QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 October 2004
doi: 10.1242/jcs.01482

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lee, H.-H. Articles by Chang, Z.-F. Search for Related Content PubMed PubMed Citation Articles by Lee, H.-H. Articles by Chang, Z.-F.

Nuclear efflux of heterogeneous nuclear ribonucleoprotein C1/C2 in apoptotic cells: a novel nuclear export dependent on Rho-associated kinase activation

¹ Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No.1 Jen Ai Road Sec.1, Taipei, 100, Taiwan, Republic of China
² Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, No.1 Jen Ai Road Sec.1, Taipei, 100, Taiwan, Republic of China
³ Institute of Chemistry, Academia Sinica, 128, Academia Sinica Road, Nankang, Taipei, 115, Taiwan, Republic of China

View larger version (65K): [in a new window]   Fig. 1. A proteomic screen for ROCK-regulated changes during PMA-induced apoptosis. D2 cells were pre-treated with or without 20 µM Y27632 for 1 hour in serum-free medium on hydrogel-coated dishes prior to treatment with 32.4 nM PMA. (A) After PMA treatment for 8 hours, cell viability was determined by the Trypan Blue exclusion method. NT, cells without either PMA or Y27632 treatment. (B) Phase-contrast micrographs of D2 cells after 4 hours of PMA treatment as indicated. (C) Following treatment with PMA for 4 hours, the cytosolic proteins of cells treated with and without Y27632 were prepared and labeled with Cy3 and Cy5, respectively. Two sets of these labeled lysates each containing 100 µg of proteins were pooled together and separated on a single 2DE gel. Green indicates Cy3-tagged proteins, red Cy5-tagged proteins and yellow colocalization of the spots. (D) A higher power image of spots corresponding to hnRNP C1/C2. (E) Whole cell extracts of D2 cells treated as in C were separated by 2DE and the gels were silver stained. Red dashed line outlines the spots corresponding to hnRNP C1/C2.

View larger version (43K): [in a new window]   Fig. 2. HnRNP C1/C2 are translocated to cytoplasm in PMA-induced pro-apoptotic cells in a manner dependent on ROCK-mediated cytoskeleton rearrangement. (A) D2 cells were treated without (NT) or with 32.4 nM PMA in serum-containing medium on regular dishes. Cells remained in suspension (SUS) and attached to the culture dish (ATT) were harvested at the time indicated. Whole cell lysates, cytosolic and nuclear fractions were prepared and separated by SDS-PAGE, followed by western blot analysis with antibodies against hnRNP C1/C2, Sp1 and TK1. (B) D2 cells treated with 32.4 nM PMA as described above for 4 hour were fixed and incubated with antibody against hnRNP C1/C2, followed by visualization with TRITC-conjugated anti-goat antibody. A parallel PMA-induced suspension culture with 50 µM zVAD-fmk incubation was subjected to the same analysis. Nuclei were stained with DAPI. Scale bar: 8 µm. (C) D2 cells were incubated in serum-free medium on hydrogel-coated dishes and treated with 32.4 nM PMA for 4 hours in the presence of reagents as indicated (0.5 µM latruculin B, LB; 20 µM zDEVD-fmk; 20 µM Y27632; 50 µg/ml cycloheximide, CHX; 5 µg/ml actinomycin D, Act D). (D) D2 cells were treated as (C) in the presence of 50 µM blabbistatin for 4 hours. The cytosolic and nuclear fractions were prepared for Western blot analysis as described above.

View larger version (117K): [in a new window]   Fig. 3. No nuclear leakage occurs in PMA-induced pro-apoptotic cells. (A) Electron micrograph of D2 cells that were treated with 32.4 nM PMA for 2 hours in the presence of 50 µM zVAD-fmk. Cells were then washed with PBS and fixed with 4% paraformaldehyde and 1% glutaraldehyde at 4°C for overnight. N, nucleus; C, cytoplasm; a, heterochromatin; b, nuclear envelope; c, nuclear pore complexes. (B) Western blot of the cytosolic and nuclear fractions, as described in the legend to Fig. 2C, with antibodies against hnRNP C1/C2, hnRNP A2/B1, lamin A/C and TK1.

View larger version (48K): [in a new window]   Fig. 4. The cytosolic distribution of hnRNP C1/C2 in TNF-induced apoptotic NIH3T3 cells is also dependent on ROCK activation. NIH3T3 mouse fibroblasts on 35 mm dishes were transfected with 1 µg of pDsRed-hnRNP C1. Cells were serum-starved for 12 hours, after which cells were pre-treated with cycloheximide (20 µg/ml) for 1 hour, followed by incubation with or without Y27632 (20 µM) or zVAD-fmk (50 µM) as indicated for another 1 hour. Apoptosis was initiated by adding 25 ng/ml TNF to the medium for 3 hours. The nuclei were stained with Hoechst.

View larger version (43K): [in a new window]   Fig. 5. Expression of the dominant active form of ROCK(CAT) induces cytosolic translocation of hnRNP C1/C2. (A) HEK293T cells on 35 mm culture dishes were transfected with 1 µg of expression plasmid of ROCK(CAT) or control vector overnight. Cells were fixed and stained with antibodies against hnRNP C1/C2 and c-myc, followed by visualization with TRITC-conjugated anti-goat and FITC-conjugated anti-mouse antibodies. Nuclear staining was performed with DAPI. (B) The cytosolic and nuclear fractions from cells as described in A were prepared for western blot analysis with antibodies against hnRNP C1/C2 and hnRNP A2/B1. (C) HEK293T cells were co-transfected with 0.5 µg of expression plasmid of GFP-RhoAV14 or GFP-RhoAV14 E40L or pEGFP and 0.5 µg of pDsRed-hnRNP C1. Cells were stained with Hoechst and observed by fluorescence microscopy.

View larger version (46K): [in a new window]   Fig. 6. Mapping the sequence region of hnRNP C1 required for ROCK-induced cytosolic translocation. (A) Schematic presentation of various domains of hnRNP C1 and the constructs of its deletion mutants. HnRNP C1 contains a RNA binding domain (RBD: 1-104), a nuclear retention sequence (NRS: 88-165), and a C-terminal acidic region. (B) HEK293T cells on 35 mm dishes were co-transfected with 0.5 µg of expression plasmid of ROCK(CAT) or control vector together with 1 µg of pDsRed-hnRNP C1 or its deletion mutants as indicated. After expression for 20 hours, cells were fixed, stained with DAPI and observed by fluorescence microscopy for nuclei and DsRed detection.

View larger version (51K): [in a new window]   Fig. 7. The C-terminal 40 amino acids confer nuclear protein ROCK-responsive translocation. (A) HEK293T cells were transfected with 1 µg of pYFP-Nuc or pYFP-Nuc-C40 in combination with 0.5 µg of ROCK(CAT) expression plasmid or control vector as indicated. Cells were fixed and stained with DAPI and observed by fluorescence microscopy for nuclear and YFP expression. (B) The amino acid sequence of the C-terminal 40 amino acids of hnRNP C1 (residues 251-290).