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.01477

This Article Summary Full Text Full Text (PDF) Supplementary Material 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 Yang, Z. Articles by Chan, E. K. L. Search for Related Content PubMed PubMed Citation Articles by Yang, Z. Articles by Chan, E. K. L. Social Bookmarking                         What's this?

GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation

Zheng Yang^1,*,, Andrew Jakymiw^1,*, Malcolm R. Wood², Theophany Eystathioy³, Robert L. Rubin⁴, Marvin J. Fritzler³ and Edward K. L. Chan^1,

¹ Department of Oral Biology, University of Florida, PO Box 100424, Gainesville, FL 32610-0424, USA
² The Core Microscopy Facility, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
³ Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive, Calgary, Alberta, T2N 4N1, Canada
⁴ Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, 915 Camino de Salud NE, Albuquerque, NM 87131, USA

View larger version (63K): [in a new window]   Fig. 1. The number and size of GWBs varies during the cell cycle. (A-C) HEp-2 cells were co-stained with the index human anti-GW182 serum (A, green; nuclei counterstained with DAPI, blue) and rabbit anti-CENP-F antibody (B, red). CENP-F is known to be expressed predominantly in cells in late S or G2 phase and throughout mitosis (B, arrows). (C) The merged image of GW182 and CENP-F. GWBs show cell-cycle-dependent expression, with the largest bodies (arrowheads) detected primarily within cells in the late S or G2 phase. The number of GWBs also is higher in cells with the strongest expression of CENP-F. Staining of GW182 in cells at anaphase (a), telophase (t), and G1 stages are indicated by double-arrows (A). GWBs are sometimes observed adjacent to the nuclear membrane (thin arrowheads). Scale bar: 10 µm. (D) Western blot analysis of the index human anti-GW182 serum (lane 1) and a control normal human serum (lane 2) using HEp-2 cell lysates, separated by SDS-PAGE using a 12.5% gel and transferred to a nitrocellulose membrane.

View larger version (168K): [in a new window]   Fig. 2. Immunogold electron microscopy localization of GWBs in the cytoplasm of HeLa cells during interphase. Frozen sections of fixed and gelatin-embedded HeLa cells were incubated with the index human anti-GW182 serum diluted 1:400 and then post-immunolabeled with protein A-gold (10 nm). (A-E) Representative gold-labeled cytoplasmic structures with diameters which vary from 100-300 nm. The gold labels are clustered on electron dense fibrils or strands, 8 to 10 nm in diameter, which are more obvious in some images (arrowheads in A,B,D,E). These fibrils appear to form the matrix that the gold decorates. Some gold particles are observed on the periphery of the cluster (arrows in A,C) while the majority of the gold is found clearly within the center of these structures.

View larger version (168K): [in a new window]   Fig. 3. Immunogold electron microscopy localization of GWBs adjacent to the nuclear membrane in HeLa cells during interphase. (A) Two clusters of gold particles (arrows) adjacent to the cytoplasmic face of the nuclear envelope. Note that they are some distance from nuclear pores (arrowheads). The enlarged images of the two clusters are shown in B and C.

View larger version (84K): [in a new window]   Fig. 4. Disassembly of GWBs before mitosis and reassembly after mitosis was preceded by the reassembly of the nucleolus. HEp-2 cells were double stained with the index human anti-GW182 serum (green, A,E,I,M) and mouse monoclonal anti-fibrillarin antibody 72B9 (red, B,F,J,N). (C,G,K,O) DAPI counterstain; (D,H,L,P) merged images. Cells in anaphase (A-D) or telophase (E-P) show few or no GWBs while diffuse cytoplasmic staining is still visible (A,E). In daughter cells showing maturing nucleoli (I-L, M-P), newly assembled GWBs (arrowheads) are observed. Arrows indicate pre-nucleolar bodies in B and reassembling nucleoli in N.

View larger version (164K): [in a new window]   Fig. 5. Immunogold electron microscope images of GWBs in mitotic HeLa cells. Samples were prepared and processed as in Fig. 2. (A) Low magnification of mitotic chromosomes (M). (B,C) Higher magnification views of the boxed areas indicated in A. Arrows show clusters of gold particles adjacent to a mitotic chromosome.

View larger version (66K): [in a new window]   Fig. 6. Expression of GW182 in HeLa cells synchronized by double thymidine block. (A) Cells were processed for IIF at 0, 3, 6 and 9 hours after release from thymidine block and stained with human anti-PCNA serum or the index human anti-GW182 serum. At 0 and 3 hours, most cells stained with anti-PCNA showed the fine nuclear speckled distribution (arrowheads) characteristic of cells in early and mid S phase. Most cells had small GWBs. At 6 and 9 hours, apparently larger GWBs (long arrows) were observed along with the characteristic nucleolar distribution of PCNA in late S and G2 phase (arrows). Scale bar; 10 µm. (B) Western blot analysis of GW182 showed increased expression from early S phase (0 hour), mid S (3 hours), late S (6 hours) to G2 phase (9 hours). Furthermore, slower migrating, diffuse bands (arrowhead) were observed at the later stages suggestive of post-translational modifications, such as phosphorylation.

View larger version (79K): [in a new window]   Fig. 7. Increased expression of GWBs and GW182 in proliferating 3T3 cells after release from serum starvation. (A) Cells were analyzed by IIF at different time points after refeeding, as indicated. At 0 hours, there was little or no staining from human anti-GW182 serum. Smaller discrete GWBs (arrows) were observed beginning at 3 and 6 hours after refeeding. Larger and more numerous GWBs were observed at the subsequent time points (arrowheads). Scale bar: 10 µm. (B) Cells were harvested at different time points and supernatant fractions were separated in a 5% gel by SDS-PAGE and transferred to a nitrocellulose membrane that was later probed with a human anti-GW182 serum.

View larger version (49K): [in a new window]   Fig. 8. Enhanced expression of GW182 in proliferating mouse splenocytes and isolated T cells. Isolated mouse spleen cells or purified T cells were cultured in vitro either in the presence or absence of 5µg/ml ConA for 2 to 4 days. (A-D) Representative anti-GW182 IIF images of isolated T cells from day 3. Cells treated with ConA (C) showed cell aggregation and abundant small (arrowheads) and large (arrows) GWBs. Untreated cells were negative for GW182 (A). (B,D) DAPI counterstaining of the cell nuclei shown in A and C, respectively. Scale bar: 10 µm. (E) Western blot comparison of GW182 expression in cells treated with or without ConA.

View larger version (126K): [in a new window]   Fig. 9. Fusion of GWBs. (A) A continuous sequence of live cell images showing the fusion of two GFP-labeled GWBs into one (arrows) in a HEp-2 cell transfected with a plasmid construct expressing a GFP-GW182 fusion protein. Original magnification x1,500. (See also movie 2 in supplementary material.) (B) ImmunoEM images of protein A-gold-labeled cytoplasmic structures that may be GWBs undergoing fusion (or division). Dotted lines encircle the potential GWBs undergoing fusion similar to the type observed in A.

View larger version (88K): [in a new window]   Fig. 10. GW182 is the GWB `matrix/scaffold' protein as revealed in gene knockdown experiments. GW182 (A) and LSm4 (B) in GWBs are detected by staining with respective antibodies and Alexa Fluor 555-conjugated secondary antibody (red; i,v). GW182 siRNA construct-transfected cells are marked by green fluorescent protein, via cotransfection with phrGFP, which is detected both in the nucleus and cytoplasm (ii,vi). GWBs are detected in non-transfected cells (arrows, i,v) and cells transfected with pSHAG vector control (arrowheads, v,viii) but are absent in cells with the siRNA construct (arrowheads, i,iv). (iv,viii) Merged images. Nuclei are counterstained with DAPI (iii,vii). Scale bar: 10 µm.

View larger version (93K): [in a new window]   Fig. 11. HeLa cells selected for GW182 gene knockdown demonstrated a reduction in GW182 protein levels and few or no GWBs. (A) Western blot analysis of GW182 expression using the index human anti-GW182 serum in GW182-knockdown cells (721A9), control selected cells (721B9), and control untreated HeLa cells. Tubulin antibody was used to check for equal loading of samples. Cell lysates were run on a 10% polyacrylamide gel and transferred to nitrocellulose prior to immunoblotting. (B) GW182 and LSm4 in GWBs were detected by staining with respective antibodies and Alexa Fluor-conjugated secondary antibodies (i-iv). Arrowheads in ii and iv indicate 721A9 cells with few or no GWBs; arrows indicate cells with normal GWB expression (i,iii), and reduced GWB expression (ii,iv). Scale bar: 10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter