Figure 1.
Localization signals efficiently change the intracellular location of CTF.
(A) Recombinant Cav2.1 C-terminal fragment (rCTF)s used in this study. Definition of rCTF (amino acid(AA) #1954–2506 in Cav2.1 [GenBank AB035726] [18]), artificial nuclear localization or export signals (NLS or NES), two different polyglutamine (Qn) (Q13; Q28) and recognition sites of three different antibodies (1C2 against expanded polyQ, A6RPT-#5803 against Cav2.1 CTF, c-Myc antibody against Myc-tag) are shown. (B) The rCTF-Q13 is predominantly expressed in the cytoplasm of PC12 cells. The NLS and NES efficiently localize the tagged rCTF to the designed location [A6RPT-#5803 antibody (red), Hoechst 33258 (blue)] (scale bar: 10 µm). (C) The NLS and NES efficiently shift rCTFs with expanded polyQ (Q28) into the nucleus and cytoplasm respectively (scale bars: 10 µm). (D) The proportion of the subcellular localization in each rCTF expressed. The rCTF-polyQ is predominantly, though not exclusively, expressed in the cytoplasm of PC12 cells. The localization signals change subcellular localization of rCTFs-polyQ effectively. (N; the cells expressing rCTF exclusively in the nucleus; N-c: the cells expressing rCTF predominantly in the nucleus than in the cytoplasm; n-C: the cells expressing rCTF predominantly in the cytoplasm than in the nucleus; C: the cells expressing rCTF exclusively in the cytoplasm) (For D: ***:p<0.001;ANOVA. Error bars indicate ± SEM.).
Figure 2.
Characterization of inducible PC12 cells stably expressing rCTFs.
Only the cell lines expressing rCTF-Q28-NES, rCTF-Q28-NLS and rCTF-Q13-NES are shown here (Other cells are shown upon request). (A) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by quantitative real-time PCR (qRT-PCR). The rCTF mRNA level starts to be detected inDay3 and gradually increases with a time-dependent manner. The beta-actin expression level was used as an internal control. (B) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by Western blot using the A6RPT-#5803 antibody. Protein expression starts to be detected on the fourth day after the Dox removal (“Day4”) and reaches abundant level in Day6. Anti beta-tubulin antibody was used as internal control. (C) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q28-NLS (lower row). The NES-tag faithfully anchored rCTF to the cytoplasm, whereas the NLS-tag efficiently directed rCTF to the nucleus. (D) Western blot analysis on protein extracts from stable cell lines expressing rCTF-Q28-NLS and rCTF-Q28-NES confirming efficient intracellular localizations (A6RPT-#5803: anti-Cav2.1CTF, beta-tubulin: a cytoplasmic protein marker, Histone H3: a nuclear protein marker). (E) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q13-NES (lower row). In PC12 cells expressing rCTF-Q28-NES, cytoplasmic aggregates are detected by both A6RPT-#5803 and 1C2. In rCTF-Q13-NES cells, rCTF-aggregates are recognized by the anti-Cav2.1 antibody A6RPT-#5803, but not by 1C2, a monoclonal antibody specific for expanded polyQ. (For C&E, scale bars: 10 µm).
Figure 3.
The rCTF is toxic to cells when expressed in the cytoplasm.
(A) Cell death in transiently over-expressed HEK293T cells assessed by LDH level at 72 hours after transfection. The rCTF-polyQ-NES exhibits stronger cytotoxicity than rCTF-polyQ-NLS. (B) Time course of cell death in inducible rCTF-Q28-NES PC12 cell line. Cell toxicity is detected from Day5 and become prominent in Day6. (C) The cell death at Day6 compared in 6 inducible PC12 cell lines (rCTF-Q13, rCTF-Q13-NLS, rCTF-Q13-NES, rCTF-Q28, rCTF-Q28-NLS, rCTF-Q28-NES). The cell line expressing rCTF-Q28-NES exerts the strongest cell death. Y-axis shows differences of LDH values between (Dox(−)) and (Dox(+)) states. Cells do not show obvious cell death in Dox(+) states. (D&E) TUNEL positive cells are dramatically increased when doxycyclin is removed (Dox(−)). Eight randomly selected microscope fields were counted for TUNEL positive cells and total cell numbers. (For A to C and E: *: p<0.05; **: p<0.01; ***:p<0.001;ANOVA for A to C. Mann-Whitney's U test for E. Error bars indicate ± SEM.).
Figure 4.
CREB co-localizes with intracytoplasmic CTF aggregates and quantity of CREB is decreased in cultured cell model.
(A) In non-transfected PC12 cells, CREB and p-CREB-immunofluorescence labeling is strong in the nucleus, while the cytoplasm shows only weak and diffuse immunofluorescence. (B) In PC12 cells over-expressing rCTF-Q28-NES, the cytoplasmic CTF aggregates co-localize with CREB (upper row) and p-CREB (lower row), showing focally strong immunofluorescence in their cell bodies (arrows). (C&D) In contrast to the Dox(+) stable PC12 cells (upper row in C&D), co-localizations of CREB (lower row in C) or p-CREB (lower row in D) with cytoplasmic aggregates are also observed in Dox(−) stable PC12 cells expressing rCTF-Q28-NES (arrows). (E) The quantities of CREB and p-CREB in the nucleus were both decreased when the rCTF was expressed in the cytoplasm, but not so when the rCTF was targeted in the nucleus. (For A-D: scale bars: 10 µm).
Figure 5.
CREB and Cav2.1 aggregates co-localize in SCA6 human Purkinje cells.
(A) In a control brain affected with Parkinson's disease, the immunoreactivity against CREB is present but weak and homogeneous in the neuronal cytoplasm of two Purkinje cells. (B) In SCA6 human Purkinje cells, focally accentuated immunoreactive structures (arrows) are seen in the cytoplasm. (C) In ten control brains, a vast majority of Purkinje cells showed weak and diffuse immunoreactivity for the anti-CREB antibody as demonstrated in the Figure 5 A. In contrast, aggregate-like CREB-immunoreactive structures were seen in the Purkinje cells from three SCA6 cerebella. Please refer to the Materials and Methods for the detailed description on the criteria of CREB-immunoreactivities. (D) On double immunofluorescence analysis using a rabbit polyclonal CREB antibody and 1C2, a mouse monoclonal antibody against expanded polyQ tracts, microscopic Cav2.1 aggregates in the cytoplasm of the SCA6 Purkinje cells indeed co-localized with CREB (arrows). (For A&B: scale bars: 50 µm; for D&E: scale bar: 10 µm). (E) In three SCA6 cerebella, approximately 50% of Purkinje cells containing 1C2-positive polyQ aggregates in the cytoplasm showed co-localization of the polyQ aggregates and CREB. In contrast, the Purkinje cells in control cerebella did not show 1C2-positive aggregates resulting in no co-localizations.