![]() In PC12 cells expressing rCTF-Q28-NES, cytoplasmic aggregates are detected by both A6RPT-#5803 and 1C2. (E) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q13-NES (lower row). (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-Ca v2.1CTF, beta-tubulin: a cytoplasmic protein marker, Histone H3: a nuclear protein marker). The NES-tag faithfully anchored rCTF to the cytoplasm, whereas the NLS-tag efficiently directed rCTF to the nucleus. (C) Immunofluorescence cytochemistry in Day6 stable PC12 cell lines expressing rCTF-Q28-NES (upper row) and rCTF-Q28-NLS (lower row). Anti beta-tubulin antibody was used as internal control. Protein expression starts to be detected on the fourth day after the Dox removal (“Day4”) and reaches abundant level in Day6. (B) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by Western blot using the A6RPT-#5803 antibody. The beta-actin expression level was used as an internal control. The rCTF mRNA level starts to be detected inDa圓 and gradually increases with a time-dependent manner. (A) A timeline of rCTF expression in PC12 cells stably expressing rCTF-Q28-NES by quantitative real-time PCR (qRT-PCR). Only the cell lines expressing rCTF-Q28-NES, rCTF-Q28-NLS and rCTF-Q13-NES are shown here ( Other cells are shown upon request). (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. The localization signals change subcellular localization of rCTFs-polyQ effectively. The rCTF-polyQ is predominantly, though not exclusively, expressed in the cytoplasm of PC12 cells. (D) The proportion of the subcellular localization in each rCTF expressed. (C) The NLS and NES efficiently shift rCTFs with expanded polyQ (Q28) into the nucleus and cytoplasm respectively (scale bars: 10 µm). The NLS and NES efficiently localize the tagged rCTF to the designed location (scale bar: 10 µm). (B) The rCTF-Q13 is predominantly expressed in the cytoplasm of PC12 cells. Definition of rCTF (amino acid(AA) #1954–2506 in Ca v2.1 ), 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 Ca v2.1 CTF, c-Myc antibody against Myc-tag) are shown. (A) Recombinant Ca v2.1 C-terminal fragment (rCTF)s used in this study. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). The human α1A voltage-dependent calcium channel (Cav2.1) is a pore-forming essential subunit embedded in the plasma membrane. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |