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

doi: 10.1242/10.1242/jcs.00052

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 Leach, C. Articles by Brautigan, D. L. Search for Related Content PubMed PubMed Citation Articles by Leach, C. Articles by Brautigan, D. L.

Domains of type 1 protein phosphatase inhibitor-2 required for nuclear and cytoplasmic localization in response to cell-cell contact

Center for Cell Signaling, University of Virginia School of Medicine, Box 800577-MSB7225, Charlottesville VA 22908, USA

View larger version (87K): [in a new window]   Fig. 1. Inh2 localization corresponding to cell density. PC3 (A,B) and HeLa (C,D) cells were grown to either low (A,C) or high density (B,D) on fibronectin-coated coverslips and examined by immunofluorescence using an antibody against human Inh2. To confirm Inh2 distribution by western blotting, high-density (HD) or low-density (LD) HeLa cells were permeabilized with 0.01% digitonin to yield a cytosolic fraction, labeled C, and a nuclear fraction, labeled N. The top panel shows western blotting using an antibody to Inh2 and the bottom panel shows blotting with an antibody to RCC1 the Ran activator protein, a constitutively nuclear protein.

View larger version (63K): [in a new window]   Fig. 2. Endogenous PP1 has a similar localization to Inh2. HeLa cells were grown to either low (A) or high density (B) on coverslips and examined by immunofluorescence using an antibody against PP1.

View larger version (21K): [in a new window]   Fig. 3. FACS analysis of cells cultured at different densities. HeLa cells growing at low or high density were trypsinized and stained with propidium iodide as described in Materials and Methods. The phase of the cell cycle was determined on the basis of the intensity of the propidium iodide staining using CellQuest software.

View larger version (51K): [in a new window]   Fig. 4. Inh2 relocalization occurs within minutes of altering cell density. HeLa cells growing at high density were trypsinized and replated at low density on fibronectin-coated coverslips. After 20 (A,B,C), 40 or 60 minutes (D,E,F), cells were fixed with 3% paraformaldehyde and examined by immunofluorescence microscopy using an antibody to Inh2. Panels A and D show the staining of endogenous Inh2 in green. Panels B and E show the DAPI staining of DNA in red. Panels C and F show the merged image of the Inh2 staining with DAPI staining in red with the overlay of the two stains yielding yellow. Cells were scored as having nuclear staining brighter than, equal to, or dimmer than cytoplasmic staining. The bottom graph shows the combined result of three independent experiments with more than 100 cells being tallied in each experiment. The dashed line shows the percentage of cells with predominant nuclear staining and the solid line shows the percentage of cells with predominant cytoplasmic staining.

View larger version (40K): [in a new window]   Fig. 5. Separate domains of Inh2 are required for cytoplasmic retention and nuclear import. HeLa cells expressing myc-tagged Inh2 proteins were cultured at either low (B,D,F,H,J) or high density (A,C,E,G,I) on fibronectin-coated coverslips. A and B show a phase image for I and J and are representative of all panels. C and D show a near full-length Inh2 [0-197]; E and F are an N-terminally truncated form (residues 78-197); G and H are a C-terminal truncation (residues 0-119) and I and J are of a central construct consisting of residues 78-150. K shows a schematic of the truncations used.

View larger version (63K): [in a new window]   Fig. 6. Residues 78-119 are sufficient for cytoplasmic retention of a heterologous protein. HeLa cells cultured at low density and expressing either GFP (A) or a fusion of residues 78-119 of Inh2 with GFP (B) were visualized for GFP fluorescence.

View larger version (45K): [in a new window]   Fig. 7. Inhibitor-2 is actively imported into the nucleus. A and B show low-density HeLa cells expressing either an Inh2GFP3 fusion (A) or an Inh2GFP3 fusion in which lysines 142 and 144 have been mutated to alanine, termed NLS mutGFP3 (B). Cells were visualized with the fluorescence of GFP and the nuclear, cytoplasmic and total fluorescence were quantified. The graph shows the import index for GFP3, Inh2GFP3 fusion, NLS MutGFP3 or Inh2[Thr72Ala]GFP3 fusion.

View larger version (56K): [in a new window]   Fig. 8. Inhibitor-2 exits the nucleus by passive diffusion. HeLa cells were electroporated with either Inh2GFP3 or Inh2HA and grown at low density for 6 hours. The cells were then trypsinized and plated onto fibronectin-coated coverslips for 4 hours. The coverslips were then fixed and visualized either for GFP fluorescence or by indirect immunofluorescence techniques directed against the triple HA tag (orange). A shows the GFP fluorescence and B shows a merge of the HA immunofluorescence (orange) and DAPI staining of the DNA (Blue).