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First published online September 17, 2008
doi: 10.1242/10.1242/jcs.033266

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Zinc as a translation regulator in neurons: implications for P-body aggregation

Department of Neurobiology, Weizmann Institute of Science, 76100 Rehovot, Israel

View larger version (30K): [in this window] [in a new window]   Fig. 1. Zinc treatment induces disassembly of polysomes. (A) P19 neurons were treated with 100 M zinc for the indicated times. The cell lysate was loaded onto a 15-45% linear sucrose gradient. Gradient fractions were collected from the top, and equal volume samples were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and analysed for ribosomal proteins distribution. (B) RNA was isolated from the gradient fractions and separated on 1% agarose gel. (C) Statistical analysis of the ribosomal proteins content in fractions 4-6. Bars represent mean ± s.e.m., n 3. *P<0.05 using Student's t-test. (D) Statistical analysis of the ribosomal proteins content in fractions 7-10. Bars represent mean ± s.e.m., n 3. *P<0.05, Student's t-test.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Zinc treatment impairs the association between IMP1 and HuD with polysomes. P19 neurons were treated with 100 M zinc for the indicated times. The cell lysates were loaded on a 15-45% linear sucrose gradient. Gradient fractions were collected and equal volume samples were separated and analysed on SDS-PAGE using IMP1, HuD and ribosomal proteins antibodies.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Zinc application induces the aggregation of Dcp1a protein. P19 neurons were treated with 100 M zinc for 1 hour. Cells were lysed and extracts were centrifuged for 10 minutes at 1000 g. Supernatants with an equal amount of protein were centrifuged for 10 minutes at 20,000 g. (A) Dcp1a protein levels in 20K supernatants in lysates of zinc-treated cells (Z1-Z3) compared with control cell lysates (C1-C3). (B) Dcp1a protein levels in 20K pellets (resuspended in equal volumes of lysis buffer) of zinc-treated cells (Z1-Z3) compared with control cell pellets (C1-C3). (C) Statistical analysis of Dcp1a protein levels in 20K supernatants of zinc-treated cell lysates and control cell lysates, normalized to -actin protein levels. Bars represent mean ± s.e.m., n 3. *P<0.01, Student's t-test. (D) Statistical analysis of Dcp1a protein levels in 20K pellets of zinc-treated cells and in control cells, normalized to -actin protein levels. Bars represent mean ± s.e.m., n 3. *P<0.01, Student's t-test.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Zinc application induces the aggregation of Dcp1a protein in dense sucrose gradient fractions. P19 neurons were treated with 100 M zinc for the indicated times. Cell lysate was loaded on 15-45% linear sucrose gradient and gradient fractions were collected from the top and analysed for Dcp1a protein distribution. (A) SDS-PAGE of equal volume samples of the gradient fractions. (B) Statistical analysis of Dcp1a protein levels in gradient fractions 6-10 of cells treated with zinc for 2 hours, compared with control cells. Results were normalized to 2% of the cell lysate Bars represent mean ± s.e.m., n 3. *P<0.05, Student's t-test.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Dcp1a aggregation in response to zinc treatment is RNA dependent. P19 neurons were treated with 100 M zinc and harvested. Cell lysates were incubated with 1 g/l RNase A for 30 minutes at room temperature, prior to sucrose gradient centrifugation. Gradient fractions were collected and subjected to western blot analysis. Western blot analysis (A) using Dcp1a antibody and (B) using HuD antibody, as a control.

View larger version (50K): [in this window] [in a new window]   Fig. 6. Overexpression of Dcp1a in HEK293 cells does not induce disassembly of polysomes. (A) HEK293 cells were transfected with GFP-Dcp1a or GFP, and analysed for the presence of P-bodies using Dcp1a antibody. Scale bars: 2 m. Cell extracts of HEK293 cells transfected with GFP-Dcp1a were loaded on 15-45% sucrose gradient. Gradient fractions were collected (see Fig. 4) and examined using western blot analysis. (B) Dcp1a distribution in gradient fractions of GFP-Dcp1a or GFP (control) transfected cells. (C) Ribosomal proteins distribution in gradient fractions of GFP-Dcp1a or GFP (control) transfected cells.

View larger version (66K): [in this window] [in a new window]   Fig. 7. HuD, but not IMP1, colocalizes with Dcp1a in HEK293 cells. (A) GFP-HuD construct (a) was transfected into HEK293 cells, followed by immunostaining with anti-Dcp1a antibody (b). Colocalization in P-bodies is indicated with white arrows (c). GFP-IMP1 construct was transfected into HEK293 cells (d), followed by immunostaining with anti-Dcp1a antibody (e). P-bodies are indicated with a yellow arrow (f). (B) Lysates of HEK293 cells transfected with GFP-Dcp1a were incubated with GST beads coupled to GST-tagged HuD protein.

View larger version (95K): [in this window] [in a new window]   Fig. 8. Endogenous colocalization of HuD with Dcp1a in P19 neurons. Day 8 (post retinoic acid) P19 neurons were immunostained using antibodies against HuD and Dcp1a proteins. Fluorescein isothiocyanate (FITC)- and Cy3-conjugated secondary antibodies were used to label (A) Dcp1a and (B) HuD, respectively. (C) Merged image of HuD and Dcp1a. Arrows indicate colocalization of both proteins (D) 3D reconstruction of the merged image, produced with the LSM 510 software. Scale bars: 1 m.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Endogenous colocalization of HuD and Dcp1a in growth-cone-like structures of differentiated PC12 cells. PC12 cells were neuronally differentiated by incubation with 50 ng/ml NGF for 4 days. Cells were then fixed and immunostained using antibodies against HuD and Dcp1a. Fluorescein isothiocyanate (FITC)- and Cy3-conjugated secondary antibodies were used to label (A) Dcp1a and (B) HuD, respectively. (C) Merged image of both Dcp1a and HuD. Arrows indicate colocalization of both proteins. (D) Phase image of differentiated PC12 cells.

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