Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins Abstract Jaw1/LRMP is characterized as a Type II integral membrane protein that is localized to endoplasmic reticulum, however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with Klarsicht/ANC-1/Syne/homology (KASH) proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with Sad-1/UNC-84 (SUN) proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules. Jaw1/LRMP, KASH proteins, LINC complex, nuclear envelope, SUN proteins The nucleus is an organelle that contains packaged DNA, where it is encapsulated with a nuclear envelope (NE) consisting of double lipid bilayers, i.e. an inner nuclear membrane (INM) and an outer nuclear membrane (ONM) that are separated by the perinuclear space (PNS). For the modulation of gene expression in response to changes in the extracellular environment and flexible nuclear stiffness during cell migration, the maintenance or the regulation of dynamic changes in nuclear shape has been reported to be an important factor (1–3). Furthermore, positioning the nucleus to the precise location in cells is also important for cellular polarity, migration and differentiation (4). The linker of nucleus and cytoskeletons (LINC) complex consists of Klarsicht/ANC-1/Syne/homology (KASH) proteins that are localized to the ONM and Sad-1/UNC-84 (SUN) proteins localized to the INM, which form a complex via the interaction of these protein in the PNS. KASH proteins bind to microtubules or the actin-based cytoskeleton network on the cytosolic face and SUN proteins connect with the nuclear lamina in the nucleoplasm (5, 6). This cytoskeletal network that is produced via the LINC complex makes it possible to transduce the mechanical forces between the cytosol and nucleus for maintaining both the shape and position of the nucleus. Five KASH proteins, Nesprin1-4 and KASH5 and four SUN proteins, SUN1-4 have been identified to date (4, 7). The depletion of these components in the LINC complex results in aberrant nuclear shapes and alternations in the position of the nucleus in the cell, eventually leading to defects in cellular migration, cellular stiffness and cellular integrity (8–12). Furthermore, these nuclear physiological dysfunctions are possible causes of Emery–Dreifuss Muscular Dystrophy (13), infertility (14, 15) and Hutchinson–Gilford Progeria syndrome (16). Jaw1 has been identified as a Type II integral membrane protein that is localized on the endoplasmic reticulum (ER), and is specifically expressed in some immune cells and in Type II cells in taste buds (17–19). Jaw1 consists of an N-terminal coiled-coil domain oriented toward the cytosol, a single trans-membrane domain and a carboxyl-terminal luminal domain. The coiled-coil of Jaw1 has been reported to be interacted with Type III inositol 1,4,5-triphosphate receptor (IP3R3), a calcium-gate channel on ER membrane (19). Recent reports indicated that Jaw1 is localized on the ER and the NE which is continuous to the ER network. Furthermore, it is known that the Jaw1 carboxyl-terminal region has a partial homology to the PPPX motif, four amino acids of carboxyl-terminus in KASH proteins (20). Based on these data, Jaw1 has been hypothesized to function as a potential member of KASH protein (20–23). However, the interaction between Jaw1 and SUN proteins and physiological function of Jaw1 at nuclear membrane are unknown. In this context, we focussed on the issue whether Jaw1 functions as a new member of KASH protein and what the role of Jaw1 is at nuclear membrane. The findings reported herein indicate that Jaw1 has a role in maintaining nuclear shape. We confirmed that Jaw1 is localized to the ONM in addition to the ER in mouse melanoma cell line B16F10 cells. We also found that the siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape. Furthermore, co-immunoprecipitation experiments indicated that Jaw1 interacts with SUN proteins and microtubules. Based on these findings, we propose that Jaw1 functions as a component of the LINC complex and is involved in the maintenance of nuclear shape. Materials and Methods Production of Jaw1 polyclonal antibody The N-terminal region encoding amino acids (204–351) of human Jaw1(NM_006152.3) was amplified by polymerase chain reaction (PCR) using following primers: forward 5′-CACACGAATTCAGTGAGAACACTTCTGCT-3′, reverse 5′-CACGTCGACCTAAATTTGGCAGTCATCATCATC-3′ and inserted into pMAL cRI vector (New England Biolabs (NEB)) containing Tobacco etch virus (TEV) protease recognition site downstream of the maltose-binding protein (MBP). The MBP fused protein was overexpressed in BL21(DE3) and purified using a amylose resin (NEB). After cutting the MBP tag with a TEV protease, the solution was passed over the amylose resin again to trap the cut MBP tag, resulting in the isolation of the purified antigen. The prepared antigen was used to immunize rats. Primary immunization was performed by mixture with ADJUVANT COMPLETE FREUND (BD) and following ones were performed by mixture with ADJUVANT INCOMPLETE FREUND (BD). After the several immunizations, whole serum was collected and the anti-Jaw1 antibody was purified using an NHS-column coupled with the above purified antigen. For equilibration and washing the column, 0.02 M Tris buffer (pH 7.2) containing 0.5 M NaCl was used. Furthermore, the antibody was eluted with 0.2 M glycine, pH 3.0 and neutralized with 1 M Tris, pH 9.0. This experimental protocol was approved by the Animal Care and Use Committee of Tokyo University of Agriculture and Technology. Cells and cell culture B16F10 and HEK293T cells were incubated in MEM supplemented with 10% foetal bovine serum (FBS) and 5.84 mg/ml l-glutamine and in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma) and 5.84 mg/ml l-glutamine, respectively, and maintained at 37°C in 5% CO2. Cloning and plasmids Mouse Jaw1 cDNA (NM_008511.3) was amplified from total cDNA (reverse transcribed from total RNA derived mouse (C57/B6J) spleen) using Clone Amp HiFi PCR Premix (TaKaRa) with following primers: forward 5′-CTGGTGCCAAGCTTGGTACCATGCTCTGTGTAAAAGGTCC-3′, reverse 5′-CTGGACTAGTGGATCCTCACACTGGCAGTGGTC-3′. For the production of pcDNA5 FRT/TO HA FLAG Jaw1, the PCR product was inserted into the 3′ end of HA-FLAG tandem tag coding region within the pcDNA5 FRT/TO HA FLAG vector (digested with KpnI/BamHI) using an In-Fusion HD cloning Kit (TaKaRa). For the production of pTagGFP Jaw1, pcDNA5 FRT/TO HA FLAG Jaw1 was digested with KpnI/BamHI and the fragment was ligated into pTagGFP2-C (Evrogen) digested with the same restriction enzymes. For the generation of pTagGFP Jaw1ΔC-KASH5 C-lum, the DNA fragment of luminal region-truncated Jaw1 non-containing stop codon was first amplified from pcDNA5 FRT/TO HA FLAG Jaw1 with the following primers: forward 5′-CTCAAGCTTCGAATTCTATGCTCTGTGTAAAAGGTCCC-3′, reverse 5′-CCGCGGTACCGTCGACGAAGAGCTGACCTGTGAGG-3′. The PCR product was then inserted into pTagGFP2-C digested with EcoRI/SalI using In-Fusion HD cloning Kit. The DNA cassette coding KASH5 luminal region was then produced using the following annealing primers: sense 5′-TCGACAGCCAGTCCCCGCCACCCACCTGGCCTCACCTGCAGCTCTACTACCTACAGCCGCCACCAGTGTGAG-3′, antisense 5′-GATCCTCACACTGGTGGCGGCTGTAGGTAGTAGAGCTGCAGGTGAGGCCAGGTGGGTGGCGGGGACTGGCTG-3′ was ligated into above vector (digested with SalI/BamHI), resulting in pTagGFP Jaw1ΔC-KASH5 C-lum. For the generation of pTagGFP Jaw1RQ, pTagGFP Jaw1 was performed for quick change using following primers: forward 5′-GCTTCCCTCCCTAGAAATTTAGGGAACGTAGGCCTGGTGTCAGGCATGGAA-3′, reverse 5′-CCTACGTTCCCTAAATTTCTAGGGAGGGAAGCAATGGTCACTCTTCG-3′. To generate pcDNA5/FRT/TO HA FLAG SUN1 and pcDNA5/FRT/TO HA FLAG SUN2, EGFP-C1 SUN1 and EGFP-C1 SUN2 (kindly provided by Howard, J. Worman (24)) were performed for PCR with following primers: forward 5′-TTCTGGTGCCAAGCTTATGGACTTTTCTCGGCTGCAC-3′, reverse 5′-TGGACTAGTGGATCCCTACTGGATGGGCTCTCCGT-3′ for SUN1 and forward 5′-TTCTGGTGCCAAGCTTCTCGAGATGTCGAGACGAAGCCAG-3′, reverse 5′-GCCCTCTAGACTCGACCGGTCTAGTGGGCAGGCTCTCC-3′ for SUN2. Each PCR product was then inserted into pcDNA5 FRT/TO HA FLAG digested with HindIII/BamHI or HindIII/XhoI, respectively, using In-Fusion HD cloning Kit. Furthermore, EGFP-C1 SUN2 was digested with HindIII/XbaI and the fragment was ligated into reconstructed pTagRFP vector, resulting into pTagRFP SUN2. For the production of the reconstructed pTagRFP vector, the PCR product coding RFP was amplified from pTagRFP-N (Evrogen) with the following primers: forward 5′-CGTCAGATCCGCTAGCATGGTGTCTAAGGGCGAAG-3′, reverse 5′-GAAGCTTGAGCTCGAGAATTAAGTTTGTGCCCCAGTTTG-3′: and was inserted into pTagGFP-C digested with NheI/XhoI using In-Fusion HD cloning Kit. Transfection Plasmids were introduced into B16F10 or HEK293T cells using Lipofectamine 2000 Reagents (Invitrogen) according to the manufacturer’s instructions. Confocal microscopy For the observing the fluorescence protein tagged proteins or changes in nuclear shape, B16F10 cells were grown on collagen-coated eight wells chamber slides. After transfection, the cells were fixed in 4% paraformaldehyde for 10 min. After washing with PBS, the PBS containing Hoechst33342 was then added. Twenty minutes later, the cells were washed with PBS and mounted with VECTOR SHIELD (Vector laboratories, Inc.). For the immunostaining of B16F10 cells transiently expressing FLAG Jaw1, the fixed cells were permeabilized with 0.2% Triton X-100/PBS for 30 min, blocked with 3% bovine serum albumin (BSA) diluted with PBS for 1 h and reacted with an anti-FLAG mouse monoclonal antibody (1:200) (Sigma) diluted in 1% BSA/PBS for 1 h. After washing three times with 0.1% BSA/PBS, the cells were incubated with Alexa Fluor 488-labelled rabbit anti-mouse IgG (1:500) (Life technologies) diluted in 1% BSA/PBS containing Hoechst33342 for 1 h. After washing with 0.1% BSA/PBS three times, the cells were mounted, as described above. Images were examined using confocal microscopy (Zeiss, LSM710) (Objective lens; Zeiss Plan Apo-chromat 63×1.4 NA). RNA interference siRNA-mediated Jaw1 knockdown assays in B16F10 cells were performed using the Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. An siRNA oligo specific for mouse Jaw1 (MSS275372) and scramble control RNA were purchased from Invitrogen. After treatment for 24 h, the medium was refreshed and the incubation continued for an additional 48 h. Nuclear shape was then observed by staining with Hoechst33342 using confocal microscopy. RT-PCR Total RNA was isolated from B16F10 cells grown in 60 mm dishes (treated with control or siRNA) using RNAiso Plus (TaKaRa) according to the manufacturer’s instructions. Four hundred nanograms of total RNA were reverse transcribed using PrimeScript RT Master Mix (TaKaRa). Subsequent PCR was performed using SYBR Premix EX Taq II (Tli RNaseH Plus) (TaKaRa) with following primers for Jaw1: #1, forward 5′-GTGACTGGTTTACCTTGGAG-3′, reverse 5′-CAGAGAGTTAAAAGACCTGTCGTTCTGC-3′; #2, forward 5′-GCTGCTTATGGAGACTACACGAG-3′, reverse 5′-ATACTCTTCTCCAGCTTCTT-3′. Co-immunoprecipitation For the co-immunoprecipitation Jaw1 and SUN proteins, pcDNA5 FRT/TO HA FLAG SUN1 or SUN2 were co-transfected with pTagGFP, pTagGFP Jaw1 or pTagGFP Jaw1ΔC-KASH5 C-lum into HEK293T cells grown in 60 mm dishes. After treatment for 24 h, the medium was refreshed, and the incubation continued for an additional 24 h. The cells were peeled off using cell scraper and centrifuged at 1,000 ×g for 10 min at 4°C. The pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.5% NP-40) containing 1 µl protease inhibitor cocktail (nacalai tasque). The lysates were sonicated for 10 min on ice and centrifuged at 12,000 ×g for 30 min at 4°C. A portion of the supernatant was used as input and the remainder was added to anti-FLAG M2 beads (Sigma) equilibrated with incubation buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% NP-40) at 4°C for overnight. The beads were then collected by brief centrifugation and washed five times with washing buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl). For elution, the beads were incubated with elution buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl) containing 250 µg/ml FLAG peptide (Sigma) for 30 min on ice. After a brief centrifugation, the supernatants were mixed with SDS-PAGE buffer, heat blocked at 95°C for 5 min and subjected to western blotting. For the co-immunoprecipitation Jaw1 and α-tubulin, pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells grown in 100 mm dishes. After incubation for 24 h, the medium was refreshed and the incubation continued for an additional 24 h. The preparation for the lysates and the co-immunoprecipitation was performed, as described above. For examining Jaw1 oligomerization, pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected into HEK293T cells with pTagGFP or pTagGFP Jaw1. After treatment for 24 h, the medium was refreshed and the incubation continued for an additional 24 h. Lysate preparation and co-immunoprecipitation was performed as described above. Western blotting B16F10 cells (or transiently expressing FLAG Jaw1, GFP, GFP Jaw1RQ) were peeled off with cell scraper and the cells were collected by centrifugation at 1,000 ×g for 10 min at 4°C. The pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40). The lysates were sonicated for 10 min on ice and centrifuged at 12,000 ×g for 30 min at 4°C. The supernatants were mixed with SDS-PAGE buffer and heat blocked at 95°C for 5 min. These samples and the co-immunoprecipitation solutions were subjected to western blotting following SDS-PAGE. Polyvinylidene fluoride (PVDF) membranes were blocked in 3% skim milk (FUJIFILM WAKO Pure Chemical Corp. (WAKO)) diluted Tris-buffered saline (TBS) (20 mM Tris–HCl pH 7.6 and 137 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 h and reacted with primary antibodies: the purified anti-Jaw1 rat antibody (1:500), an anti-FLAG mouse monoclonal antibody (1:500) (Sigma), an anti-GFP mouse monoclonal antibody (1:500) (nacalai tasque) or an anti-α-tubulin mouse monoclonal antibody (1:500) (WAKO) diluted with 1% skim milk/TBS-T. After washing the membrane with TBS-T, horseradish peroxidase (HRP)-conjugated anti rat IgG (1:5,000) or HRP-conjugated anti mouse IgG (1:5,000) (GE Healthcare) was added. After that, the membranes were washed with TBS-T. Immunostar Zeta (WAKO) was used as substrates and the bands were detected using LAS3000. Transmission electron microscopy For observing nuclear shape using transmission electron microscope, the samples were prepared as previously described (25). B16F10 cells grown in a 35-mm dish (treated with control or siRNA) were prefixed by treatment with 2% paraformaldehyde and 2% glutaraldehyde in 30 mM HEPES buffer (pH 7.4) for 4 h at room temperature. Post-fixation was then performed in an 1% OsO4 mixture containing 0.8% K3(Fe(CN)6) in 30 mM HEPES buffer (pH 7.4) for 1 h at room temperature. After washing with hyperpure water, the cells were stained en bloc with EM stainer (Nisshin EM) and dehydrated in ethanol, and then embedded in Quetol 812 (Nisshin EM). The blocks were sectioned using an ultramicrotome (Leica EM UC7; Leica). The sections were then observed by electron microscopy (JEM-1400; JEOL). Rescue experiments B16F10 cells grown on eight wells chamber slides or 60 mm dishes were treated with siRNA for 24 h. After the medium was refreshed and incubated for an additional 24 h, the pTagGFP or pTagGFP Jaw1RQ cells were transfected. After incubation for 24 h, the cells were subjected to western blotting or the nuclear shapes were observed by staining with Hoechst33342 using confocal microscopy (Zeiss, LSM710). Statistical analysis The collected data were analysed and graphically presented using Microsoft Excel. Statistical significance was determined by Student’s t-test. Results Localization of Jaw1 at ONM Although it has been reported that Jaw1 is specifically expressed in immune organs and taste buds (17–19), the database suggests that Jaw1 is also expressed in other human tissues including melanomas at medium or low level (THE HUMAN PROTEIN ATLAS, PATHOLOGY ATLAS, Melanoma, Antibody HPA018505; LRMP). Therefore, we tested whether Jaw1 is expressed in B16F10 cells, a mouse melanoma cell line. Jaw1 mRNA was detected by RT-PCR using two independent primer sets (Fig. 1B). This indicates that Jaw1 is expressed in B16F10 cells, consistent with the above database. However, we failed to detect a specific band corresponding to endogenous Jaw1 by western blotting, probably because of its relatively low level of expression. Fig. 1 View largeDownload slide Expression and localization of Jaw1 at the ONM in B16F10 cells. (A) Schematic representation of Jaw1. Coiled-coil, coiled-coil domain, grey box; single trans-membrane domain. (B) The expression of Jaw1 mRNA in B16F10 cells was detected by RT-PCR using two independent primer sets (#1 and #2). The image was acquired following electrophoresis in 2% agarose gel. (C) pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and incubated for 24 h. After refreshing the medium, the cells were incubated for an additional 24 h and the lysates were subjected to western blotting. FLAG Jaw1 bands were detected using anti-FLAG mouse monoclonal antibody. Shaded triangle; full length and non-shaded triangle; carboxyl terminal-cleaved form. (D) Amino acid sequence alignment of luminal region of Jaw1 and other mouse KASH proteins (nesprin1–4, KASH5). Amino acids conserved between Jaw1 and KASH proteins are shaded in yellow and among the KASH proteins except for Jaw1 in purple. (E) pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected with pTagRFP SUN2 into B16F10 cells and the localization was observed using confocal microscopy. After 24 h from transfection, the medium was refreshed and incubated again for 24 h. Nucleus were stained with Hoechst33342. FLAG Jaw1 was detected primary antibody: anti-FLAG mouse monoclonal antibody and secondary antibody: Alexa Fluor 488-labelled rabbit anti-mouse IgG antibody. Scale bar: 10 μm. Along the arrow in the magnified image of (E), a representative line plot profile was shown (n=3). Blue line, Hoechst signal; red line, RFP signal and Green; Alexa Fluor 488 signal (FLAG). Fig. 1 View largeDownload slide Expression and localization of Jaw1 at the ONM in B16F10 cells. (A) Schematic representation of Jaw1. Coiled-coil, coiled-coil domain, grey box; single trans-membrane domain. (B) The expression of Jaw1 mRNA in B16F10 cells was detected by RT-PCR using two independent primer sets (#1 and #2). The image was acquired following electrophoresis in 2% agarose gel. (C) pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and incubated for 24 h. After refreshing the medium, the cells were incubated for an additional 24 h and the lysates were subjected to western blotting. FLAG Jaw1 bands were detected using anti-FLAG mouse monoclonal antibody. Shaded triangle; full length and non-shaded triangle; carboxyl terminal-cleaved form. (D) Amino acid sequence alignment of luminal region of Jaw1 and other mouse KASH proteins (nesprin1–4, KASH5). Amino acids conserved between Jaw1 and KASH proteins are shaded in yellow and among the KASH proteins except for Jaw1 in purple. (E) pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected with pTagRFP SUN2 into B16F10 cells and the localization was observed using confocal microscopy. After 24 h from transfection, the medium was refreshed and incubated again for 24 h. Nucleus were stained with Hoechst33342. FLAG Jaw1 was detected primary antibody: anti-FLAG mouse monoclonal antibody and secondary antibody: Alexa Fluor 488-labelled rabbit anti-mouse IgG antibody. Scale bar: 10 μm. Along the arrow in the magnified image of (E), a representative line plot profile was shown (n=3). Blue line, Hoechst signal; red line, RFP signal and Green; Alexa Fluor 488 signal (FLAG). In order to investigate the intracellular localization of Jaw1, we prepared an N-terminal HA-FLAG tandem tagged Jaw1 (FLAG Jaw1) construct. Consistent with a previous report (26), western blotting showed two bands for Jaw1: the full length and the carboxyl terminal-cleaved form, in cells expressing FLAG Jaw1 (Fig. 1C). As previously reported (20), alignment of the amino acid sequence of Jaw1 with KASH proteins showed that the Jaw1 carboxyl-terminal has a partial homology to the PPPX motif, four amino acids of carboxyl-terminus in KASH domain (20) (Fig. 1D). Therefore, it has been hypothesized that the full length Jaw1 functions at nuclear membrane as a KASH protein via interaction with SUN proteins. In order to confirm the localization of Jaw1, FLAG Jaw1 and RFP tagged SUN2 (RFP SUN2) were co-transfected into B16F10 cells. Confocal microscopic image showed that both FLAG Jaw1 was localized to RFP SUN2-positive NE and the ER network (Fig. 1E), consistent with the previous report (20). Importantly, a line plot profile of the confocal microscopic image showed that the peak for FLAG Jaw1 is slightly outside of RFP SUN2 in the nuclear membrane. Furthermore, the distance between the two peaks for Jaw1 and RFP SUN2 was ∼100 nm, which is near to the total of two distances: the distance (∼50 nm) between ONM and INM, as previously reported (6), and the molecular lengths of N-terminal tagged FLAG Jaw1 and RFP SUN2. Therefore, these results suggest that Jaw1 localized on the ONM. Aberrant nuclear shape in Jaw1-depleted B16F10 cells A defect in components of LINC complex causes nuclear abnormalities (8). Based on the partial homology of the Jaw1 carboxyl-terminal region to KASH proteins and the localization of Jaw1 on the ONM, we investigated the effects of Jaw1 depletion on nuclear shape by siRNA-mediated Jaw1 knockdown in B16F10 cells. Hoechst DNA staining was used to evaluate the nuclear shape in the cells. The confocal microscopic images showed that a significant number of nuclei in the Jaw1 KD B16F10 cells were misshapen or contained nuclear lobes, while most of the nuclei in control cells appeared to have a normal ellipse shape (Fig. 2A). These aberrant nuclear shapes were observed in 30% of the total Jaw1 KD B16F10 cells (Fig. 2B). Furthermore, the abnormalities of the nuclear shape in Jaw1 KD B16F10 cells were confirmed by transmission electron microscopy (TEM). As shown in Fig. 2C, nuclear lobes and misshapen nuclei were also observed in the electron micrographs of Jaw1 KD B16F10 cells. To verify the effect of siRNA against Jaw1 on nuclear shape, rescue experiments were carried out. For this purpose, we prepared an siRNA-resistant Jaw1 construct (GFP Jaw1RQ) and confirmed its expression by western blotting (Fig. 2D). B16F10 cells were transfected with siRNA followed by the transfection with plasmid encoding GFP alone or GFP Jaw1RQ, and then stained them with Hoechst33342 to evaluate nuclear shape. Approximately 30% of the cells had aberrant nuclear shapes in GFP expressing control cells. In sharp contrast, almost all the cells expressing GFP Jaw1RQ had normal nuclear shapes (Fig. 2E and F), indicating that Jaw depletion does, in fact, induce aberrant nuclear shapes. Collectively, these results indicate that Jaw1 plays a role in maintaining nuclear shape in B16F10 cells. Fig. 2 View largeDownload slide The effects of Jaw1 knockdown on nuclear shape. B16F10 cells were treated with an siRNA against Jaw1 (Jaw1 KD) or a scrambled control RNA (NT). (A) Nuclear shape was observed using confocal microscopy after staining the DNA with Hoechst33342. Scale bar: 20 μm. Three magnified images of the boxes surrounded with red dot lines (gray in black and white) are shown in the side. Scale bar: 10 μm. (B) Counting of cells having aberrant nuclear shapes of (A) (n>300). In the graph, the proportion of cells with aberrant nuclear shape is shown based on the average of three independent experiments per condition; error bars show SE; ***P <0.005. (C) The cells were observed by TEM. Arrow, blebs of nuclei; Scale bar: 2 μm (left and middle), 500 nm (right). The images of nuclear shape which NE is bordered are shown in the bottom. Rescue experiments. B16F10 cells were treated with siRNA and pTagGFP or pTagGFP Jaw1RQ were transfected. (D) The lysates were subjected to western blotting. GFP Jaw1RQ bands were detected using purified anti-Jaw1 rat antibody and HRP-conjugated anti-rat IgG antibody. (E) The nuclear shape was observed by confocal microscopy. Top: overlay between green; GFP and violet, Hoechst33342. Bottom: the signal of Hoechst33342 was transformed into white. Arrowheads: aberrant nuclei (yellow (gray in black and white), GFP-positive; white, GFP-negative). Arrows: normal nuclei of GFP-positive cells. Scale bar: 20 μm. (F) The proportion of the cells having aberrant nuclear shape out of the number of the cells expressing GFP or GFP Jaw1RQ (n>200) was shown. The cell number is counted up to (n>200) as a total from the experiments divided into three times. Fig. 2 View largeDownload slide The effects of Jaw1 knockdown on nuclear shape. B16F10 cells were treated with an siRNA against Jaw1 (Jaw1 KD) or a scrambled control RNA (NT). (A) Nuclear shape was observed using confocal microscopy after staining the DNA with Hoechst33342. Scale bar: 20 μm. Three magnified images of the boxes surrounded with red dot lines (gray in black and white) are shown in the side. Scale bar: 10 μm. (B) Counting of cells having aberrant nuclear shapes of (A) (n>300). In the graph, the proportion of cells with aberrant nuclear shape is shown based on the average of three independent experiments per condition; error bars show SE; ***P <0.005. (C) The cells were observed by TEM. Arrow, blebs of nuclei; Scale bar: 2 μm (left and middle), 500 nm (right). The images of nuclear shape which NE is bordered are shown in the bottom. Rescue experiments. B16F10 cells were treated with siRNA and pTagGFP or pTagGFP Jaw1RQ were transfected. (D) The lysates were subjected to western blotting. GFP Jaw1RQ bands were detected using purified anti-Jaw1 rat antibody and HRP-conjugated anti-rat IgG antibody. (E) The nuclear shape was observed by confocal microscopy. Top: overlay between green; GFP and violet, Hoechst33342. Bottom: the signal of Hoechst33342 was transformed into white. Arrowheads: aberrant nuclei (yellow (gray in black and white), GFP-positive; white, GFP-negative). Arrows: normal nuclei of GFP-positive cells. Scale bar: 20 μm. (F) The proportion of the cells having aberrant nuclear shape out of the number of the cells expressing GFP or GFP Jaw1RQ (n>200) was shown. The cell number is counted up to (n>200) as a total from the experiments divided into three times. Interaction of Jaw with SUN proteins and microtubules KASH proteins are defined as components of the LINC complex through interaction with SUN proteins. In order to investigate the interaction between Jaw1 and SUN proteins, the plasmids coding N-terminal HA-FLAG tandem tagged SUN1or SUN2 (hereafter referred to as FLAG SUN1 or FLAG SUN2) and GFP Jaw1 were used (Fig. 3A). As a positive control, GFP Jaw1ΔC-KASH5 C-lum, in which the Jaw1 luminal region is replaced by the KASH domain of KASH5 was used. HEK293T cells were transfected as indicated and the lysates were co-immunoprecipitated by treatment with anti-FLAG beads. As predicted, GFP Jaw1ΔC-KASH5 C-lum (positive control) was co-immunoprecipitated by FLAG SUN1 or SUN2, while GFP was not (Fig. 3B and C). Importantly, GFP Jaw1 was co-immunoprecipitated by FLAG SUN1 or SUN2 (Fig. 3B and C). These results indicate that Jaw1 has an affinity for SUN proteins, the same as KASH proteins. Fig. 3 View largeDownload slide Interaction of Jaw1 with SUN proteins and microtubules and oligomerization among Jaw1 molecules. (A) Schematic representations of GFP Jaw1 and GFP Jaw1ΔC-KASH5 C-lum, the luminal region of Jaw1 is replaced by that of mouse KASH5. Interaction between Jaw1 and SUN1 or SUN2, pTagGFP, pTagGFP Jaw1ΔC-KASH5 C-lum or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG SUN1 (B) or pcDNA5 FRT/TO HA FLAG SUN2 (C) into HEK293T cells. After incubation for 24 h, the medium was refreshed and incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation using anti-FLAG beads. For western blotting, anti-FLAG mouse monoclonal antibody and anti-GFP mouse monoclonal antibody as primary antibodies and HRP-conjugated anti-mouse IgG antibody as a secondary antibody were used. (D) Interaction between Jaw1 and microtubules. pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and the cells were incubated for 24 h. After the refreshing the medium, the cells were incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation and western blotting using an anti-FLAG mouse monoclonal antibody and anti-α-tubulin monoclonal antibody were used. (E) Oligomerization of Jaw1 molecules. pTagGFP or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG Jaw1 into HEK293T cells. After that it was followed by the procedures of (B, C). Fig. 3 View largeDownload slide Interaction of Jaw1 with SUN proteins and microtubules and oligomerization among Jaw1 molecules. (A) Schematic representations of GFP Jaw1 and GFP Jaw1ΔC-KASH5 C-lum, the luminal region of Jaw1 is replaced by that of mouse KASH5. Interaction between Jaw1 and SUN1 or SUN2, pTagGFP, pTagGFP Jaw1ΔC-KASH5 C-lum or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG SUN1 (B) or pcDNA5 FRT/TO HA FLAG SUN2 (C) into HEK293T cells. After incubation for 24 h, the medium was refreshed and incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation using anti-FLAG beads. For western blotting, anti-FLAG mouse monoclonal antibody and anti-GFP mouse monoclonal antibody as primary antibodies and HRP-conjugated anti-mouse IgG antibody as a secondary antibody were used. (D) Interaction between Jaw1 and microtubules. pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and the cells were incubated for 24 h. After the refreshing the medium, the cells were incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation and western blotting using an anti-FLAG mouse monoclonal antibody and anti-α-tubulin monoclonal antibody were used. (E) Oligomerization of Jaw1 molecules. pTagGFP or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG Jaw1 into HEK293T cells. After that it was followed by the procedures of (B, C). It has been reported that KASH proteins interact with cytoskeletons such as actin filaments, microtubules or intermediate filaments, through its cytosolic region (4). We previously used co-immunoprecipitation and mass spectrometry to identify proteins that interact with Jaw1. This comprehensive analysis indicated the existence of an interaction between Jaw1 and microtubules (data not shown). Therefore, we confirmed whether Jaw1 interacts with microtubules similar to that for KASH proteins. Co-immunoprecipitation assays in B16F10 cells confirmed an interaction between FLAG Jaw1 and α-tubulin (Fig. 3D), suggesting that Jaw1 is associated with microtubules. These data suggest that Jaw1 interacts with SUN proteins and microtubules, and functions as a component of the LINC complex. It is known that SUN proteins interact with KASH proteins in a trimer, resulting in the formation of a heterohexamer (27–29). Furthermore, it has been reported that some KASH proteins form oligomeric structures (30). Therefore, we examined the issue of whether Jaw1 forms an oligomer. As shown in Fig. 3E, GFP Jaw1 was co-immunoprecipitated by FLAG Jaw1, while GFP was not co-immunoprecipitated by FLAG Jaw1. This result indicates that Jaw1 interacts with SUN proteins and microtubules potentially in the form of an oligomer. Collectively, our data suggest that Jaw1, similar to KASH proteins, functions as a component of the LINC complex in the form of an oligomer. Discussion In this study, we performed a functional analysis of Jaw1/LRMP in B16F10 cells. Confocal imaging showed that Jaw1 is localized on the nuclear membrane and the ER network, as previously reported (20). Line plot profiling of the confocal microscopic images suggested that Jaw1 is localized on the ONM. Knockdown-rescue experiments indicated that Jaw1is important for maintaining nuclear shape. Co-immunoprecipitation assays showed that Jaw1 interacts with SUN1, SUN2 and microtubules. Furthermore, we also found that Jaw1 has the potential to form oligomers. We therefore propose that Jaw1 functions as a component of the LINC complex. Jaw1 interacts with microtubules on the cytosolic face and with SUN proteins in the PNS via the KASH domain, as shown in Fig. 4. The Jaw1-mediated physical linkage across the NE would enable a nuclear shape to be maintained in the form of a normal ellipse. Fig. 4 View largeDownload slide A model for Jaw1 function as a KASH protein. A model showing the function of Jaw1 as a KASH protein. Jaw1 interacts with SUN1 and SUN2 in the PNS and bind to microtubules on cytosolic face in the form of oligomer. This LINC complex bridging across the NE functions for the maintenance of the nuclear shape. Fig. 4 View largeDownload slide A model for Jaw1 function as a KASH protein. A model showing the function of Jaw1 as a KASH protein. Jaw1 interacts with SUN1 and SUN2 in the PNS and bind to microtubules on cytosolic face in the form of oligomer. This LINC complex bridging across the NE functions for the maintenance of the nuclear shape. Five mammalian KASH proteins have been identified to date: Nesprin1–4 that is expressed ubiquitously and KASH5 that is expressed in reproductive organs specifically in testis and ovary (4, 7). Nesprin1 and Nesprin2 directly bind to actin filaments via their actin-binding domains (31), Nesprin3 binds to intermediate filaments via Plectin (30, 32) and Nesprin4 and KASH5 are connected to microtubules via motor proteins such as kinesin and dynein respectively (7, 12). The findings reported herein indicate that Jaw1 has a role in maintaining nuclear shape via its interaction with microtubules; however, it remains unclear whether Jaw1 interacts with microtubules directly or indirectly through other molecules. Furthermore, we observed the abnormal nuclear shape in Jaw1 KD B16F10 cells, which is caused by the acute reduction of the expression level of Jaw1 by siRNA-mediated knockdown. However, Jaw1 has a potential redundancy with Nesprin1–4 that is expressed ubiquitously to maintain the nuclear shape. This point should be examined in future studies if a more complete understanding of the mechanism responsible for how Jaw1 functions at nuclear membrane. In this study, we confirmed that the Jaw1 carboxyl-terminal is cleaved post-translationally, consistent with a previous report (26). Since the carboxyl-terminal KASH domain is required for its interaction with SUN proteins, it would be possible that full length Jaw1 functions at nuclear membrane as a KASH protein, on the other hand, carboxyl-terminal cleaved form resides at the ER membranes has other roles. To test the hypothesis, we plan to attempt a subcellular fractionation of the nucleus and the ER membranes in cells expressing epitope-tagged Jaw1. Although Jaw1 has been identified as a protein that is expressed abundantly in lymphoid organs and immune cells (17, 18), we found that Jaw1 is also expressed in B16F10 mouse melanoma cells at a transcriptional level. As shown in the database (THE HUMAN PROTEIN ATLAS, TISSUE ATLAS, LRMP), Jaw1 is also expressed in other human tissues including the brain, lungs, pancreas, urinary bladder, ovaries, skin etc. at lower or medium levels. Therefore, we propose that Jaw1 is expressed in many tissues at different levels. 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Virol . 91 , e02303 – e02316 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ER endoplasmic reticulum GFP, green fluorescence protein; INM inner nuclear membrane KASH Klarsicht/ANC-1/Syne/homology LINC linker of nucleus and cytoskeletons LRMP lymphoid restricted membrane protein NE nuclear envelope ONM outer nuclear membrane PNS perinuclear space SUN Sad-1/UNC-84 © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Jaw1/LRMP has a role in maintaining nuclear shape via interaction with SUN proteins

The Journal of Biochemistry , Volume Advance Article – Jun 6, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy053
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Abstract

Abstract Jaw1/LRMP is characterized as a Type II integral membrane protein that is localized to endoplasmic reticulum, however, its physiological functions have been poorly understood. An alignment of amino acid sequence of Jaw1 with Klarsicht/ANC-1/Syne/homology (KASH) proteins, outer nuclear membrane proteins, revealed that Jaw1 has a partial homology to the KASH domain. Here, we show that the function of Jaw1 is to maintain nuclear shape in mouse melanoma cell line. The siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape, and the defect was rescued by ectopic expression of siRNA-resistant Jaw1. Since co-immunoprecipitation assay indicates that Jaw1 interacts with Sad-1/UNC-84 (SUN) proteins that are inner nuclear proteins and microtubules, this study suggests that Jaw1 has a role in maintaining nuclear shape via interactions with SUN proteins and microtubules. Jaw1/LRMP, KASH proteins, LINC complex, nuclear envelope, SUN proteins The nucleus is an organelle that contains packaged DNA, where it is encapsulated with a nuclear envelope (NE) consisting of double lipid bilayers, i.e. an inner nuclear membrane (INM) and an outer nuclear membrane (ONM) that are separated by the perinuclear space (PNS). For the modulation of gene expression in response to changes in the extracellular environment and flexible nuclear stiffness during cell migration, the maintenance or the regulation of dynamic changes in nuclear shape has been reported to be an important factor (1–3). Furthermore, positioning the nucleus to the precise location in cells is also important for cellular polarity, migration and differentiation (4). The linker of nucleus and cytoskeletons (LINC) complex consists of Klarsicht/ANC-1/Syne/homology (KASH) proteins that are localized to the ONM and Sad-1/UNC-84 (SUN) proteins localized to the INM, which form a complex via the interaction of these protein in the PNS. KASH proteins bind to microtubules or the actin-based cytoskeleton network on the cytosolic face and SUN proteins connect with the nuclear lamina in the nucleoplasm (5, 6). This cytoskeletal network that is produced via the LINC complex makes it possible to transduce the mechanical forces between the cytosol and nucleus for maintaining both the shape and position of the nucleus. Five KASH proteins, Nesprin1-4 and KASH5 and four SUN proteins, SUN1-4 have been identified to date (4, 7). The depletion of these components in the LINC complex results in aberrant nuclear shapes and alternations in the position of the nucleus in the cell, eventually leading to defects in cellular migration, cellular stiffness and cellular integrity (8–12). Furthermore, these nuclear physiological dysfunctions are possible causes of Emery–Dreifuss Muscular Dystrophy (13), infertility (14, 15) and Hutchinson–Gilford Progeria syndrome (16). Jaw1 has been identified as a Type II integral membrane protein that is localized on the endoplasmic reticulum (ER), and is specifically expressed in some immune cells and in Type II cells in taste buds (17–19). Jaw1 consists of an N-terminal coiled-coil domain oriented toward the cytosol, a single trans-membrane domain and a carboxyl-terminal luminal domain. The coiled-coil of Jaw1 has been reported to be interacted with Type III inositol 1,4,5-triphosphate receptor (IP3R3), a calcium-gate channel on ER membrane (19). Recent reports indicated that Jaw1 is localized on the ER and the NE which is continuous to the ER network. Furthermore, it is known that the Jaw1 carboxyl-terminal region has a partial homology to the PPPX motif, four amino acids of carboxyl-terminus in KASH proteins (20). Based on these data, Jaw1 has been hypothesized to function as a potential member of KASH protein (20–23). However, the interaction between Jaw1 and SUN proteins and physiological function of Jaw1 at nuclear membrane are unknown. In this context, we focussed on the issue whether Jaw1 functions as a new member of KASH protein and what the role of Jaw1 is at nuclear membrane. The findings reported herein indicate that Jaw1 has a role in maintaining nuclear shape. We confirmed that Jaw1 is localized to the ONM in addition to the ER in mouse melanoma cell line B16F10 cells. We also found that the siRNA-mediated knockdown of Jaw1 caused a severe defect in nuclear shape. Furthermore, co-immunoprecipitation experiments indicated that Jaw1 interacts with SUN proteins and microtubules. Based on these findings, we propose that Jaw1 functions as a component of the LINC complex and is involved in the maintenance of nuclear shape. Materials and Methods Production of Jaw1 polyclonal antibody The N-terminal region encoding amino acids (204–351) of human Jaw1(NM_006152.3) was amplified by polymerase chain reaction (PCR) using following primers: forward 5′-CACACGAATTCAGTGAGAACACTTCTGCT-3′, reverse 5′-CACGTCGACCTAAATTTGGCAGTCATCATCATC-3′ and inserted into pMAL cRI vector (New England Biolabs (NEB)) containing Tobacco etch virus (TEV) protease recognition site downstream of the maltose-binding protein (MBP). The MBP fused protein was overexpressed in BL21(DE3) and purified using a amylose resin (NEB). After cutting the MBP tag with a TEV protease, the solution was passed over the amylose resin again to trap the cut MBP tag, resulting in the isolation of the purified antigen. The prepared antigen was used to immunize rats. Primary immunization was performed by mixture with ADJUVANT COMPLETE FREUND (BD) and following ones were performed by mixture with ADJUVANT INCOMPLETE FREUND (BD). After the several immunizations, whole serum was collected and the anti-Jaw1 antibody was purified using an NHS-column coupled with the above purified antigen. For equilibration and washing the column, 0.02 M Tris buffer (pH 7.2) containing 0.5 M NaCl was used. Furthermore, the antibody was eluted with 0.2 M glycine, pH 3.0 and neutralized with 1 M Tris, pH 9.0. This experimental protocol was approved by the Animal Care and Use Committee of Tokyo University of Agriculture and Technology. Cells and cell culture B16F10 and HEK293T cells were incubated in MEM supplemented with 10% foetal bovine serum (FBS) and 5.84 mg/ml l-glutamine and in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma) and 5.84 mg/ml l-glutamine, respectively, and maintained at 37°C in 5% CO2. Cloning and plasmids Mouse Jaw1 cDNA (NM_008511.3) was amplified from total cDNA (reverse transcribed from total RNA derived mouse (C57/B6J) spleen) using Clone Amp HiFi PCR Premix (TaKaRa) with following primers: forward 5′-CTGGTGCCAAGCTTGGTACCATGCTCTGTGTAAAAGGTCC-3′, reverse 5′-CTGGACTAGTGGATCCTCACACTGGCAGTGGTC-3′. For the production of pcDNA5 FRT/TO HA FLAG Jaw1, the PCR product was inserted into the 3′ end of HA-FLAG tandem tag coding region within the pcDNA5 FRT/TO HA FLAG vector (digested with KpnI/BamHI) using an In-Fusion HD cloning Kit (TaKaRa). For the production of pTagGFP Jaw1, pcDNA5 FRT/TO HA FLAG Jaw1 was digested with KpnI/BamHI and the fragment was ligated into pTagGFP2-C (Evrogen) digested with the same restriction enzymes. For the generation of pTagGFP Jaw1ΔC-KASH5 C-lum, the DNA fragment of luminal region-truncated Jaw1 non-containing stop codon was first amplified from pcDNA5 FRT/TO HA FLAG Jaw1 with the following primers: forward 5′-CTCAAGCTTCGAATTCTATGCTCTGTGTAAAAGGTCCC-3′, reverse 5′-CCGCGGTACCGTCGACGAAGAGCTGACCTGTGAGG-3′. The PCR product was then inserted into pTagGFP2-C digested with EcoRI/SalI using In-Fusion HD cloning Kit. The DNA cassette coding KASH5 luminal region was then produced using the following annealing primers: sense 5′-TCGACAGCCAGTCCCCGCCACCCACCTGGCCTCACCTGCAGCTCTACTACCTACAGCCGCCACCAGTGTGAG-3′, antisense 5′-GATCCTCACACTGGTGGCGGCTGTAGGTAGTAGAGCTGCAGGTGAGGCCAGGTGGGTGGCGGGGACTGGCTG-3′ was ligated into above vector (digested with SalI/BamHI), resulting in pTagGFP Jaw1ΔC-KASH5 C-lum. For the generation of pTagGFP Jaw1RQ, pTagGFP Jaw1 was performed for quick change using following primers: forward 5′-GCTTCCCTCCCTAGAAATTTAGGGAACGTAGGCCTGGTGTCAGGCATGGAA-3′, reverse 5′-CCTACGTTCCCTAAATTTCTAGGGAGGGAAGCAATGGTCACTCTTCG-3′. To generate pcDNA5/FRT/TO HA FLAG SUN1 and pcDNA5/FRT/TO HA FLAG SUN2, EGFP-C1 SUN1 and EGFP-C1 SUN2 (kindly provided by Howard, J. Worman (24)) were performed for PCR with following primers: forward 5′-TTCTGGTGCCAAGCTTATGGACTTTTCTCGGCTGCAC-3′, reverse 5′-TGGACTAGTGGATCCCTACTGGATGGGCTCTCCGT-3′ for SUN1 and forward 5′-TTCTGGTGCCAAGCTTCTCGAGATGTCGAGACGAAGCCAG-3′, reverse 5′-GCCCTCTAGACTCGACCGGTCTAGTGGGCAGGCTCTCC-3′ for SUN2. Each PCR product was then inserted into pcDNA5 FRT/TO HA FLAG digested with HindIII/BamHI or HindIII/XhoI, respectively, using In-Fusion HD cloning Kit. Furthermore, EGFP-C1 SUN2 was digested with HindIII/XbaI and the fragment was ligated into reconstructed pTagRFP vector, resulting into pTagRFP SUN2. For the production of the reconstructed pTagRFP vector, the PCR product coding RFP was amplified from pTagRFP-N (Evrogen) with the following primers: forward 5′-CGTCAGATCCGCTAGCATGGTGTCTAAGGGCGAAG-3′, reverse 5′-GAAGCTTGAGCTCGAGAATTAAGTTTGTGCCCCAGTTTG-3′: and was inserted into pTagGFP-C digested with NheI/XhoI using In-Fusion HD cloning Kit. Transfection Plasmids were introduced into B16F10 or HEK293T cells using Lipofectamine 2000 Reagents (Invitrogen) according to the manufacturer’s instructions. Confocal microscopy For the observing the fluorescence protein tagged proteins or changes in nuclear shape, B16F10 cells were grown on collagen-coated eight wells chamber slides. After transfection, the cells were fixed in 4% paraformaldehyde for 10 min. After washing with PBS, the PBS containing Hoechst33342 was then added. Twenty minutes later, the cells were washed with PBS and mounted with VECTOR SHIELD (Vector laboratories, Inc.). For the immunostaining of B16F10 cells transiently expressing FLAG Jaw1, the fixed cells were permeabilized with 0.2% Triton X-100/PBS for 30 min, blocked with 3% bovine serum albumin (BSA) diluted with PBS for 1 h and reacted with an anti-FLAG mouse monoclonal antibody (1:200) (Sigma) diluted in 1% BSA/PBS for 1 h. After washing three times with 0.1% BSA/PBS, the cells were incubated with Alexa Fluor 488-labelled rabbit anti-mouse IgG (1:500) (Life technologies) diluted in 1% BSA/PBS containing Hoechst33342 for 1 h. After washing with 0.1% BSA/PBS three times, the cells were mounted, as described above. Images were examined using confocal microscopy (Zeiss, LSM710) (Objective lens; Zeiss Plan Apo-chromat 63×1.4 NA). RNA interference siRNA-mediated Jaw1 knockdown assays in B16F10 cells were performed using the Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer’s instructions. An siRNA oligo specific for mouse Jaw1 (MSS275372) and scramble control RNA were purchased from Invitrogen. After treatment for 24 h, the medium was refreshed and the incubation continued for an additional 48 h. Nuclear shape was then observed by staining with Hoechst33342 using confocal microscopy. RT-PCR Total RNA was isolated from B16F10 cells grown in 60 mm dishes (treated with control or siRNA) using RNAiso Plus (TaKaRa) according to the manufacturer’s instructions. Four hundred nanograms of total RNA were reverse transcribed using PrimeScript RT Master Mix (TaKaRa). Subsequent PCR was performed using SYBR Premix EX Taq II (Tli RNaseH Plus) (TaKaRa) with following primers for Jaw1: #1, forward 5′-GTGACTGGTTTACCTTGGAG-3′, reverse 5′-CAGAGAGTTAAAAGACCTGTCGTTCTGC-3′; #2, forward 5′-GCTGCTTATGGAGACTACACGAG-3′, reverse 5′-ATACTCTTCTCCAGCTTCTT-3′. Co-immunoprecipitation For the co-immunoprecipitation Jaw1 and SUN proteins, pcDNA5 FRT/TO HA FLAG SUN1 or SUN2 were co-transfected with pTagGFP, pTagGFP Jaw1 or pTagGFP Jaw1ΔC-KASH5 C-lum into HEK293T cells grown in 60 mm dishes. After treatment for 24 h, the medium was refreshed, and the incubation continued for an additional 24 h. The cells were peeled off using cell scraper and centrifuged at 1,000 ×g for 10 min at 4°C. The pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.5% NP-40) containing 1 µl protease inhibitor cocktail (nacalai tasque). The lysates were sonicated for 10 min on ice and centrifuged at 12,000 ×g for 30 min at 4°C. A portion of the supernatant was used as input and the remainder was added to anti-FLAG M2 beads (Sigma) equilibrated with incubation buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.1% NP-40) at 4°C for overnight. The beads were then collected by brief centrifugation and washed five times with washing buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl). For elution, the beads were incubated with elution buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl) containing 250 µg/ml FLAG peptide (Sigma) for 30 min on ice. After a brief centrifugation, the supernatants were mixed with SDS-PAGE buffer, heat blocked at 95°C for 5 min and subjected to western blotting. For the co-immunoprecipitation Jaw1 and α-tubulin, pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells grown in 100 mm dishes. After incubation for 24 h, the medium was refreshed and the incubation continued for an additional 24 h. The preparation for the lysates and the co-immunoprecipitation was performed, as described above. For examining Jaw1 oligomerization, pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected into HEK293T cells with pTagGFP or pTagGFP Jaw1. After treatment for 24 h, the medium was refreshed and the incubation continued for an additional 24 h. Lysate preparation and co-immunoprecipitation was performed as described above. Western blotting B16F10 cells (or transiently expressing FLAG Jaw1, GFP, GFP Jaw1RQ) were peeled off with cell scraper and the cells were collected by centrifugation at 1,000 ×g for 10 min at 4°C. The pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40). The lysates were sonicated for 10 min on ice and centrifuged at 12,000 ×g for 30 min at 4°C. The supernatants were mixed with SDS-PAGE buffer and heat blocked at 95°C for 5 min. These samples and the co-immunoprecipitation solutions were subjected to western blotting following SDS-PAGE. Polyvinylidene fluoride (PVDF) membranes were blocked in 3% skim milk (FUJIFILM WAKO Pure Chemical Corp. (WAKO)) diluted Tris-buffered saline (TBS) (20 mM Tris–HCl pH 7.6 and 137 mM NaCl) containing 0.1% Tween-20 (TBS-T) for 1 h and reacted with primary antibodies: the purified anti-Jaw1 rat antibody (1:500), an anti-FLAG mouse monoclonal antibody (1:500) (Sigma), an anti-GFP mouse monoclonal antibody (1:500) (nacalai tasque) or an anti-α-tubulin mouse monoclonal antibody (1:500) (WAKO) diluted with 1% skim milk/TBS-T. After washing the membrane with TBS-T, horseradish peroxidase (HRP)-conjugated anti rat IgG (1:5,000) or HRP-conjugated anti mouse IgG (1:5,000) (GE Healthcare) was added. After that, the membranes were washed with TBS-T. Immunostar Zeta (WAKO) was used as substrates and the bands were detected using LAS3000. Transmission electron microscopy For observing nuclear shape using transmission electron microscope, the samples were prepared as previously described (25). B16F10 cells grown in a 35-mm dish (treated with control or siRNA) were prefixed by treatment with 2% paraformaldehyde and 2% glutaraldehyde in 30 mM HEPES buffer (pH 7.4) for 4 h at room temperature. Post-fixation was then performed in an 1% OsO4 mixture containing 0.8% K3(Fe(CN)6) in 30 mM HEPES buffer (pH 7.4) for 1 h at room temperature. After washing with hyperpure water, the cells were stained en bloc with EM stainer (Nisshin EM) and dehydrated in ethanol, and then embedded in Quetol 812 (Nisshin EM). The blocks were sectioned using an ultramicrotome (Leica EM UC7; Leica). The sections were then observed by electron microscopy (JEM-1400; JEOL). Rescue experiments B16F10 cells grown on eight wells chamber slides or 60 mm dishes were treated with siRNA for 24 h. After the medium was refreshed and incubated for an additional 24 h, the pTagGFP or pTagGFP Jaw1RQ cells were transfected. After incubation for 24 h, the cells were subjected to western blotting or the nuclear shapes were observed by staining with Hoechst33342 using confocal microscopy (Zeiss, LSM710). Statistical analysis The collected data were analysed and graphically presented using Microsoft Excel. Statistical significance was determined by Student’s t-test. Results Localization of Jaw1 at ONM Although it has been reported that Jaw1 is specifically expressed in immune organs and taste buds (17–19), the database suggests that Jaw1 is also expressed in other human tissues including melanomas at medium or low level (THE HUMAN PROTEIN ATLAS, PATHOLOGY ATLAS, Melanoma, Antibody HPA018505; LRMP). Therefore, we tested whether Jaw1 is expressed in B16F10 cells, a mouse melanoma cell line. Jaw1 mRNA was detected by RT-PCR using two independent primer sets (Fig. 1B). This indicates that Jaw1 is expressed in B16F10 cells, consistent with the above database. However, we failed to detect a specific band corresponding to endogenous Jaw1 by western blotting, probably because of its relatively low level of expression. Fig. 1 View largeDownload slide Expression and localization of Jaw1 at the ONM in B16F10 cells. (A) Schematic representation of Jaw1. Coiled-coil, coiled-coil domain, grey box; single trans-membrane domain. (B) The expression of Jaw1 mRNA in B16F10 cells was detected by RT-PCR using two independent primer sets (#1 and #2). The image was acquired following electrophoresis in 2% agarose gel. (C) pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and incubated for 24 h. After refreshing the medium, the cells were incubated for an additional 24 h and the lysates were subjected to western blotting. FLAG Jaw1 bands were detected using anti-FLAG mouse monoclonal antibody. Shaded triangle; full length and non-shaded triangle; carboxyl terminal-cleaved form. (D) Amino acid sequence alignment of luminal region of Jaw1 and other mouse KASH proteins (nesprin1–4, KASH5). Amino acids conserved between Jaw1 and KASH proteins are shaded in yellow and among the KASH proteins except for Jaw1 in purple. (E) pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected with pTagRFP SUN2 into B16F10 cells and the localization was observed using confocal microscopy. After 24 h from transfection, the medium was refreshed and incubated again for 24 h. Nucleus were stained with Hoechst33342. FLAG Jaw1 was detected primary antibody: anti-FLAG mouse monoclonal antibody and secondary antibody: Alexa Fluor 488-labelled rabbit anti-mouse IgG antibody. Scale bar: 10 μm. Along the arrow in the magnified image of (E), a representative line plot profile was shown (n=3). Blue line, Hoechst signal; red line, RFP signal and Green; Alexa Fluor 488 signal (FLAG). Fig. 1 View largeDownload slide Expression and localization of Jaw1 at the ONM in B16F10 cells. (A) Schematic representation of Jaw1. Coiled-coil, coiled-coil domain, grey box; single trans-membrane domain. (B) The expression of Jaw1 mRNA in B16F10 cells was detected by RT-PCR using two independent primer sets (#1 and #2). The image was acquired following electrophoresis in 2% agarose gel. (C) pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and incubated for 24 h. After refreshing the medium, the cells were incubated for an additional 24 h and the lysates were subjected to western blotting. FLAG Jaw1 bands were detected using anti-FLAG mouse monoclonal antibody. Shaded triangle; full length and non-shaded triangle; carboxyl terminal-cleaved form. (D) Amino acid sequence alignment of luminal region of Jaw1 and other mouse KASH proteins (nesprin1–4, KASH5). Amino acids conserved between Jaw1 and KASH proteins are shaded in yellow and among the KASH proteins except for Jaw1 in purple. (E) pcDNA5 FRT/TO HA FLAG Jaw1 was co-transfected with pTagRFP SUN2 into B16F10 cells and the localization was observed using confocal microscopy. After 24 h from transfection, the medium was refreshed and incubated again for 24 h. Nucleus were stained with Hoechst33342. FLAG Jaw1 was detected primary antibody: anti-FLAG mouse monoclonal antibody and secondary antibody: Alexa Fluor 488-labelled rabbit anti-mouse IgG antibody. Scale bar: 10 μm. Along the arrow in the magnified image of (E), a representative line plot profile was shown (n=3). Blue line, Hoechst signal; red line, RFP signal and Green; Alexa Fluor 488 signal (FLAG). In order to investigate the intracellular localization of Jaw1, we prepared an N-terminal HA-FLAG tandem tagged Jaw1 (FLAG Jaw1) construct. Consistent with a previous report (26), western blotting showed two bands for Jaw1: the full length and the carboxyl terminal-cleaved form, in cells expressing FLAG Jaw1 (Fig. 1C). As previously reported (20), alignment of the amino acid sequence of Jaw1 with KASH proteins showed that the Jaw1 carboxyl-terminal has a partial homology to the PPPX motif, four amino acids of carboxyl-terminus in KASH domain (20) (Fig. 1D). Therefore, it has been hypothesized that the full length Jaw1 functions at nuclear membrane as a KASH protein via interaction with SUN proteins. In order to confirm the localization of Jaw1, FLAG Jaw1 and RFP tagged SUN2 (RFP SUN2) were co-transfected into B16F10 cells. Confocal microscopic image showed that both FLAG Jaw1 was localized to RFP SUN2-positive NE and the ER network (Fig. 1E), consistent with the previous report (20). Importantly, a line plot profile of the confocal microscopic image showed that the peak for FLAG Jaw1 is slightly outside of RFP SUN2 in the nuclear membrane. Furthermore, the distance between the two peaks for Jaw1 and RFP SUN2 was ∼100 nm, which is near to the total of two distances: the distance (∼50 nm) between ONM and INM, as previously reported (6), and the molecular lengths of N-terminal tagged FLAG Jaw1 and RFP SUN2. Therefore, these results suggest that Jaw1 localized on the ONM. Aberrant nuclear shape in Jaw1-depleted B16F10 cells A defect in components of LINC complex causes nuclear abnormalities (8). Based on the partial homology of the Jaw1 carboxyl-terminal region to KASH proteins and the localization of Jaw1 on the ONM, we investigated the effects of Jaw1 depletion on nuclear shape by siRNA-mediated Jaw1 knockdown in B16F10 cells. Hoechst DNA staining was used to evaluate the nuclear shape in the cells. The confocal microscopic images showed that a significant number of nuclei in the Jaw1 KD B16F10 cells were misshapen or contained nuclear lobes, while most of the nuclei in control cells appeared to have a normal ellipse shape (Fig. 2A). These aberrant nuclear shapes were observed in 30% of the total Jaw1 KD B16F10 cells (Fig. 2B). Furthermore, the abnormalities of the nuclear shape in Jaw1 KD B16F10 cells were confirmed by transmission electron microscopy (TEM). As shown in Fig. 2C, nuclear lobes and misshapen nuclei were also observed in the electron micrographs of Jaw1 KD B16F10 cells. To verify the effect of siRNA against Jaw1 on nuclear shape, rescue experiments were carried out. For this purpose, we prepared an siRNA-resistant Jaw1 construct (GFP Jaw1RQ) and confirmed its expression by western blotting (Fig. 2D). B16F10 cells were transfected with siRNA followed by the transfection with plasmid encoding GFP alone or GFP Jaw1RQ, and then stained them with Hoechst33342 to evaluate nuclear shape. Approximately 30% of the cells had aberrant nuclear shapes in GFP expressing control cells. In sharp contrast, almost all the cells expressing GFP Jaw1RQ had normal nuclear shapes (Fig. 2E and F), indicating that Jaw depletion does, in fact, induce aberrant nuclear shapes. Collectively, these results indicate that Jaw1 plays a role in maintaining nuclear shape in B16F10 cells. Fig. 2 View largeDownload slide The effects of Jaw1 knockdown on nuclear shape. B16F10 cells were treated with an siRNA against Jaw1 (Jaw1 KD) or a scrambled control RNA (NT). (A) Nuclear shape was observed using confocal microscopy after staining the DNA with Hoechst33342. Scale bar: 20 μm. Three magnified images of the boxes surrounded with red dot lines (gray in black and white) are shown in the side. Scale bar: 10 μm. (B) Counting of cells having aberrant nuclear shapes of (A) (n>300). In the graph, the proportion of cells with aberrant nuclear shape is shown based on the average of three independent experiments per condition; error bars show SE; ***P <0.005. (C) The cells were observed by TEM. Arrow, blebs of nuclei; Scale bar: 2 μm (left and middle), 500 nm (right). The images of nuclear shape which NE is bordered are shown in the bottom. Rescue experiments. B16F10 cells were treated with siRNA and pTagGFP or pTagGFP Jaw1RQ were transfected. (D) The lysates were subjected to western blotting. GFP Jaw1RQ bands were detected using purified anti-Jaw1 rat antibody and HRP-conjugated anti-rat IgG antibody. (E) The nuclear shape was observed by confocal microscopy. Top: overlay between green; GFP and violet, Hoechst33342. Bottom: the signal of Hoechst33342 was transformed into white. Arrowheads: aberrant nuclei (yellow (gray in black and white), GFP-positive; white, GFP-negative). Arrows: normal nuclei of GFP-positive cells. Scale bar: 20 μm. (F) The proportion of the cells having aberrant nuclear shape out of the number of the cells expressing GFP or GFP Jaw1RQ (n>200) was shown. The cell number is counted up to (n>200) as a total from the experiments divided into three times. Fig. 2 View largeDownload slide The effects of Jaw1 knockdown on nuclear shape. B16F10 cells were treated with an siRNA against Jaw1 (Jaw1 KD) or a scrambled control RNA (NT). (A) Nuclear shape was observed using confocal microscopy after staining the DNA with Hoechst33342. Scale bar: 20 μm. Three magnified images of the boxes surrounded with red dot lines (gray in black and white) are shown in the side. Scale bar: 10 μm. (B) Counting of cells having aberrant nuclear shapes of (A) (n>300). In the graph, the proportion of cells with aberrant nuclear shape is shown based on the average of three independent experiments per condition; error bars show SE; ***P <0.005. (C) The cells were observed by TEM. Arrow, blebs of nuclei; Scale bar: 2 μm (left and middle), 500 nm (right). The images of nuclear shape which NE is bordered are shown in the bottom. Rescue experiments. B16F10 cells were treated with siRNA and pTagGFP or pTagGFP Jaw1RQ were transfected. (D) The lysates were subjected to western blotting. GFP Jaw1RQ bands were detected using purified anti-Jaw1 rat antibody and HRP-conjugated anti-rat IgG antibody. (E) The nuclear shape was observed by confocal microscopy. Top: overlay between green; GFP and violet, Hoechst33342. Bottom: the signal of Hoechst33342 was transformed into white. Arrowheads: aberrant nuclei (yellow (gray in black and white), GFP-positive; white, GFP-negative). Arrows: normal nuclei of GFP-positive cells. Scale bar: 20 μm. (F) The proportion of the cells having aberrant nuclear shape out of the number of the cells expressing GFP or GFP Jaw1RQ (n>200) was shown. The cell number is counted up to (n>200) as a total from the experiments divided into three times. Interaction of Jaw with SUN proteins and microtubules KASH proteins are defined as components of the LINC complex through interaction with SUN proteins. In order to investigate the interaction between Jaw1 and SUN proteins, the plasmids coding N-terminal HA-FLAG tandem tagged SUN1or SUN2 (hereafter referred to as FLAG SUN1 or FLAG SUN2) and GFP Jaw1 were used (Fig. 3A). As a positive control, GFP Jaw1ΔC-KASH5 C-lum, in which the Jaw1 luminal region is replaced by the KASH domain of KASH5 was used. HEK293T cells were transfected as indicated and the lysates were co-immunoprecipitated by treatment with anti-FLAG beads. As predicted, GFP Jaw1ΔC-KASH5 C-lum (positive control) was co-immunoprecipitated by FLAG SUN1 or SUN2, while GFP was not (Fig. 3B and C). Importantly, GFP Jaw1 was co-immunoprecipitated by FLAG SUN1 or SUN2 (Fig. 3B and C). These results indicate that Jaw1 has an affinity for SUN proteins, the same as KASH proteins. Fig. 3 View largeDownload slide Interaction of Jaw1 with SUN proteins and microtubules and oligomerization among Jaw1 molecules. (A) Schematic representations of GFP Jaw1 and GFP Jaw1ΔC-KASH5 C-lum, the luminal region of Jaw1 is replaced by that of mouse KASH5. Interaction between Jaw1 and SUN1 or SUN2, pTagGFP, pTagGFP Jaw1ΔC-KASH5 C-lum or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG SUN1 (B) or pcDNA5 FRT/TO HA FLAG SUN2 (C) into HEK293T cells. After incubation for 24 h, the medium was refreshed and incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation using anti-FLAG beads. For western blotting, anti-FLAG mouse monoclonal antibody and anti-GFP mouse monoclonal antibody as primary antibodies and HRP-conjugated anti-mouse IgG antibody as a secondary antibody were used. (D) Interaction between Jaw1 and microtubules. pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and the cells were incubated for 24 h. After the refreshing the medium, the cells were incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation and western blotting using an anti-FLAG mouse monoclonal antibody and anti-α-tubulin monoclonal antibody were used. (E) Oligomerization of Jaw1 molecules. pTagGFP or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG Jaw1 into HEK293T cells. After that it was followed by the procedures of (B, C). Fig. 3 View largeDownload slide Interaction of Jaw1 with SUN proteins and microtubules and oligomerization among Jaw1 molecules. (A) Schematic representations of GFP Jaw1 and GFP Jaw1ΔC-KASH5 C-lum, the luminal region of Jaw1 is replaced by that of mouse KASH5. Interaction between Jaw1 and SUN1 or SUN2, pTagGFP, pTagGFP Jaw1ΔC-KASH5 C-lum or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG SUN1 (B) or pcDNA5 FRT/TO HA FLAG SUN2 (C) into HEK293T cells. After incubation for 24 h, the medium was refreshed and incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation using anti-FLAG beads. For western blotting, anti-FLAG mouse monoclonal antibody and anti-GFP mouse monoclonal antibody as primary antibodies and HRP-conjugated anti-mouse IgG antibody as a secondary antibody were used. (D) Interaction between Jaw1 and microtubules. pcDNA5 FRT/TO HA FLAG Jaw1 was transfected into B16F10 cells and the cells were incubated for 24 h. After the refreshing the medium, the cells were incubated for an additional 24 h. The lysates were subjected to co-immunoprecipitation and western blotting using an anti-FLAG mouse monoclonal antibody and anti-α-tubulin monoclonal antibody were used. (E) Oligomerization of Jaw1 molecules. pTagGFP or pTagGFP Jaw1 was co-transfected with pcDNA5 FRT/TO HA FLAG Jaw1 into HEK293T cells. After that it was followed by the procedures of (B, C). It has been reported that KASH proteins interact with cytoskeletons such as actin filaments, microtubules or intermediate filaments, through its cytosolic region (4). We previously used co-immunoprecipitation and mass spectrometry to identify proteins that interact with Jaw1. This comprehensive analysis indicated the existence of an interaction between Jaw1 and microtubules (data not shown). Therefore, we confirmed whether Jaw1 interacts with microtubules similar to that for KASH proteins. Co-immunoprecipitation assays in B16F10 cells confirmed an interaction between FLAG Jaw1 and α-tubulin (Fig. 3D), suggesting that Jaw1 is associated with microtubules. These data suggest that Jaw1 interacts with SUN proteins and microtubules, and functions as a component of the LINC complex. It is known that SUN proteins interact with KASH proteins in a trimer, resulting in the formation of a heterohexamer (27–29). Furthermore, it has been reported that some KASH proteins form oligomeric structures (30). Therefore, we examined the issue of whether Jaw1 forms an oligomer. As shown in Fig. 3E, GFP Jaw1 was co-immunoprecipitated by FLAG Jaw1, while GFP was not co-immunoprecipitated by FLAG Jaw1. This result indicates that Jaw1 interacts with SUN proteins and microtubules potentially in the form of an oligomer. Collectively, our data suggest that Jaw1, similar to KASH proteins, functions as a component of the LINC complex in the form of an oligomer. Discussion In this study, we performed a functional analysis of Jaw1/LRMP in B16F10 cells. Confocal imaging showed that Jaw1 is localized on the nuclear membrane and the ER network, as previously reported (20). Line plot profiling of the confocal microscopic images suggested that Jaw1 is localized on the ONM. Knockdown-rescue experiments indicated that Jaw1is important for maintaining nuclear shape. Co-immunoprecipitation assays showed that Jaw1 interacts with SUN1, SUN2 and microtubules. Furthermore, we also found that Jaw1 has the potential to form oligomers. We therefore propose that Jaw1 functions as a component of the LINC complex. Jaw1 interacts with microtubules on the cytosolic face and with SUN proteins in the PNS via the KASH domain, as shown in Fig. 4. The Jaw1-mediated physical linkage across the NE would enable a nuclear shape to be maintained in the form of a normal ellipse. Fig. 4 View largeDownload slide A model for Jaw1 function as a KASH protein. A model showing the function of Jaw1 as a KASH protein. Jaw1 interacts with SUN1 and SUN2 in the PNS and bind to microtubules on cytosolic face in the form of oligomer. This LINC complex bridging across the NE functions for the maintenance of the nuclear shape. Fig. 4 View largeDownload slide A model for Jaw1 function as a KASH protein. A model showing the function of Jaw1 as a KASH protein. Jaw1 interacts with SUN1 and SUN2 in the PNS and bind to microtubules on cytosolic face in the form of oligomer. This LINC complex bridging across the NE functions for the maintenance of the nuclear shape. Five mammalian KASH proteins have been identified to date: Nesprin1–4 that is expressed ubiquitously and KASH5 that is expressed in reproductive organs specifically in testis and ovary (4, 7). Nesprin1 and Nesprin2 directly bind to actin filaments via their actin-binding domains (31), Nesprin3 binds to intermediate filaments via Plectin (30, 32) and Nesprin4 and KASH5 are connected to microtubules via motor proteins such as kinesin and dynein respectively (7, 12). The findings reported herein indicate that Jaw1 has a role in maintaining nuclear shape via its interaction with microtubules; however, it remains unclear whether Jaw1 interacts with microtubules directly or indirectly through other molecules. Furthermore, we observed the abnormal nuclear shape in Jaw1 KD B16F10 cells, which is caused by the acute reduction of the expression level of Jaw1 by siRNA-mediated knockdown. However, Jaw1 has a potential redundancy with Nesprin1–4 that is expressed ubiquitously to maintain the nuclear shape. This point should be examined in future studies if a more complete understanding of the mechanism responsible for how Jaw1 functions at nuclear membrane. In this study, we confirmed that the Jaw1 carboxyl-terminal is cleaved post-translationally, consistent with a previous report (26). Since the carboxyl-terminal KASH domain is required for its interaction with SUN proteins, it would be possible that full length Jaw1 functions at nuclear membrane as a KASH protein, on the other hand, carboxyl-terminal cleaved form resides at the ER membranes has other roles. To test the hypothesis, we plan to attempt a subcellular fractionation of the nucleus and the ER membranes in cells expressing epitope-tagged Jaw1. Although Jaw1 has been identified as a protein that is expressed abundantly in lymphoid organs and immune cells (17, 18), we found that Jaw1 is also expressed in B16F10 mouse melanoma cells at a transcriptional level. As shown in the database (THE HUMAN PROTEIN ATLAS, TISSUE ATLAS, LRMP), Jaw1 is also expressed in other human tissues including the brain, lungs, pancreas, urinary bladder, ovaries, skin etc. at lower or medium levels. Therefore, we propose that Jaw1 is expressed in many tissues at different levels. However, the question arises as to why Jaw1 expression levels are so different between lymphoid organs and other tissues. It is known that the expression levels of the LINC components are varied along with the differentiation and maturation in granulocytes and macrophages (33). Furthermore, cellular polarity and cell proliferation following activation are regulated via a mechanism mediated by the LINC complex (34, 35). Eventually, the physical network based on cytoskeletons via LINC complex has an important role in cell migration, polarity, differentiation and proliferation. Therefore, the issue of how Jaw1 functions as a KASH protein and whether a carboxyl-terminal event is involved in above cellular events will be our next research focus. Acknowledgements We wish to thank Ms Ea Kristine Clarisse B. Tulin for critical reading of the manuscript and Ms Ayana Umeda for technical supports. We also wish to thank to Dr Howard J. 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Virol . 91 , e02303 – e02316 Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations ER endoplasmic reticulum GFP, green fluorescence protein; INM inner nuclear membrane KASH Klarsicht/ANC-1/Syne/homology LINC linker of nucleus and cytoskeletons LRMP lymphoid restricted membrane protein NE nuclear envelope ONM outer nuclear membrane PNS perinuclear space SUN Sad-1/UNC-84 © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of BiochemistryOxford University Press

Published: Jun 6, 2018

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