAGAT-3′; and reverse, 5′-CATCATGCAGTATACCCTG-3′. SLAIN1 primers were previously described (Smith et al., 2010
Protein purification, peptide preparation, pull-down assays, and protein analysis
GST fusions of the N-terminal and C-terminal fragments of SLAIN2 were generated in pGEX-4T-1. GST-EB1, -EB2, -EB3, –EB1-N (amino acids 1–124), and –EB1-C (amino acids 125–268) were generated in pGEX-3X using full-length mouse EB1 and EB2 (long isoform) and human EB3 (long isoform) cDNAs (Komarova et al., 2005
). GST–CLIP-170-N (amino acids 1–310 of rat CLIP-170) was described previously (Lansbergen et al., 2004
). GST pull-downs, IPs, and Western blotting were performed according to Komarova et al. (2005)
and Lansbergen et al. (2004)
. Treatments with λ phosphatase (New England Biolabs, Inc.) were performed on beads after GST pull-down assays or IP. All GST fusions were expressed in BL21 E. coli
and purified with glutathione–Sepharose 4B (GE Healthcare) according to the manufacturer’s instructions. The His6-tagged SLAIN2 fragment (amino acids 1–43) was inserted in the vector PSTCm1 (Olieric et al., 2010
). It was expressed in BL21 (DE3; Agilent Technologies) and purified by immobilized metal affinity chromatography on Ni2+
-Sepharose (GE Healthcare) followed by size exclusion chromatography using a separation column (Superdex 200 10/300 GL; GE Healthcare). Identity of the protein was confirmed by liquid chromatography/mass spectrometry. BioGFP-SLAIN2 was purified from HEK293T cells. 70% confluent HEK293T cells were cotransfected with the constructs BioGFP-SLAIN2 and BirA using Lipofectamine 2000. 1 d after transfection, cells were lysed in a buffer containing 20 mM Tris-HCl, 100 mM KCl, 1% Triton X-100, and protease inhibitors (Complete; Roche) and purified with Mutein beads (Roche) according to the manufacturer’s instructions.
Human CLIP-170 and p150Glued
fragments CLIPCG1 (residues 56–128), CLIPCG2 (residues 210–282), CLIPCG12 (residues 48–300), and p150CG (residues 18–111) were cloned into pETG20A-MTA using the Gateway cloning system (Invitrogen) and purified as previously described in Weisbrich et al. (2007)
. In brief, transformed E. coli
strains BL21 (DE3; Agilent Technologies; for CLIPCG1, CLIPCG2, and p150CG) and C41 (DE3; Lucigen; for CLIPCG12) were grown at 37°C in Luria-Bertani media to an OD600
of 0.7. Expression was induced with 1mM IPTG and performed overnight at 20°C. The His6-tagged fusion proteins were affinity purified by immobilized metal affinity chromatography on Ni2+
-Sepharose at 4°C. Proteolytic cleavage to remove the His6 tag was performed at 4°C using human thrombin (Sigma-Aldrich). Cleaved proteins were subjected to a second Ni2+
-Sepharose column and further purified by size exclusion chromatography on Superdex 75 (CLIPCG1, CLIPCG2, and p150CG) or Superdex 200 columns (CLIPCG12; GE Healthcare) equilibrated in PBS (137 mM NaCl, 2.7 mM KCl, 8.3 mM Na2
, and 1.47 mM KH2
, pH 7.4). Throughout the CG12 purification, reducing conditions (1 mM β-mercaptoethanol) were maintained. The homogeneity of the recombinant proteins was assessed by SDS-PAGE, and their identity was confirmed by mass spectral analysis.
The SLAIN2c (residues 569–581 of human SLAIN2), SLAIN2c-W576A, and SLAIN2c-ΔY581 peptides were purchased from United Peptide. The purity of the peptides was verified by reversed-phase analytical HPLC, and their identities were assessed by mass spectral analysis.
Size exclusion chromatography coupled to multiangle light scattering was performed on a three-angle detector (DAWN EOS; Wyatt) followed by a refractometer (Optilab Rex; Wyatt). Protein solutions (100 µl of 1–15 mg/ml) were injected on a size exclusion chromatography column (Superdex 200 10/300 GL) equilibrated with PBS. Molecular weights were calculated by using the ASTRA V version 184.108.40.206 software package (Wyatt).
Far-UV circular dichroism spectroscopy was performed on a spectrometer (Chirascan-plus; Applied Photophysics) equipped with a temperature-controlled quartz cell of 0.1-cm path length. A ramping rate of 1°C/min was used to record thermal unfolding profiles. Midpoints of the transitions (Tm’s) were taken as the maximum of the derivative (d): d[θ]222/dT, in which T is the temperature.
ITC experiments were performed in PBS at 25°C on a calorimeter (iTC200; MicroCal) machine. The sample cell was filled with 160–240 µM CAP-Gly solutions. The syringe was filled with 2.2–2.6 mM SLAIN2c peptide solutions. In the experiment with CLIPCG12, the buffer was supplemented with 1 mM β-mercaptoethanol. 2.6-µl SLAIN2c aliquots from the stirred syringe were injected 14–28 times into the sample cell. To determine the binding stoichiometry and the equilibrium dissociation constant (Kd) of the binding, isotherms were fitted using a nonlinear least squares minimization method provided with the ITC calorimeter. Exact concentrations of protein solutions were determined by absorbance at 280 nm in 6 M GuHCl for the CAP-Gly domains or by quantitative amino acid analysis for the SLAIN2c peptide.
Crystal structure determination
For crystallization, CLIPCG1 and SLAIN2c in PBS were mixed in a 1:1.2 ratio to reach a final complex concentration of 24 mg/ml. Crystals were obtained at 20°C by the hanging-drop vapor diffusion method from a 1:1 mixture of the complex solution and a reservoir composed of 36% PEG 6000 and 100 mM citric acid, pH 4.5.
X-ray diffraction data were collected at 100 K at beamline X06DA of the Swiss Light Source (Villigen PSI). The structure was solved by molecular replacement using the CLIP-170 CAP-Gly structure as a search model (Protein Data Bank accession no. 2E3I). Data processing and refinement statistics are summarized in Table S1. The atomic coordinates of the CLIPCG1–SLAIN2c complex have been deposited in the Protein Data Bank (accession no. 3RDV).
GST pull-down assays followed by mass spectrometry and streptavidin bead pull-down assays from HeLa cells followed by mass spectrometry were performed as previously described by Grigoriev et al. (2007)
. 1D SDS-PAGE gel lanes were cut into 2-mm slices using an automatic gel slicer and subjected to an in-gel reduction with dithiothreitol, alkylation with iodoacetamide, and digestion with trypsin (sequencing grade; Promega) essentially as previously described by Wilm et al. (1996)
. Nanoflow liquid chromatography with tandem mass spectrometry was performed on a capillary liquid chromatography system (1100 series; Agilent Technologies) coupled to either an LTQ Orbitrap or an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific) both operating in positive mode and equipped with a nanospray source. Peptide mixtures were trapped on a reversed-phase column (ReproSil C18; Dr. Maisch GmbH; column dimensions were 1.5 cm × 100 µm, packed in house) at a flow rate of 8 µl/min. Peptide separation was performed on another ReproSil C18 reversed-phase column (column dimensions were 15 cm × 50 µm, packed in house) using a linear gradient from 0 to 80% B (A, 0.1% formic acid; B, 80% [vol/vol] acetonitrile and 0.1% formic acid) in 70 min and at a constant flow rate of 200 nl/min using a splitter. The column eluent was directly sprayed into the electrospray ionization source of the mass spectrometer. Mass spectra were acquired in continuum mode; fragmentation of the peptides was performed in the data-dependent mode. Peak lists were automatically created from raw data files using the Mascot Distiller software (version 2.1; Matrix Science). The Mascot search algorithm (version 2.2) was used for searching against the International Protein Index database (release number IPI_mouse_20100507.fasta or IPI_human_20100507.fasta). The peptide tolerance was typically set to 10 ppm for Orbitrap data and to 2 D for ion trap data. The fragment ion tolerance was set to 0.8 D. A maximum number of two missed cleavages by trypsin were allowed, and carbamidomethylated cysteine and oxidized methionine were set as fixed and variable modifications, respectively. The Mascot score cut-off value for a positive protein hit was set to 60. Individual peptide tandem mass spectrometry spectra with Mascot scores <40 were checked manually and either interpreted as valid identifications or discarded. Proteins present in the negative controls (pull-down assays with either GST or bioGFP alone) were omitted from the table.
Phosphorylated peptides were selectively enriched in an offline chromatographic manner using a TiO2
(Titansphere) packed fused silica capillary that is used as a trap, which acts as an 1D separation step in a 2D chromatography system (Pinkse et al., 2004
). Phosphorylated peptides were separated from nonphosphorylated peptides by trapping them under acidic conditions on the TiO2
column and ultimately desorbed under alkaline conditions, dried, and dissolved in 0.1 M formic acid. Subsequently, nanoflow liquid chromatography with tandem mass spectrometry was performed on an capillary liquid chromatography system (1100 series) coupled to a mass spectrometer (LTQ Orbitrap) operating in positive mode and equipped with a nanospray source as described in the previous paragraph. The Mascot search algorithm (version 2.2) was used for searching against the International Protein Index database (release number IPI_human_20100507).
Antibodies and immunofluorescent cell staining
Rabbit antibodies against SLAIN1/2 were raised against a bacterially purified GST-SLAIN2 N terminus. We used rabbit polyclonal antibodies against GFP (Abcam), CLASP1 (Mimori-Kiyosue et al., 2005
), CLASP2, CLIP-170 (Akhmanova et al., 2001
), EB3 (Stepanova et al., 2003
), phosphorylated histone H3 (Ser 10; Millipore), cyclin B1 (GNS1; Santa Cruz Biotechnology, Inc.), and ch-TOG (a gift from L. Cassimeris, Lehigh University, Bethelehem, PA; Charrasse et al., 1998
); mouse monoclonal antibodies against GFP and HA tag (Roche), EB1 (BD), β-tubulin, acetylated tubulin (Sigma-Aldrich), p150Glued
(BD), and actin (Millipore); and the rat monoclonal antibody against EB1/3 (clone 15H11; Absea) and HA tag (Roche). The following secondary antibodies were used: alkaline phosphatase–conjugated anti–rabbit, anti–mouse, or anti–rat antibodies (Sigma-Aldrich); goat anti–rabbit, anti–mouse, and anti–rat IgG (IRDye 800CW; LI-COR Biosciences); and Alexa Fluor 350–, Alexa Fluor 488–, and Alexa Fluor 598–conjugated goat antibodies against rabbit, rat, and mouse IgG (Invitrogen).
Cultured cells were fixed with −20°C methanol for 15 min in the case of EB1/3, ch-TOG, CLASP1/2, and p150Glued labeling. In the case of EB1, SLAIN1/2, CLIP-170, acetylated tubulin, and β-tubulin labeling, cells were fixed with −20°C methanol for 15 min and postfixed in 4% PFA in PBS for 15 min at RT. Cells were rinsed with 0.15% Triton X-100 in PBS; subsequent washing and labeling steps were performed in PBS supplemented with 1% bovine serum albumin and 0.15% Tween 20. At the end, slides were rinsed in 100% ethanol, air dried, and mounted in mounting medium (Vectashield; Vector Laboratories).
Image acquisition and processing
Images of fixed cells were collected with a microscope (DMRBE; Leica) equipped with PL Fluotar 100× 1.3 NA or 40× 1.00–0.50 NA oil objectives with a FITC/EGFP filter 41012 (Chroma Technology Corp.) and Texas red filter 41004 (Chroma Technology Corp.) and a charge-coupled device (CCD) camera (ORCA-ER-1394; Hamamatsu Photonics).
Live-cell imaging was performed on an inverted research microscope (Eclipse Ti-E; Nikon) with a Perfect Focus System (Nikon), equipped with CFI Apo total internal reflection fluorescence (TIRF) 100× 1.49 NA oil objective (Nikon) and an EMCCD camera (QuantEM 512SC; Roper Scientific) and controlled with MetaMorph 7.5 software (Molecular Devices). The 16-bit images were projected onto the CCD chip with intermediate lens 2.5× at a magnification of 0.065 µm/pixel. To keep cells at 37°C, we used a stage-top incubator (model INUG2E-ZILCS; Tokai Hit); cells were imaged in the normal culture medium. The microscope was equipped with a TIRF-E motorized TIRF illuminator modified by Roper Scientific (PICT-IbiSA; Institut Curie). For regular imaging, we used a mercury lamp (HBO-100W/2; Osram) for excitation or 491-nm 50-mW Calypso (Cobolt) and 561-nm 50-mW Jive (Cobolt) lasers. We used an ET-GFP filter set (Chroma Technology Corp.) for imaging of proteins tagged with GFP and an ET-mCherry filter set (Chroma Technology Corp.) for imaging of proteins tagged with mCherry. For simultaneous imaging of green and red fluorescence, we used an ET-mCherry/GFP filter set together with an imaging system (Dual View DV2; Roper Scientific) equipped with dichroic filter (565 DCXR; Chroma Technology Corp.) and an emission filter (HQ530/30m; Chroma Technology Corp.).
The FRAP assay was performed using a FRAP scanning system (I-Las/I-Launch [Roper Scientific]; PICT-IBiSA [Institut Curie]) installed on the same microscope and with the lasers mentioned in the previous paragraph at 100% laser power.
For imaging of mitotic cells, we used a spinning-disc microscope (CSU-X1-A1; Yokogawa) equipped with a 405/491/561 triple band mirror and GFP, mCherry, and GFP/mCherry emission filters (Chroma Technology Corp.) installed on an inverted research microscope (Eclipse Ti-E), which is almost identical to the one described previously in this section. The 16-bit images were projected onto the CCD chip with a 2.0× intermediate lens at a magnification of 0.068 µm/pixel.
Images were prepared for publication using MetaMorph and Photoshop (Adobe). All images were modified by adjustments of levels and contrast; for images of live cells, averaging of several consecutive frames was performed in some cases. In addition to adjustments of levels and contrast, Unsharp Mask and Blur filters (Photoshop) were applied to tubulin images. Maximum intensity projection, kymograph analysis, and various quantifications were performed in MetaMorph. Statistical analysis was performed using a nonparametric Mann-Whitney U test in Statistica for Windows (Microsoft) and SigmaPlot (Systat Software, Inc.).
Measurement of parameters of MT dynamics
Two HeLa cell lines stably expressing GFP– or mCherry–α-tubulin were used for the analysis at 72 h after transfection with SLAIN2 and ch-TOG siRNAs. The data were pooled because no significant differences were observed between the two lines for the measured parameters. Live-cell images were collected with 30, 10, and 2 frames per second. Initial analysis revealed no significant differences in the measured values. Therefore, all the data were averaged to obtain movies with a 0.5-s time interval for the final analysis. Parameters of MT growth were confirmed by independent measurements using HeLa cells stably expressing EB3-GFP. We applied kymograph analysis in order to distinguish very short episodes of growth and shortening, which are relevant for describing the phenotypes of SLAIN2 and ch-TOG depletion. This led to much higher values for transition frequencies than those commonly determined using MT life history plots or particle-tracking algorithms for EB-GFP videos. For comparison, a similar analysis was performed in Swiss 3T3 fibroblasts stably expressing EB3-GFP after siRNA transfection or in CHO cells after transient cotransfection of EB3-GFP and shRNA constructs. As we only focused on MT dynamics in internal cell regions, we did not analyze the frequency and duration of pausing that is mostly associated with region-specific cortical MT stabilization. For measurements of instantaneous growth and shortening rates, the velocity of MT end displacements that were longer than 0.5 μm were taken into account. Statistical analysis was performed using the Mann-Whitney U test.
Online supplemental material
Fig. S1 provides details of the mass spectrometry data on the identification of SLAIN2 and ch-TOG as EB1-binding partners and illustrates dimer formation by the SLAIN2 N terminus and the specificity of anti-SLAIN1/2 antibodies. Fig. S2 provides additional data on the SLAIN2–CLIP-170 interaction. Fig. S3 provides details of the mass spectrometry analysis of SLAIN-binding partners, RT-PCR–based data on expression of SLAIN1 and 2 and characterization of SLAIN2-depleted cells. Fig. S4 provides additional data on SLAIN2-mediated ch-TOG binding to EB1 and to MT tips and the dominant-negative properties of the ch-TOG C terminus. Fig. S5 shows MT density after depletion or disruption of the SLAIN2–ch-TOG complex and MT organization and dynamics in SLAIN2 and ch-TOG–depleted cells. Video 1 shows that GFP-SLAIN2 tracks growing MT ends in interphase HeLa cells. Video 2 shows that SxIP-like motifs of SLAIN2 contribute to its plus end–tracking behavior. Video 3 shows that GFP–ch-TOG colocalizes with mCherry-SLAIN2 at the plus ends of growing MTs. Video 4 shows MT plus end growth visualized with EB3-GFP in control, SLAIN2, and ch-TOG–depleted cells. Video 5 shows that GFP-SLAIN2 does not track MT plus ends in metaphase cells. Video 6 shows that GFP-SLAIN2 does not track MT plus ends in anaphase cells. Video 7 shows that GFP-SLAIN2 reassociates with MT plus ends in late telophase. Table S1 provides information on x-ray data collection and refinement statistics. Table S2 provides the values and statistical analysis of the MT dynamics parameters. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201012179/DC1