Technical Reference #1608

1608.

PinX1 is recruited to the mitotic chromosome periphery by Nucleolin and facilitates chromosome congression Na Li; Kai Tuan; Feng Yan; Yuda Huo; Tongge Zhu; Xing Liu; Zhen Guo; Xuebiao Yao, University of Science and Technology of China, Biochemical and Biophysical Research, 384(1608), (2009)

Abstract:
Mitotic chromosome movements are orchestrated by interactions between spindle microtubules andchromosomes. It is well known that kinetochore is the major site where microtubule-chromosomeattachment occurs. However the functions of other domains of ch

Keywords:
Mitotic chromosome periphery; chromosome congression; nucleolin; PinX1

Materials & Methods:
Cell culture and synchronization. HeLa cells (American Type Culture Collection Rockville MD) were maintained as sub-confluent monolayers in Dulbecco’s modified Eagle’s medium (Invitrogen) with 10% fetal bovine serum (Hyclone Logan UT) and 100 units/ ml penicillin plus 100 lg/ml streptomycin (Invitrogen) at 37 C with 8% CO2. Cells were synchronized at G1/S with 5 mM thymidine for 1216 h and then washed with phosphate-buffered saline (PBS) 5 times and cultured in thymidine-free medium for 10 h. To collect mitotic cells for in vitro studies cells were synchronized with 0.1 lg/ml nocodazole for 16 h. Plasmids construction and protein expression. The cDNA of PinX1 (NM_017884) was kindly provided by Dr. Kunping Lu (Harvard). To generate green fluorescent protein (GFP)-tagged and bacterial expression constructs of PinX1 and deletions PCR-amplified cDNAs were cloned into pEGFP-C1 (Clontech) and pGEX-5X-3 (Amersham Bioscience) vectors by EcoRI and SalI. The bacterial expression construct of Nucleolin MBP-1234-RGG was a generous gift from Dr. Nancy Maizels (Yale). The GST-tagged proteins and the MBP-1234-RGG were expressed and purified as previously reported [1819]. Antibodies and RNA interference. The following antibodies were used: anti-PinX1 (Abnova) anti-Nucleolin (Abcam) anti-cyclin B (BD). siRNA targeting PinX1 (SI00684971) and Nucleolin (SI02654925) were purchased from Qiagen. All the siRNAs and plasmids were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen). In vitro pulldown and immunoprecipitation. GST-PinX1-bound Sepharose beads were used as an affinity matrix to isolate interacting proteins from mitotic HeLa cell lysate. Briefly 5 107 HeLa cells were synchronized by nocodazole and collected by mitotic shake off. The mitotic cells were washed once with PBS and lysed in lysis buffer (50 mMHepes pH 6.9 150 mMNaCl 2 mMEGTA 0.1% Triton X-100 1 mMphenylmethylsulfonyl fluoride 10 g/ml leupeptin and 10 g/ml pepstatin A). After centrifugation the supernatant was first pre-cleared by incubating with GST-bound Sepharose beads for 2 h and then incubated with GST-PinX1-bound Sepharose beads for 4 h. All the incubations were carried out at 4 C to avoid protein degradation. Beads were washed 4 times with lysis buffer and boiled in SDS sample buffer followed by fractionation of bound proteins on 8% SDS–PAGE gel. The protein band of interest was removed for mass spectrometric analyses as described previously [20]. For immunoprecipitation control IgG and Nucleolin mAb were first conjugated to protein A/G (Pierce) according to the manufacturer’s protocol and then incubated with mitotic HeLa cell lysate for 4 h. After extensive washes beads were boiled in SDS sample buffer. The bound proteins were fractionated on 10% SDS–PAGE gel and transferred to nitrocellulose membrane. The membrane was divided into three strips and probed with antibodies against Nucleolin PinX1 and tubulin respectively. Immunofluorescence microscopy and time-lapse microscopy. Cells were seeded onto sterile acid-treated 12-mm coverslips in 24 well plates (Corning Glass Works Corning NY). The next day the cells were transfected with 1 ll of Lipofectamine 2000 pre-mixed with plasmids or siRNAs described above. If not specified 48 h after transfection cells were rinsed with PHEM buffer (100 mM PIPES 20 mM Hepes pH 6.9 5 mM EGTA 2 mM MgCl2 and 4 M glycerol) and fixation in freshly prepared 3.7% formaldehyde for 5 min. After permeabilization and rinsed three times in PBS cells were blocked with PBST (0.05% Tween 20 in PBS) with 1% bovine serum albumin (Sigma) followed by incubation with various primary antibodies in a humidified chamber for 1 h. After three washes in PBST primary antibodies were visualized by FITC (fluorescein isothiocyanate) or rhodamine conjugated goat anti-mouse or rabbit IgG. DNA was stained with DAPI (406-diamidino-2-phenylindole). Slides were examined with a DeltaVision Deconvolution microscopy (Applied Precision Inc.). Images were processed using DeltaVision SoftWorx software. Images for display were generated by projecting the sum of the optical sections using the maximum-intensity method. For time-lapse microscopy cells were cultured in a glass-bottom culture dish (MatTek MA) with Leibovitz’s L-15 medium (Invitrogen) at 37 C and examined with a DeltaVision microscopy system. Images were acquired at 2 min intervals and presented in Photoshop.

Microscopic Technique
Time Lapse Microscopy

Cell Type(s)
HeLa