The sperm-oocyte switch in the C. elegans hermaphrodite is controlled through steady-state levels of the fem-3 mRNA

Post-transcriptional control regulates many aspects of germline development in the Caenorhabditis elegans hermaphrodite. This nematode switches from spermatogenesis to oogenesis and is, therefore, capable of self-fertilization. This sperm-oocyte switch requires 3′ UTR-mediated repression of the fem-3 mRNA. Loss of fem-3 repression results in continuous spermatogenesis in hermaphrodites. Although several factors regulating fem-3 have been identified, little is known about the mechanisms that control fem-3. Here, we investigate the steady-state levels of the fem-3 transcript and the expression pattern of its protein product. We show that FEM-3 is exclusively present in germ cells that are committed to spermatogenesis. We found that in fem-3(gf)/+ heterozygotes, mutant fem-3 gain-of-function transcripts are more abundant than their wild-type counterpart. Furthermore, we show that the penetrance of the fem-3(gf) allele correlates with inefficient FBF binding and extended poly(A) tail size of fem-3 mRNAs. Finally, we show that wild-type and gain-of-function mutated fem-3 mRNAs associate equally well with polyribosomes. We propose that the fem-3 mRNA is regulated through stabilization rather than through translatability

Subcellular localization of BiFC signals.

<p>HEK293 cells were imaged by confocal microscopy. (A) Cotransfection of bFos-YC and bJun-YN generates a BiFC signal distributed throughout the cell nucleus. (B) Cotransfection of APP-YC, HA-Fe65 and Tip 60-YN results in multiple spherical nuclear BiFC signals from AFT complexes. (C) In some cells, cotransfection also results in formation of extranuclear BiFC signals (arrow), which is consistent with an interaction of Tip60 and AICD/Fe65 also outside the nucleus as previously reported by us [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076094#B8" target="_blank">8</a>]. (D) Cotransfection with APP-YC and APP-YN results in perinuclear fluorescence (E+F). Colocalization of the APP/APP-BiFC signal with calnexin and TGN46 is consistent with the localization of APP/APP-dimers in the ER/Golgi. Nuclei were counterstained with DAPI. Length of bar: 13 µm (A, C, E, F) or 10 µm (B, D).</p

AFT complex formation and the BiFC principle.

<p>(A) Spherical nuclear AFT complexes in HEK293 cells cotransfected with APP-Citrine, CFP-Tip60 and HA-Fe65 (arrow). Cells that lack CFP-Tip60 accumulate neither AICD-Citrine nor HA-Fe65 in the nucleus (arrowhead). Nuclei were counterstained with DAPI. Length of bar: 20 µm. (B) Schematic depiction of the BiFC-based AFT complex detection system, where APP and Tip60 are fused to YFP halves. Since Fe65 serves as an adaptor between APP and Tip60, fluorescence complementation only occurs in the presence of all three proteins. (C) Fluorescence maturation in AFT-BiFC. Fluorescence maturation at 30°C was allowed for increasing time periods before FC quantification (n=3 per timepoint, error bars represent SEM). If samples were maintained at 37°C, no maturation occurred (samples labeled 37°C).</p

AFT-BiFC requires the presence of Fe65.

<p>(A) After cotransfection of APP-YC, Tip 60-YN and HA-Fe65 the BiFC signal can be detected in a subset of cells in the confocal microscope. In contrast, cotransfection of APP-YC and Tip 60-YN alone does not lead to fluorescence complementation, which indicates absence of direct interaction between AICD and Tip60. Transfection of APP-YC and Tip 60-YN together with the AICD-binding protein MINT1/X11 that traps AICD in the cytosol also does not generate a BiFC signal. Lower panels show BiFC overlay with DAPI nuclear staining. Length of bar: 60 µm. (B) Representative BiFC-FC scatter plots of individual samples. Percentages refer to gated cells. Fluorescence intensity and forward scatter are depicted in arbitrary units. (C) BiFC-FC quantification of HEK293 cells cotransfected with APP-YC and Tip 60-YN together with or without HA-Fe65 or with MINT1/X11 (n=6 vs. 5 vs. 6, error bars represent SEM, *** p<0.005, U-test). The approximately 1% BiFC-positive cells in the APP-Tip60 and APP-X11-Tip60 conditions are most likely due to background autofluorescence of HEK cells as well as fluorescence complementation mediated by endogenous Fe65.</p

Visualization and quantification of APP intracellular domain-mediated nuclear signaling by bimolecular fluorescence complementation

BACKGROUND: The amyloid precursor protein (APP) intracellular domain (AICD) is released from full-length APP upon sequential cleavage by either α- or β-secretase followed by γ-secretase. Together with the adaptor protein Fe65 and the histone acetyltransferase Tip60, AICD forms nuclear multiprotein complexes (AFT complexes) that function in transcriptional regulation. OBJECTIVE: To develop a medium-throughput machine-based assay for visualization and quantification of AFT complex formation in cultured cells. METHODS: We used cotransfection of bimolecular fluorescence complementation (BiFC) fusion constructs of APP and Tip60 for analysis of subcellular localization by confocal microscopy and quantification by flow cytometry (FC). RESULTS: Our novel BiFC-constructs show a nuclear localization of AFT complexes that is identical to conventional fluorescence-tagged constructs. Production of the BiFC signal is dependent on the adaptor protein Fe65 resulting in fluorescence complementation only after Fe65-mediated nuclear translocation of AICD and interaction with Tip60. We applied the AFT-BiFC system to show that the Swedish APP familial Alzheimer's disease mutation increases AFT complex formation, consistent with the notion that AICD mediated nuclear signaling mainly occurs following APP processing through the amyloidogenic β-secretase pathway. Next, we studied the impact of posttranslational modifications of AICD on AFT complex formation. Mutation of tyrosine 682 in the YENPTY motif of AICD to phenylalanine prevents phosphorylation resulting in increased nuclear AFT-BiFC signals. This is consistent with the negative impact of tyrosine phosphorylation on Fe65 binding to AICD. Finally, we studied the effect of oxidative stress. Our data shows that oxidative stress, at a level that also causes cell death, leads to a reduction in AFT-BiFC signals. CONCLUSION: We established a new method for visualization and FC quantification of the interaction between AICD, Fe65 and Tip60 in the nucleus based on BiFC. It enables flow cytometric analysis of AICD nuclear signaling and is characterized by scalability and low background fluorescence