The amyloid precursor protein (APP), a central molecule in Alzheimer's disease (AD), has physiological roles in cell adhesion and signaling, migration, neurite outgrowth and synaptogenesis. Intracellular adapter proteins mediate the function of transmembrane proteins. Fe65 (also known as APBB1) is a major APP-binding protein. Regulated intramembrane proteolysis (RIP) by γ-secretase releases the APP intracellular domain (AICD), together with the interacting proteins, from the membrane. We studied the impact of the Fe65 family (Fe65, and its homologs Fe65L1 and Fe65L2, also known as APBB2 and APBB3, respectively) on the nuclear signaling function of the AICD. All Fe65 family members increased amyloidogenic processing of APP, generating higher levels of β-cleaved APP stubs and AICD. However, Fe65 was the only family member supporting AICD translocation to nuclear spots and its transcriptional activity. Using a recently established transcription assay, we dissected the transcriptional activity of Fe65 and provide strong evidence that Fe65 represents a transcription factor. We show that Fe65 relies on the lysine acetyltransferase Tip60 (also known as KAT5) for nuclear translocation. Furthermore, inhibition of APP cleavage reduces nuclear Tip60 levels, but this does not occur in Fe65-knockout cells. The rate of APP cleavage therefore regulates the nuclear translocation of AICD–Fe65–Tip60 (AFT) complexes, to promote transcription by Fe65.

Alterations in the complex metabolism of APP have been linked to Alzheimer's disease (AD). Proteolytic processing of APP generates several fragments, of which the Aβ peptide is currently the most pursued therapeutic target – in line with the amyloid cascade hypothesis (Hardy and Selkoe, 2002; Sevigny et al., 2016). In the opposing non-amyloidogenic cleavage pathway, Aβ is destroyed and a secreted APP fragment (sAPPα) is generated that has diverse positive functions on neurons (Müller et al., 2017). The choice between the two cleavage pathways determines the risk of developing AD, as shown by genetic mutations in APP (Bertram and Tanzi, 2008). APP has properties of a cell adhesion molecule (Soba et al., 2005), and its cleavage can be regulated by interaction with extracellular matrix components or neighboring cells. A major determinant of amyloidogenic cleavage is the subcellular localization of APP (Rajendran et al., 2008) and this is regulated by proteins binding to the APP intracellular domain (AICD).

Fe65 (also known as APBB1) is the most prominent AICD-binding protein, frequently found in yeast two hybrid screens using AICD as bait (Bressler et al., 1996; Cao and Sudhof, 2001). As with APP, Fe65 has two homologs, the Fe65-like proteins Fe65L1 and Fe65L2 (also known as APBB2 and APBB3, respectively). The importance of Fe65 in mediating APP function is revealed by the similar phenotypes of APP- and Fe65 family-knockout (KO) mice, which show cortical dysplasia caused by altered neuroblast migration (Guénette et al., 2006; Herms et al., 2004; McLoughlin and Miller, 2008) and deficits in formation of the neuromuscular synapse (Strecker et al., 2016; Wang et al., 2005). In C. elegans, knockdown of the orthologs of Fe65 or APP also reveals similar negative effects in the regulation of pharyngeal pumping (Zambrano et al., 2002). Fe65 function is thus strongly connected with APP/AICD signaling functions. Fe65 dominates AICD function even in the presence of other AICD-binding proteins such as MINT1 (also known as X11α and APBA1) or Jip1 (also known as MAPK8IP1), as seen in pull-down assays or localization of AICD to different nuclear compartments (Konietzko et al., 2010; Lau et al., 2000). The crystal structure of AICD bound to the phosphotyrosine-binding domain 2 (PTB2) of Fe65 revealed an extraordinary extended interaction interface, three times larger than the known peptide–PTB domain complexes (Radzimanowski et al., 2008), lending an explanation for the predominance of APP–Fe65 interaction among the plethora of interaction partners for AICD (Müller et al., 2008).

Fe65 contains two PTB domains and a WW domain that can simultaneously bind different proteins, and it can thus be viewed as a scaffolding protein (Chow et al., 2015a). Full-length APP can anchor Fe65 at the membrane (Minopoli et al., 2001), forming a scaffold, for instance with the ApoE receptor family (Herz and Beffert, 2000), and γ-secretase-mediated cleavage of APP releases the AICD–Fe65 complex, enabling it to translocate to the nucleus (Kimberly et al., 2001; Kinoshita et al., 2002). In the nucleus, AICD–Fe65 localizes together with the lysine acetyltransferase Tip60 (also known as KAT5) in spherical AFT spots that represent sites of transcription (Konietzko et al., 2010; von Rotz et al., 2004). Fusion of yeast Gal4 DNA-binding domains to APP or AICD has revealed that transactivation activity can be dramatically enhanced by co-expression of Fe65 (Cao and Sudhof, 2001). Further studies have shown varying results for AICD–Fe65 signaling and reports on transcriptional activity of the other Fe65 family members are sparse. We therefore studied the Fe65 family with respect to nuclear signaling and transcription, and performed detailed analysis of the role of different domains and motifs. We conclude that of the family, only Fe65 is a transcription factor whose activity in the nucleus is regulated by APP processing.

The predicted NLS of Fe65 is not functional for nuclear import

We aligned the three Fe65 family member amino acid sequences, revealing the described conserved WW, PTB1 and PTB2 domains (see Fig. 7A). Fe65 and Fe65L1 are extended N-terminal to the WW domain by around 250 and 290 amino acids, respectively. Only Fe65 contains a unique stretch of 15 negatively charged amino acids, with six more in close vicinity, that contains only one positively charged lysine. We have added this motif (denoted A) in the Fe65 scheme. For a more accurate discussion we have termed the N- and C-terminal parts bracketing these motifs and domains Nt and Ct, and the linkers in between from L1 to L3 (Fig. 1A, scaled to amino acid numbering of Fe65). We made various Fe65 deletion or mutation constructs as shown in Fig. 1A. The Fe65 mutants include progressive deletions of the N-terminus or C-terminus, deletion of single domains or selected point mutations. All constructs were tagged at the N-terminus with a streptavidin-binding peptide (SBP) followed by a Myc tag.

Fig. 1.

The predicted NLS of Fe65 is not functional in nuclear import. (A) Overview of the different Fe65 constructs used in this study. Fe65 is drawn scaled to amino acid numbering based on reported domains and alignments. The acidic region (A), the WW (W), the PTB1 and PTB2 domains are highlighted in grey. The N-terminal (Nt) and C-terminal (Ct) regions as well as the linker (L1, L2, 3) regions are shown in white. Internal deletions are drawn as a line, point mutations as a cross. (B) Mutation of the predicted NLS in Fe65 does not prevent nuclear AFT spot formation. The Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells, together with APP-Cit and CFP-Tip60, and imaged by confocal microscopy. Scale bar: 10 μm. Enlarged nucleus is magnified by a factor of 2. (C) SBP-myc-tagged Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells together with either myc-Tip60 or APP-3HA, followed by streptavidin-based purification. Blots show cell lysates (L) and eluates (E) after purification. Fe65 was detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies, and full-length APP (flAPP) and APP CTF with anti-HA antibodies. (D) The nuclear export import inhibitor leptomycin B (LMB) results in nuclear accumulation of Fe65 with mutations in the predicted NLS. Scale bar: 10 μm. (E) LMB analysis of all Fe65 mutants revealed that only simultaneous deletion of the WW domain and L2 prevents nuclear import of Fe65. Outline of DAPI-stained nuclei is overlaid in white. Scale bar: 5 μm.

Fig. 1.

The predicted NLS of Fe65 is not functional in nuclear import. (A) Overview of the different Fe65 constructs used in this study. Fe65 is drawn scaled to amino acid numbering based on reported domains and alignments. The acidic region (A), the WW (W), the PTB1 and PTB2 domains are highlighted in grey. The N-terminal (Nt) and C-terminal (Ct) regions as well as the linker (L1, L2, 3) regions are shown in white. Internal deletions are drawn as a line, point mutations as a cross. (B) Mutation of the predicted NLS in Fe65 does not prevent nuclear AFT spot formation. The Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells, together with APP-Cit and CFP-Tip60, and imaged by confocal microscopy. Scale bar: 10 μm. Enlarged nucleus is magnified by a factor of 2. (C) SBP-myc-tagged Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells together with either myc-Tip60 or APP-3HA, followed by streptavidin-based purification. Blots show cell lysates (L) and eluates (E) after purification. Fe65 was detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies, and full-length APP (flAPP) and APP CTF with anti-HA antibodies. (D) The nuclear export import inhibitor leptomycin B (LMB) results in nuclear accumulation of Fe65 with mutations in the predicted NLS. Scale bar: 10 μm. (E) LMB analysis of all Fe65 mutants revealed that only simultaneous deletion of the WW domain and L2 prevents nuclear import of Fe65. Outline of DAPI-stained nuclei is overlaid in white. Scale bar: 5 μm.

We wanted to verify the predicted C-terminal nuclear localization signal (NLS) in Fe65 and mutated two lysine residues to alanine. We co-transfected fluorescently tagged APP-Citrine (Cit) and CFP-Tip60 with wild-type (wt) Fe65 or the Fe65 K701/703A mutant into HEK293 cells to observe the formation of nuclear AFT spots using confocal microscopy as described previously (Konietzko et al., 2010; von Rotz et al., 2004). Although the mutation of the putative NLS should prevent nuclear translocation, we clearly detected nuclear AFT spots (Fig. 1B). We analyzed whether the mutant Fe65 was still able to interact with APP and Tip60. HEK293 lysates co-expressing SBP-tagged Fe65 K701/703A mutant or wt, together with either Tip60 or APP, were subjected to streptavidin-based purification. The Fe65 K701/703A mutant interacted with APP and Tip60 to similar degree to wt Fe65, in line with its capacity to form AFT spots (Fig. 1C). Fe65 undergoes nuclear export that can be inhibited by leptomycin B (LMB), leading to nuclear accumulation due to ongoing nuclear import of Fe65 (von Rotz et al., 2004). LMB treatment resulted in clear nuclear accumulation of the Fe65 K701/703A mutant, further revealing that this putative NLS is not necessary for nuclear import (Fig. 1D). We subjected all Fe65 constructs to LMB analysis (Fig. 1E). The N-terminal deletion series revealed lack of nuclear import starting with ΔN357, a construct that still contains the predicted NLS, again showing its non-functionality. As the ΔN253 construct accumulated in the nucleus after LMB treatment, the NLS would be expected to reside in the WW-L2 region of Fe65. Sole deletion of all motifs, including the WW domain, or the L2 and Ct regions did not interfere with nuclear import of Fe65. Thus, we could not identify a single motif directing nuclear import of Fe65 in the WW domain or L2 – deletion of both is necessary to prevent nuclear import.

The Fe65 and Tip60 interaction promotes the nuclear translocation of both proteins

The lack of a unique NLS in Fe65 prompted us to access the possibility that Fe65 uses the NLSs of Tip60 for nuclear translocation. We transfected HEK293 cells with either APP, Fe65 or Tip60 alone, or in different combinations (Fig. 2A). Fe65 was distributed throughout the cytosol and in the nucleus and also Tip60 could be detected in both compartments. Co-expression of Fe65 and Tip60 resulted in enhanced nuclear localization of both proteins and additional APP co-expression retained more Tip60 and Fe65 outside of the nucleus. We quantified the nuclear:cytoplasmic (nuc/cyt) ratio as described in the Materials and Methods (Fig. 2B). The nuc/cyt ratio of both Fe65 and Tip60 was increased by ∼2-fold when the two were co-expressed as compared to single expression, suggesting that Fe65 and Tip60 shuttle to the nucleus together in a complex. APP co-expression reduced the nuclear signals of Tip60 and Fe65. At the same time, compared to expressing APP alone, the nuc/cyt ratio of APP increased when Fe65 and Tip60 were co-expressed.

Fig. 2.

Fe65 and Tip60 interact to promote the nuclear translocation of each other. (A) Confocal microscopy of HEK293 cells transfected with HA-Fe65, myc-Tip60 or APP-Cerulean alone, or in different combinations. Every row represents one experiment. Co-expression of Fe65 and Tip60 leads to less extranuclear staining. Scale bar: 100 μm. (B) Quantification of APP, Fe65 and Tip60 in nuclei demarcated by DAPI and calculation of cytosolic levels from total measurements. Nuclear/cytoplasmic (nuc/cyt) ratios are relative measures due to the different accessibility of nuclear and cytoplasmic antigens for antibodies (mean±s.d., n=14). *P<0.05, **P<0.01 (Kruskal–Wallis with Dunn's test). (C) Identification of Fe65 domains necessary for interaction with Tip60. SBP-myc-Fe65 deletion constructs were co-expressed with CFP-Tip60. After streptavidin–dynabead isolation, proteins were eluted with biotin, and lysate and eluates were analyzed by western blotting with antibodies against the Myc tag and GFP. (D) Quantification of Tip60 in eluates, normalized to Fe65 eluate levels (mean±s.e.m., n=3). *P<0.05 (Mann–Whitney U-test). Tip60–Fe65 interaction is disrupted by deletion of the Fe65 N-terminal region (Nt-Ac-L1) or deletion of PTB1. (E) HEK293 wt and Fe65-KO cells were transfected with myc-Tip60 and treated with DAPT or control solution, followed by fractionation into cytosol/membrane (C/M) and nuclear (N) fractions. The Tip60 signal in the C/M fraction is occluded by an unspecific band (star). Fe65 is only detected in the C/M fraction of wt cells. (F) Quantification of Tip60, normalized to histone H3, in the N fraction of three independent KO cell lines versus n=3 wt cell lines (mean±s.d.). **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction).

Fig. 2.

Fe65 and Tip60 interact to promote the nuclear translocation of each other. (A) Confocal microscopy of HEK293 cells transfected with HA-Fe65, myc-Tip60 or APP-Cerulean alone, or in different combinations. Every row represents one experiment. Co-expression of Fe65 and Tip60 leads to less extranuclear staining. Scale bar: 100 μm. (B) Quantification of APP, Fe65 and Tip60 in nuclei demarcated by DAPI and calculation of cytosolic levels from total measurements. Nuclear/cytoplasmic (nuc/cyt) ratios are relative measures due to the different accessibility of nuclear and cytoplasmic antigens for antibodies (mean±s.d., n=14). *P<0.05, **P<0.01 (Kruskal–Wallis with Dunn's test). (C) Identification of Fe65 domains necessary for interaction with Tip60. SBP-myc-Fe65 deletion constructs were co-expressed with CFP-Tip60. After streptavidin–dynabead isolation, proteins were eluted with biotin, and lysate and eluates were analyzed by western blotting with antibodies against the Myc tag and GFP. (D) Quantification of Tip60 in eluates, normalized to Fe65 eluate levels (mean±s.e.m., n=3). *P<0.05 (Mann–Whitney U-test). Tip60–Fe65 interaction is disrupted by deletion of the Fe65 N-terminal region (Nt-Ac-L1) or deletion of PTB1. (E) HEK293 wt and Fe65-KO cells were transfected with myc-Tip60 and treated with DAPT or control solution, followed by fractionation into cytosol/membrane (C/M) and nuclear (N) fractions. The Tip60 signal in the C/M fraction is occluded by an unspecific band (star). Fe65 is only detected in the C/M fraction of wt cells. (F) Quantification of Tip60, normalized to histone H3, in the N fraction of three independent KO cell lines versus n=3 wt cell lines (mean±s.d.). **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction).

To analyze the molecular interaction between Fe65 and Tip60 in detail, we co-transfected Tip60 with SBP-tagged Fe65 deletion constructs and performed streptavidin purification. Western blot analysis revealed similar Tip60 expression levels in co-transfected cells and comparable expression of the Fe65 constructs in cell lysates (Fig. 2C). Quantification of pulldowns were performed, normalizing Tip60 levels in the eluate to Fe65 levels in the eluate (Fig. 2D). We observed that Tip60 pulldown was strongly reduced by deletion of the Fe65 PTB1 domain as described previously (Cao and Sudhof, 2001). In addition, deletion of the N-terminal region of Fe65 as in the ΔN253 and ΔN357 constructs, also strongly reduced Tip60 pulldown. Thus, there seems to be a widespread interaction interface between Fe65 and Tip60, whereby both the Fe65 N-terminal region and the PTB1 domain are necessary for enabling Tip60 pulldown.

We used CRISPR-Cas9 editing with three different sgRNAs to generate three independent HEK293 Fe65-KO cell lines. We did not identify an antibody that detects endogenous Tip60 and therefore transfected Tip60 into these cells. We performed inhibition of APP cleavage by γ-secretase and subcellular fractionation into cytosol/membrane and nuclear fractions. Inhibition of APP cleavage strongly attenuated nuclear Tip60 levels in wt HEK293 cells (Fig. 2E). In contrast, the Fe65-KO cell lines had lower baseline nuclear Tip60 levels that were not affected by γ-secretase inhibition. Quantification of nuclear Tip60 levels with normalization to histone 3 from all three Fe65 KO lines is shown in Fig. 2F. Tip60 levels in the cytosol/membrane fraction could not be determined, due to an unspecific band detected by the Tip60 polyclonal antibody above the Tip60 signal. Overall, these results show that processing of endogenous APP in HEK293 cells controls the subcellular localization of expressed Tip60 via the endogenously expressed Fe65. These data identify Tip60 as a component that is bound to APP–Fe65 at the membrane and that co-translocates with AICD and Fe65 to the nucleus.

Fe65 domains essential for AFT spot formation

AICD–Fe65 complexes translocate to the nucleus where they, together with the lysine acetyl transferase Tip60, form AFT complexes localized in spherical spots that represent transcription factories (Konietzko et al., 2010). Without Fe65, Tip60 localizes to speckles, representing a different nuclear compartment that does not co-localize with AFT spots. Spots and speckles can be unequivocally identified with confocal microscopy, and the occurrence of Tip60 in spots is a clear indication of the presence of Fe65 (Fig. S1).

The different Fe65 deletion constructs were co-transfected together with APP-Cit and CFP-Tip60 into HEK293 cells to identify essential domains of Fe65 for AFT spot formation (Fig. 3). Owing to the crowded nuclear environment, the antibody-mediated detection of Fe65 is not as clear, as fused fluorescent proteins and spots cannot always be resolved. Nevertheless, the relocation of Tip60 to spots clearly reveals the presence of Fe65. N-terminal Fe65 deletions until the beginning of the WW domain support AFT spot formation. The ΔN357 and ΔN531 Fe65 constructs do not translocate with AICD to the nucleus and Tip60 resides in speckles, correlating with their lack of nuclear translocation. Deletion of the WW domain or L2, together necessary for nuclear translocation, had differing effects. Fe65ΔWW localized together with Tip60 in nuclear speckles devoid of AICD. Fe65ΔL2 bound to Tip60 and induced relocation to spots that also did not harbor AICD. Deletion of the Fe65 PTB1 domain, necessary for Tip60 binding in pulldowns, still enabled association of Fe65 with Tip60 in speckles, but again no relocation to spots or nuclear co-localization of AICD could be detected. Removal of the PTB2 domain still resulted in Fe65–Tip60 localization to nuclear spots that were devoid of AICD. Finally, deletion of the C-terminal residues after the PTB2 domain in Fe65ΔCt showed AFT spot formation indistinguishable from that of wt Fe65. These data show that both the WW and PTB1 domains are necessary for spot formation.

Fig. 3.

Function of different Fe65 domains in AFT spot formation. Confocal microscopy of HEK293 cells transfected with full-length SBP-myc-Fe65 or deletion mutants, together with APP-Cit and CFP-Tip60. A representative image of each clone from a minimum of three different transfections is shown. Arrows denote a cell with nuclear AFT spots. Arrowheads denote a cell where Tip60 localizes to nuclear speckles that are devoid of Fe65 and AICD. A magnified view (factor of 2) of one cell is also shown. Nuclear signals from the fluorescent proteins are clearly resolved, whereas antibody staining of nuclear structures is restricted. Scale bar: 10 µm.

Fig. 3.

Function of different Fe65 domains in AFT spot formation. Confocal microscopy of HEK293 cells transfected with full-length SBP-myc-Fe65 or deletion mutants, together with APP-Cit and CFP-Tip60. A representative image of each clone from a minimum of three different transfections is shown. Arrows denote a cell with nuclear AFT spots. Arrowheads denote a cell where Tip60 localizes to nuclear speckles that are devoid of Fe65 and AICD. A magnified view (factor of 2) of one cell is also shown. Nuclear signals from the fluorescent proteins are clearly resolved, whereas antibody staining of nuclear structures is restricted. Scale bar: 10 µm.

Transcriptional activity of Fe65 constructs

We recently developed yeast Gal4-based cellular assays for nuclear signaling and transcriptional activity (Konietzko et al., 2019). HEK293 cells were infected with lentiviral vectors to integrate 9×UAS-Citrine reporters into the genome (denoted HEK:UAS-Cit cells). Expressing the fluorescent protein Citrine as a reporter circumvents enzymatic detection in cell lysates and enables quantification by confocal microscopy in situ. We tagged Fe65 with the Gal4 DNA-binding domain (Gal4-DBD) by replacing the SBP tag in all Fe65 constructs and used sole transfection of these constructs to determine their transcriptional activity via Citrine expression (Fig. 4A). Expression of wt Gal4-DBD-Fe65 in HEK:UAS-Cit cells induced the expression of Citrine, in contrast to lipofection alone, which only showed baseline leakage expression (Fig. 4B). Fiji software was used to measure total Citrine fluorescence and to outline and count DAPI-stained nuclei (zoom in Fig. 4B). We transfected the Fe65 constructs into different passages of HEK:UAS-Cit cells to analyze levels of Citrine expression normalized to the number of nuclei (Fig. 4C). Deletion of the Fe65 Nt (ΔN144) resulted in a significant increase in transcriptional activity that was returned to wild-type levels by additional deletion of the acidic motif and L1 (ΔN253). Further deletion of the WW domain and L2 (ΔN357) reduced Citrine expression to baseline levels and the same was seen for Fe65 ΔN531, which consists solely of PTB2 and Ct. Internal Fe65 deletions of the WW or PTB1 domains, or the L2 connecting them, caused a strong reduction in transcriptional activity of these constructs. By contrast, deletion of the PTB2 domain or Ct more than doubled the expression of Citrine. The C654F mutation in the PTB2 domain did not show a difference in transcriptional activity compared to wt Fe65. Mutation of the predicted NLS (K701/703A) significantly increased Citrine expression over wt Fe65. Transfecting a full-length Fe65 with an SBP tag replacing the Gal4-DBD showed no transcriptional activity as this construct does not bind the UAS elements preceding the Citrine reporter.

Fig. 4.

Transcriptional activity of Gal4-Fe65 constructs. (A) Schematic of the transactivation assay. HEK293 cells and N2a cells were infected with viral vectors to ensure nuclear localization of the reporter cassette that contains nine repeats of the Gal4 upstream activator sequence (UAS) driving Citrine (Cit) expression. The Gal4-DBD is fused N-terminally to the different Fe65 constructs. (B) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK:UAS-Cit cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (C) HEK:UAS-Cit cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. Total Cit intensity was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) N2a:UAS-Cit-NLS cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction).

Fig. 4.

Transcriptional activity of Gal4-Fe65 constructs. (A) Schematic of the transactivation assay. HEK293 cells and N2a cells were infected with viral vectors to ensure nuclear localization of the reporter cassette that contains nine repeats of the Gal4 upstream activator sequence (UAS) driving Citrine (Cit) expression. The Gal4-DBD is fused N-terminally to the different Fe65 constructs. (B) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK:UAS-Cit cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (C) HEK:UAS-Cit cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. Total Cit intensity was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) N2a:UAS-Cit-NLS cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction).

Lentiviral reporter vectors were improved by fusing an NLS to Citrine, which concentrates it in the nucleus (Konietzko et al., 2019). This method is more accurate as it measures Citrine fluorescence only in the DAPI-stained nuclei that are identified by Fiji software and counted for normalization. We infected mouse N2a neuroblastoma cells to derive a reporter cell line. As with the HEK:UAS-Cit cell line, the N2a:UAS-Cit-NLS cell line showed Citrine expression – now confined to the nucleus – after transfection of Gal4-Fe65 (Fig. S2). We again analyzed all Gal4-Fe65 constructs. Transcriptional activity of the different deletions closely replicated the results seen in HEK293 cells, with the exception of the ΔN253 construct that had reduced activity compared to wt Fe65 in N2a cells (Fig. 4D).

We reevaluated some of the Fe65 constructs, including ΔN253, in HEK2:UAS-Cit-NLS cells (see Fig. 6M) to give a better signal-to-noise ratio than was seen in HEK:UAS-Cit cells (Konietzko et al., 2019). Removal of the N-terminal 144 residues of Fe65 again increased transcriptional activity, whereas further deletion of the acidic motif with the adjacent L1 (ΔN253) reduced activity below wild-type levels (Fig. 5A). We also reanalyzed the effect of mutating K701/703 in Fe65, which again led to a significant increase in transcriptional activity.

Fig. 5.

UAS-Cit assay refinements and dissociation from AFT spot formation. HEK2:UAS-Cit-NLS cells were used for all transcription assays. (A) Lipofection of Fe65 deletion constructs (schemes in Fig. 1A) was followed by confocal microscopy analysis. Mean±s.d. of n=21 images from three replicates are shown. ***P<0.001 (one-way ANOVA with Bonferroni correction). (B) SBP-myc-Fe65 (SBP-Fe65) was co-transfected with mCherry-Fe65 (mChe-Fe65) wt, C654F or ΔPTB2 into HEK293 cells, followed by streptavidin-based purification. Myc and mCherry stainings were used to dissociate the different Fe65 species in lysates (L) and eluates (E). Bands marked with a star are leftover signals from the mChe-Fe65 staining. (C) UAS-Cit assay for comparison of Fe65 C-terminal deletions. Bars depict mean±s.d. of n=21 confocal microscopy images from three replicates. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (Kruskal–Wallis with Dunn's test). (D) UAS-Cit assay to compare combined N- and C-terminal deletions with sole deletion of the C-terminus. Mean±s.d. of n=21 images from three replicates are shown. ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (E) Sigmoidal dose-response in two HEK:UAS-Cit-NLS cell lines infected once or twice with reporter viruses, and transfected with increasing amounts of Gal4-Fe65 plasmids (n=6 images per concentration, mean±s.d.). (F) Transcriptional activity of Fe65 deletion constructs was analyzed with lipofection of 150 ng per construct, in contrast to the 1 µg used in all other assays. Mean±s.d. of n=21 images from three replicates are shown. *P<0.05; ***P<0.001 (one-way ANOVA with Bonferroni correction). (G) HEK2:UAS-Cit-NLS cells were transfected with Gal4-Fe65, alone or together with Cer-NLS or Gal4-myc-Cer-NLS constructs and analyzed by confocal microscopy for Cit expression. Bars depict mean±s.d. of n=7 images. ***P<0.001 (one-way ANOVA with Bonferroni correction). (H) Cit-AICD, HA-Fe65, V5-Tip60 and the Gal4-myc-Cer-NLS construct were co-transfected into HEK293 cells. Confocal microscopy revealed the formation of AFT spots detected by the Cit signal. Gal4-myc-Cer-NLS, detected by Cer fluorescence and myc staining, did not colocalize with AFT spots. Scale bar: 10 µm.

Fig. 5.

UAS-Cit assay refinements and dissociation from AFT spot formation. HEK2:UAS-Cit-NLS cells were used for all transcription assays. (A) Lipofection of Fe65 deletion constructs (schemes in Fig. 1A) was followed by confocal microscopy analysis. Mean±s.d. of n=21 images from three replicates are shown. ***P<0.001 (one-way ANOVA with Bonferroni correction). (B) SBP-myc-Fe65 (SBP-Fe65) was co-transfected with mCherry-Fe65 (mChe-Fe65) wt, C654F or ΔPTB2 into HEK293 cells, followed by streptavidin-based purification. Myc and mCherry stainings were used to dissociate the different Fe65 species in lysates (L) and eluates (E). Bands marked with a star are leftover signals from the mChe-Fe65 staining. (C) UAS-Cit assay for comparison of Fe65 C-terminal deletions. Bars depict mean±s.d. of n=21 confocal microscopy images from three replicates. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (Kruskal–Wallis with Dunn's test). (D) UAS-Cit assay to compare combined N- and C-terminal deletions with sole deletion of the C-terminus. Mean±s.d. of n=21 images from three replicates are shown. ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (E) Sigmoidal dose-response in two HEK:UAS-Cit-NLS cell lines infected once or twice with reporter viruses, and transfected with increasing amounts of Gal4-Fe65 plasmids (n=6 images per concentration, mean±s.d.). (F) Transcriptional activity of Fe65 deletion constructs was analyzed with lipofection of 150 ng per construct, in contrast to the 1 µg used in all other assays. Mean±s.d. of n=21 images from three replicates are shown. *P<0.05; ***P<0.001 (one-way ANOVA with Bonferroni correction). (G) HEK2:UAS-Cit-NLS cells were transfected with Gal4-Fe65, alone or together with Cer-NLS or Gal4-myc-Cer-NLS constructs and analyzed by confocal microscopy for Cit expression. Bars depict mean±s.d. of n=7 images. ***P<0.001 (one-way ANOVA with Bonferroni correction). (H) Cit-AICD, HA-Fe65, V5-Tip60 and the Gal4-myc-Cer-NLS construct were co-transfected into HEK293 cells. Confocal microscopy revealed the formation of AFT spots detected by the Cit signal. Gal4-myc-Cer-NLS, detected by Cer fluorescence and myc staining, did not colocalize with AFT spots. Scale bar: 10 µm.

Fig. 6.

Fe65L1 and Fe65L2 do not promote transcription. (A–J) HEK293 cells transfected with Fe65 family members, Tip60 and APP, were analyzed by confocal microscopy. Fe65L1 and Fe65L2 were transfected alone (A,G), together with APP (B,H), together with Tip60 (C,I) or with APP and Tip60 (D,J). (E,F,K) LMB treatment leads to nuclear accumulation of Fe65 and Fe65L2 but not Fe65L1. Scale bar: 10 µm. (L) Transcription assay with HEK:UAS-Cit cells imaged via confocal microscopy reveals lack of transcriptional activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (M) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK2:UAS-Cit-NLS cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (N) Transcription assay with HEK2:UAS-Cit-NLS cells confirms lack of activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction).

Fig. 6.

Fe65L1 and Fe65L2 do not promote transcription. (A–J) HEK293 cells transfected with Fe65 family members, Tip60 and APP, were analyzed by confocal microscopy. Fe65L1 and Fe65L2 were transfected alone (A,G), together with APP (B,H), together with Tip60 (C,I) or with APP and Tip60 (D,J). (E,F,K) LMB treatment leads to nuclear accumulation of Fe65 and Fe65L2 but not Fe65L1. Scale bar: 10 µm. (L) Transcription assay with HEK:UAS-Cit cells imaged via confocal microscopy reveals lack of transcriptional activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (M) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK2:UAS-Cit-NLS cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (N) Transcription assay with HEK2:UAS-Cit-NLS cells confirms lack of activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction).

To complement the N-terminal deletion constructs of Fe65, we constructed a set of C-terminal deletions (constructs depicted in Fig. 1A). Fe65ΔC254, which lacks all C-terminal sequences including the WW domain but retains the acidic motif, still possessed transcriptional activity, albeit far below wild-type levels (Fig. 5A). Additional inclusion of the WW domain (ΔC295) increased transcriptional activity over wild-type levels, yet Tip60 resided in speckles, as for the Fe65ΔC254 construct (Fig. S3). The ΔC516 construct, which only lacks the L3-PTB2-Ct part of Fe65, showed the strongest increase in reporter transcription. The ΔC516 construct includes all regions found to interact with Tip60 and leads to a dispersal of Tip60 speckles but failed to form spherical spots.

Deletion of the PTB2 increased transcriptional activity, whereas the C654F mutation did not affect it (Fig. 4). Both constructs are reported to lack APP binding (Borg et al., 1996; Cao and Sudhof, 2001; Minopoli et al., 2001), and thus sequestration of Fe65 by APP cannot explain this discrepancy. Besides APP binding, the PTB2 domain also mediates Fe65 dimer formation (Feilen et al., 2017). We hypothesized that Fe65 dimer formation could lead to Fe65 sequestration in the cytosol, akin to its sequestration at the membrane by APP. We performed pulldown experiments with differently tagged Fe65 constructs and again saw the disruption of Fe65 dimers by the PTB2 deletion, but the C654F mutation did not interfere with dimer formation (Fig. 5B).

We directly compared the ΔC516 construct, which lacks both the PTB2 domain and Ct, with the single deletions in HEK2:UAS-Cit-NLS cells. Whereas both single deletions showed an increase over wild-type Fe65 activity as seen with the other reporter cells, the ΔC516 construct revealed even higher activity, demonstrating independent inhibitory functions for the PTB2 domain and Ct (Fig. 5C). We next deleted the inhibitory Nt region in addition to PTB2-Ct, but this construct showed no further increase in activity over the ΔC516 construct alone (Fig. 5D). These experiments reveal that the central part of Fe65, from the acidic motif to the PTB1 domain, contributes to the transcriptional activity and the bracketing Nt and PTB2-Ct have an inhibitory function.

Dissociation of AFT spot formation and Gal4/UAS-Cit assay

We analyzed a DNA concentration series of Gal4-Fe65 revealing a sigmoidal response of Cit expression in two cell lines (Fig. 5E), as we reported for APP-Gal4 plus Fe65 (Konietzko et al., 2019). The 1 µg DNA used in all experiments in this study is clearly in the saturated range of the assay. We reanalyzed selected Fe65 constructs with increased or decreased transcriptional activity using only 150 ng DNA to target the linear range of the assay. The results mirrored those with 1 µg DNA with the same magnitude of change of transcriptional activity (Fig. 5F).

We designed a control construct with the Gal4-DBD fused to the fluorescent protein Cerulean (Cer; Gal4-myc-Cer-NLS) and confirmed that this construct showed no activity in the UAS-Cit assay, in contrast to Gal4-Fe65 (Fig. 5G). Gal4-Fe65 transcriptional activity is reduced upon co-expression of a control plasmid (Cer-NLS). We set this to 100% to judge the effect of the Gal4-DBD-containing construct. Co-expression of Gal4-myc-Cer-NLS, as opposed to Cer-NLS, strongly reduced Gal4-Fe65-induced Cit expression due to the competition for UAS elements integrated in the genome. We co-transfected Cit-AICD, HA-Fe65 and V5-Tip60, together with the Gal4-myc-Cer-NLS construct. In contrast to the competition seen in the UAS-Cit assay, imaging the Gal4-myc-Cer-NLS construct via Myc staining or Cer fluorescence showed no colocalization with AFT spots (Fig. 5H). These experiments show that the UAS-Cit assay merely measures transcriptional activity of Gal4-fused proteins, whereas AFT spot formation indicates the property to localize to transcription factories at endogenous promoters.

Fe65L1 and Fe65L2 do not promote transcription

We inserted the Fe65L1 and Fe65L2 sequences in the different pUKBK vectors to label them with Gal4-myc- and SBP-myc-tags. We transfected the constructs alone, or in different combinations with APP and/or Tip60 into HEK293 cells and analyzed their localization by confocal microscopy. In contrast to Fe65, Fe65L1 is excluded from the nucleus, as seen in single confocal sections (Fig. 6A). Co-expression of APP leads to relocation of diffuse Fe65L1 cytoplasmic staining to cellular sites where APP is present, in line with the reported APP–Fe65L1 interaction (Fig. 6B). No interaction could be seen with Tip60, which located to nuclear speckles, while Fe65L1 retained its diffuse cytoplasmic distribution (Fig. 6C). Co-expression of all three proteins again showed APP–Fe65L1 colocalization throughout the cell, apart from in the nucleus, where Tip60 is localized to speckles (Fig. 6D). To further show the lack of nuclear Fe65L1 translocation, we inhibited nuclear export with LMB. In contrast to Fe65, where LMB treatment leads to accumulation in the nucleus (Fig. 6E), Fe65L1 could not be detected in the nucleus (Fig. 6F).

Analysis of the third family member Fe65L2, revealed a major localization to nuclear speckles when expressed alone (Fig. 6G). Co-expression of APP completely relocated nuclear Fe65L2 to the extranuclear locations of APP, confirming the known interaction (Fig. 6H). Together with Tip60, nearly all Fe65L2 was seen to localize to the same nuclear speckle structures (Fig. 6I). With all three proteins expressed, Fe65L2 localized with APP throughout the cell and with Tip60 in nuclear speckles that revealed only a faint AICD signal (Fig. 6J). LMB treatment resulted in nuclear accumulation of Fe65L2 (Fig. 6K). Thus, all Fe65 family members bind APP but differ strongly in their nuclear localization.

Gal4-DBD-fused Fe65L1 and Fe65L2 constructs were transfected into the HEK:UAS-Cit reporter line. Confocal microscopy analysis revealed that only Fe65 shows transcriptional activity. Expression of Fe65L1 or Fe65L2 did not raise Citrine expression above baseline (Fig. 6L). We further analyzed transcriptional activity in HEK2:UAS-Cit-NLS cells (Konietzko et al., 2019). Expression of Gal4-Fe65 induced strong Citrine-NLS expression localized to the nucleus with very low leakage expression seen under control conditions (Fig. 6M). Comparison of Fe65 family members in HEK2:UAS-Cit-NLS cells again showed transcriptional activity for Fe65, not for Fe65L1 and a minor response for Fe65L2 (Fig. 6N). In conclusion, Fe65 is the sole family member with transcriptional activity.

Dissection of transcriptional function with fusion proteins

The major difference in the protein sequence of the family members is in the 250 N-terminal residues of Fe65 and Fe65L1. Fe65L2 lacks this region (Fig. 7A). To elucidate the function of the Fe65 N-terminus, we fused the 250 residues to Fe65L2 or exchanged the 300 N-terminal residues of Fe65L1 with the Fe65 sequence (Fig. 7A). Constructs with N-terminal fusion of SBP were used for confocal microscopy and N-terminal fusions of the Gal4-DBD were used to analyze transcriptional activity in HEK2:UAS-Cit-NLS cells. Co-transfection of these constructs with APP and Tip60 showed that neither was able to form AFT spots, with the Fe65L1 fusion still excluded from the nucleus and the Fe65L2 fusion residing in nuclear speckles (Fig. 7B). We analyzed the fusion proteins in the UAS-Cit assay and saw that neither acquired transcriptional activity upon the fusion of the Fe65 N-terminal residues (Fig. 7C). We have shown that the 250 N-terminal residues in Fe65 are involved in Tip60 binding (Fig. 2D). We performed pulldown experiments with the Fe65 family and the two fusion proteins to detect the interaction with Tip60. For Fe65L2 we used a CFP-Tip60 construct as the Myc-Tip60 constructs runs at the same height in SDS-PAGE (Fig. 7D). Neither Fe65L1 or Fe65L2 show co-purification of Tip60, whereas both fusion constructs were able to pulldown Tip60. These data confirm the role of the Fe65 N-terminus in binding Tip60 in addition to the PTB1 domain.

Fig. 7.

Fusion of the Fe65 N-terminus to Fe65L1 or Fe65L2 does not enable transcription. (A) Scheme of Fe65 family members and fusion proteins showing the conserved domains. Fe65L1 and Fe65L2 are adapted to Fe65 that is drawn scaled to amino acid numbering. Roughly, Fe65L1 has a 50 amino acids longer Nt and Fe65L2 starts only with the WW domain, has a L2 of half the size and a 23 residue extended Ct, all compared to Fe65. (B) Confocal microscopy of HEK293 cells transfected with the fusion constructs, together with APP-Cit and CFP-Tip60. Scale bar: 10 µm. (C) HEK2:UAS-Cit-NLS cells were transfected with the depicted constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalized by the number of nuclei and the value of full-length Fe65 set to 100%. Bars depict mean±s.d. (n=7). ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (D) HEK293 cells were co-transfected with either myc-Tip60 or CFP-Tip60 and the SBP-myc-tagged Fe65 family and fusion constructs, followed by streptavidin-based pulldown. Blots show cell lysates (L) and eluates (E) after purification. Fe65 constructs were detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies. GAPDH is only detected in lysates and not purified with the Fe65 family.

Fig. 7.

Fusion of the Fe65 N-terminus to Fe65L1 or Fe65L2 does not enable transcription. (A) Scheme of Fe65 family members and fusion proteins showing the conserved domains. Fe65L1 and Fe65L2 are adapted to Fe65 that is drawn scaled to amino acid numbering. Roughly, Fe65L1 has a 50 amino acids longer Nt and Fe65L2 starts only with the WW domain, has a L2 of half the size and a 23 residue extended Ct, all compared to Fe65. (B) Confocal microscopy of HEK293 cells transfected with the fusion constructs, together with APP-Cit and CFP-Tip60. Scale bar: 10 µm. (C) HEK2:UAS-Cit-NLS cells were transfected with the depicted constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalized by the number of nuclei and the value of full-length Fe65 set to 100%. Bars depict mean±s.d. (n=7). ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (D) HEK293 cells were co-transfected with either myc-Tip60 or CFP-Tip60 and the SBP-myc-tagged Fe65 family and fusion constructs, followed by streptavidin-based pulldown. Blots show cell lysates (L) and eluates (E) after purification. Fe65 constructs were detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies. GAPDH is only detected in lysates and not purified with the Fe65 family.

We constructed fusion proteins of Fe65 and Fe65L1 to further delineate crucial motifs and domains (Fig. S4A). We replaced the Nt region of Fe65 preceding the acidic motif with the corresponding – 48 amino acids longer – sequence from Fe65L1 and saw no effect on transcriptional activity, but AFT spot formation was abolished (Fig. S4B,C). Additional exchange of the acidic motif and L1 in Fe65 with the Fe65L1 sequence resulted in a significant reduction of Citrine reporter transcription and also no spot formation. We expressed Fe65L1 lacking the N-terminal 290 residues. In contrast to full-length Fe65L1, this construct showed transcriptional activity, albeit at quite low levels compared to Fe65, and no spot formation occurred. Replacement of the L3-PTB2-Ct domains in the truncated Fe65L1 with the Fe65 sequence further increased transcriptional activity and enabled the formation of AFT spots. The mirror construct, with the WW-L2-PTB1 sequence from Fe65 fused to the L3-PTB2-Ct of Fe65L1 revealed even stronger transcriptional activity but no AFT spot formation. Finally, replacing the acidic motif plus L1 in Fe65 with the Fe65L1 sequence resulted in a small, but significant drop in transcriptional activity. These data show that several regions of Fe65 have evolved to better support transcription. Activity is reduced by lack of the unique acidic region and exchange of the WW-L2-PTB1 central region by the Fe65L1 sequence. Furthermore, the L3-PTB2-Ct region of Fe65 is sufficient to enable spot formation when fused to a truncated Fe65L1.

The Fe65 family increases amyloidogenic APP processing and AICD levels

To determine the impact of the Fe65 family members on APP metabolism, we co-expressed them, or Cit-3HA as a control, together with a construct expressing 3myc-APP-3HA via a GAPDH promoter (Gersbacher et al., 2013). We analyzed APP fragments using western blots – due to the approximately extra 5 kDa of the 3HA tag we were able to resolve overexpressed and endogenous APP fragments using an APP C-terminal antibody (Fig. S5A). DAPT incubation was performed to verify C-terminal fragments (CTFs) and AICD bands by their increase or decrease, respectively. We stained the different APP fragments using several antibodies and quantified n=3 experiments, normalizing the levels to GAPDH. The Fe65 family members partially increased the levels of endogenous and expressed full-length APP (Fig. S5B). We did not detect a change in the of levels of the α C-terminal fragment (α-CTF) of APP, which were strongly enhanced by DAPT treatment (Fig. S5C,S6C). All three Fe65 family members enhanced the levels of the β-CTF of both endogenous and overexpressed APP (Fig. S5D,F). Furthermore, AICD levels were also strongly increased for both by all Fe65 family members (Fig. S5E,G). Identical results regarding full-length APP, β-CTF, α-CTF and AICD were seen using an anti-HA antibody to detect overexpressed APP (Fig. S6A). Analysis of medium revealed no difference in sAPPα derived from both overexpressed and endogenous APP, as well as no difference in 3myc-tagged sAPP levels (Fig. S6B). We measured the half-lives of endogenous APP by performing a cycloheximide (CHX) time series with concomitant expression of the Fe65 family members or a control construct. Both Fe65 and Fe65L2 expression resulted in a stabilization of immature and mature APP (Fig. S7A). In conclusion, the common effect of the Fe65 family members on APP metabolism is an increased amyloidogenic processing and increased AICD levels.

Fe65L2 is the sole family member with high turnover

All Fe65 family members were expressed from the same promoter, yet Fe65L2 levels were much lower (Fig. S5A). We determined the half-life of Fe65 family members in the above experiments that analyzed APP half-live by the CHX time series. Cell lysates were analyzed by SDS-PAGE and normalization was done with the highly stable GAPDH protein (Fig. 8A; Fig. S7B). Quantification of protein levels showed that Fe65 and Fe65L1 are quite stable with half-lives of 24.4 and 35.6 h, respectively. Only Fe65L2 had a high turnover with a half-life of 1.5 h, in line with the lower levels detected in Fig. S5A.

Fig. 8.

Transcriptional activity of stable Fe65 is regulated by APP cleavage. (A) Half-life of Fe65 family members as determined by CHX-mediated inhibition of protein synthesis. Data points show mean±s.e.m. for n=5 experiments. Representative western blots are shown in Fig. S7B. (B) Confocal microscopy of HEK293 cells transfected with APP-Cit, CFP-Tip60 and either wt Fe65 or Fe65 mutants with disrupted APP binding. Treatment with DAPT revealed that APP retained only wt Fe65 outside of the nucleus with Tip60 located in speckles. Even with DAPT, both Fe65 mutants can still translocate to the nucleus and form FT spots that are devoid of AICD. Scale bar: 10 μm. (C) Transcription assay with HEK2:UAS-Cit-NLS cells. Gal4-Fe65 was co-transfected with APP, AICD or an empty vector and cells treated with DAPT or control solvent. Bars depict mean±s.d. of n=21 confocal microscopy images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) Schematic overview of the role of Fe65 domains in transcription and spot formation.

Fig. 8.

Transcriptional activity of stable Fe65 is regulated by APP cleavage. (A) Half-life of Fe65 family members as determined by CHX-mediated inhibition of protein synthesis. Data points show mean±s.e.m. for n=5 experiments. Representative western blots are shown in Fig. S7B. (B) Confocal microscopy of HEK293 cells transfected with APP-Cit, CFP-Tip60 and either wt Fe65 or Fe65 mutants with disrupted APP binding. Treatment with DAPT revealed that APP retained only wt Fe65 outside of the nucleus with Tip60 located in speckles. Even with DAPT, both Fe65 mutants can still translocate to the nucleus and form FT spots that are devoid of AICD. Scale bar: 10 μm. (C) Transcription assay with HEK2:UAS-Cit-NLS cells. Gal4-Fe65 was co-transfected with APP, AICD or an empty vector and cells treated with DAPT or control solvent. Bars depict mean±s.d. of n=21 confocal microscopy images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) Schematic overview of the role of Fe65 domains in transcription and spot formation.

APP cleavage controls Fe65 nuclear signaling

We co-transfected cells to generate AFT spots and analyzed the effects of inhibiting γ-secretase, which prevents the release of AICD from APP stubs. Whereas wild-type Fe65 supported AFT spot formation under control conditions, incubation with the γ-secretase inhibitor DAPT caused Fe65 to be retained at the membrane and Tip60 was not relocated from speckles to spots (Fig. 8B). The ΔPTB2 Fe65 mutant cannot bind to APP, and DAPT was not able to prevent its translocation to the nucleus and the formation of spots with Tip60, which were consequently devoid of AICD. The C654F mutation in the PTB2 domain, which has been reported to inhibit APP binding (Minopoli et al., 2001), behaved in a similar manner to the ΔPTB2 mutant.

We further analyzed the influence of full-length APP and AICD on the transactivation activity of Gal4-Fe65 using HEK2:UAS-Cit-NLS cells. APP and AICD had opposing effects, decreasing and increasing Fe65 transactivation activity, respectively (Fig. 8C). Inhibition of γ-secretase cleavage by DAPT slightly reduced Fe65 activity under control conditions or with co-expression of AICD when compared to the respective transfections without DAPT. Inhibitor treatment resulted in a strong inhibition of Fe65 activity when APP was co-expressed. These experiments show that the turnover of APP determines the capacity of Fe65 to signal to the nucleus and activate transcription. Fig. 8D shows a scheme of Fe65 depicting the regions regulating transcription, interacting with Tip60 or APP and involved in AFT spot formation.

We directly compared the three Fe65 family members with respect to nuclear translocation, subnuclear location, transcriptional activity and influence on APP processing. We identified Fe65 as the sole family member regulating transcription and see strong evidence that Fe65 is a transcription factor regulated by APP processing. In addition, we show the role of extranuclear Tip60 in translocation of AFT complexes to the nucleus. Interestingly, Fe65 was originally identified as a transcriptional activator (Duilio et al., 1991) and was only later shown to bind APP (Borg et al., 1996; Fiore et al., 1995; Zambrano et al., 1997). In transcription assays with APP-Gal4, it was frequently reported that co-expression of Fe65 dramatically increased transcriptional activity and deleting the APP-interacting PTB2 domain of Fe65 blocked this effect (Cao and Sudhof, 2001; Konietzko et al., 2019; Wiley et al., 2007; Zambrano et al., 2004).

Using a series of Fe65 deletion constructs, we showed that constructs lacking K701/703 of the predicted NLS motif still translocate to the nucleus and form AFT spots, whereas constructs including the motif but lacking other domains do not accumulate in the nucleus or form AFT spots. We conclude that the two lysine residues are not part of a NLS but nevertheless have a functional role, as mutation to alanine increased the transcriptional activity of Fe65. In unpublished data we show that this relates to acetylation of lysine residues by Tip60 (S.P., F. Riese, L.K., N. Russi, R.M.N. and U.K., unpublished). Further experiments with Fe65 domain deletions or Fe65–Fe65L1 fusions did not reveal a confined motif for nuclear import in Fe65. The whole WW-L2-PTB1 region had to be deleted to disrupt nuclear localization. We conclude that Fe65 does not contain a classical NLS, but probably relies on a piggyback mechanism as was previously proposed (Minopoli et al., 2001).

Tip60 mediates the accumulation of Fe65 and AICD in the nucleus in AFT spots, and we had previously proposed that this is due to sequestration of AICD–Fe65 complexes undergoing nucleo-cytoplasmic shuttling by nuclear Tip60 (von Rotz et al., 2004). Nevertheless, we have also detected nucleo-cytoplasmic shuttling of Tip60 and its colocalization with APP and Fe65 in neurites (Konietzko et al., 2010). We now report that Fe65 also induces nuclear localization of Tip60; thus, these two molecules interact to promote each other's nuclear localization. Fe65, which lacks a unique NLS, therefore probably relies on the NLSs of Tip60 for nuclear import. The nuclear localization of Tip60 is controlled by APP turnover, similar to Fe65. Inhibition of γ-secretase cleavage of endogenous APP in HEK293 cells reduced nuclear Tip60 levels. This effect was abolished in Fe65-KO cells, showing that endogenous levels of APP and Fe65 in HEK293 cells are sufficient to determine the nuclear localization of ectopically expressed Tip60.

We showed that, for Fe65, the N-terminal 250 amino acids, as well as the reported PTB1 domain (Cao and Sudhof, 2001), are necessary for Tip60 binding in pulldown experiments. Thus, the Tip60–Fe65 interaction involves widespread interaction surfaces. We found no indication of Fe65L1 interaction with Tip60, whereas Fe65L2 located to speckles together with Tip60, but also showed no interaction in pulldown experiments. Adding the Fe65 250 N-terminal residues to FE65L2 or replacing the Fe65L1 N-terminal residues with the Fe65 sequence was sufficient to enable the fusion proteins to pulldown Tip60. In conclusion, Fe65 shows stronger interaction with Tip60, which results in nuclear signaling properties not shared by the family members.

Initially, yeast two-hybrid experiments had revealed that the Fe65 L2-PTB1-L3 region binds Tip60 and mutation of the NKSY motif in the PTB1 domain disrupted pulldown of Fe65 (Cao and Sudhof, 2001). Nevertheless, with immunocytochemistry, we saw colocalization of the Tip60 NKSY to NASA mutant with Fe65 and AICD in nuclear speckles (von Rotz et al., 2004). We now see a similar effect with deletion of the PTB1 domain in Fe65 – mutant Fe65 localizes with Tip60 in nuclear speckles instead of spots and is not able to pulldown Tip60. Biochemical pull-down experiments with several wash steps are harsher than fixing cells in situ. This could explain why some interaction can still be detected via colocalization using microscopy. Still, both NKSY mutation and PTB1 deletion prevent the nuclear relocation of Fe65 and Tip60 from speckles to spots.

All Fe65 family members enhanced amyloidogenic processing with increases in β-CTF and AICD levels. Increased amyloidogenic processing has been described before for Fe65 (Chow et al., 2015b; Lee et al., 2017; Sabo et al., 1999), but there are also studies proposing the opposite (Hoe et al., 2006). For Fe65L1, increased Aβ secretion and AICD generation are reported (Chang et al., 2003), and for Fe65L2 increased Aβ secretion (Tanahashi and Tabira, 2002). Taken together, the Fe65 family has an identical influence on APP processing in vitro. This in line with Fe65/Fe65L1 double KO mice showing a reduction in Aβ peptides (Guénette et al., 2006). In addition, knockin mice with the APP Y682G mutation that abolishes Fe65 binding show strongly increased sAPPα levels as well as reduced sAPPβ (Barbagallo et al., 2010). The amyloidogenic pathway mediates AICD and Fe65 nuclear signaling (Belyaev et al., 2010; Flammang et al., 2012; Goodger et al., 2009). Thus, Fe65 induces its own nuclear localization by promoting the amyloidogenic pathway.

We detect some increase in full-length APP levels upon the expression of Fe65 family members, as well as stabilization of mature and immature APP by Fe65 and Fe65L2. Stabilization of APP by Fe65 has been reported before, with stronger effects than reported here (Chow et al., 2015b). This difference might be explained by the different cells used for analysis. The HEK293 cells used in our analysis show endogenous Fe65 expression, whereas in the CHO cells used in the cited study Fe65 is barely detectable (our unpublished observations), leading to greater changes in APP turnover when Fe65 is ectopically expressed. Fe65 is predominantly expressed in neural tissue, including the adrenal gland (Sabo et al., 2003), and HEK293 cells are likely derived from the adrenal gland (Shaw et al., 2002). This might explain the high Fe65 expression in HEK293 cells compared to non-neuronal cell lines such as CHO and HeLa cells.

We directly compared all Fe65 family members with respect to transcriptional activity, using our recently developed Gal4/UAS-Cit assay (Konietzko et al., 2019). Genomic integration of reporter sequences enables detection of bona fide nuclear signaling, as opposed to the transient transfections of reporter plasmids in the commonly used luciferase assays, which mainly reside in the cytosol (discussed in Konietzko, 2012). We conclude that transcriptional activity is a unique property of Fe65. The lack of transcriptional activity for Fe65L1 (Chang et al., 2003) and Fe65L2 (Tanahashi and Tabira, 2002) have been noted before, using luciferase and chloramphenicol transferase assays. Analysis of fusion proteins containing different parts of Fe65 and Fe65L1 revealed that various regions of Fe65 have evolved to better function in transcription than the corresponding Fe65L1 sequences. This concerns the central WW-L2-PTB1 region that together contributes to transcriptional activity. The WW domain of Fe65 has been previously shown to possess transcriptional activity (Cao and Sudhof, 2004; Telese et al., 2005). Furthermore, Fe65 contains a unique acidic region in the N-terminus. Both the Fe65 deletion constructs and the Fe65/Fe65L1 fusion proteins revealed transcriptional activity of this region. In conclusion, the central part of Fe65 from the acidic region to the PTB1 domain, contributes to transcriptional activity.

The Nt region preceding the acidic motif, as well as the C-terminal PTB2 domain and Ct, have an inhibitory effect on transcription. The PTB2 domain and Ct independently act to inhibit transcription. Combined deletion further increases transcriptional activity above the similar increases seen with sole deletion of PTB2 or Ct. Deletion of Nt alone increases transcription, but combined deletion with the Fe65 Ct region does not cause a further increase. The inhibitory effect of the Ct region could be related to an intramolecular interaction with the Nt region, shielding the central transcriptionally active part of the molecule, similar to the model proposed by Cao and Sudhof (2004). The function of the PTB2 domain in suppressing transcription is discussed below.

The UAS-Cit assay has a sigmoidal response curve with increasing amounts of Gal4-Fe65 lipofection, similar to the response we have reported recently for APP-Gal4/Fe65 (Konietzko et al., 2019). There, we studied the effects of mutating amino acids known to be targets of phosphorylation in the AICD of APP-Gal4 and could only detect changes of transcriptional activity in the linear range of the assay, not at saturating 1 µg DNA. The APP G681A mutant that was shown to abolish Fe65 binding (Cao and Sudhof, 2004), disrupted APP-Gal4-induced transcriptional activity both in the linear and the saturated range. Detecting changes that only modulate transcriptional activity and are not on–off effects, and is thus only possible in the linear range. Nearly all experiments performed here were done with 1 µg Gal4-Fe65 constructs, clearly in the saturated range of the assay. Deletion of different domains in F65 influenced transcriptional activity in both directions. This is a strong indication that Fe65 represents a transcription factor – sole manipulation of Fe65 is sufficient to bidirectionally change transcriptional activity under saturating conditions.

Spherical nuclear complexes are formed upon co-expression of APP, Fe65 and Tip60, designated as AFT spots (von Rotz et al., 2004). These AFT spots localize to transcription factories (Konietzko et al., 2010) and their formation is a clear indication of the capacity of nuclear signaling by APP and Fe65 (Gersbacher et al., 2013; Goodger et al., 2009). AFT spots are highly reminiscent of the recently described phase-separated condensates that contain RNA polymerase II and other components of the transcriptional machinery (Cho et al., 2018), with the same liquid-like properties, such as spherical shape, and the ability to flow and fuse (Konietzko et al., 2010). Acidic motifs in activation domains, such as the unique region in Fe65 that is involved in transcription, have recently been connected to transcription and the formation of phase-separated condensates (Boija et al., 2018). This further supports our findings that AFT spots define nuclear loci of endogenous target gene transcription. Tip60 alone localizes to irregular speckles that represent a different nuclear compartment than the spots formed upon co-expression of Fe65. Deletion of either WW or PTB1 domains disrupted spot formation, with mutant Fe65 and Tip60 colocalizing in nuclear speckles. These constructs also showed strongly reduced transcriptional activity.

Although most constructs showed a correlation between transcriptional activity and AFT spot formation, some revealed a dissociation of the two phenomena. Deletion of L2 connecting WW and PTB1 domains did not disrupt spot formation of Fe65 with Tip60, but prevented spot localization of AICD and abolished transcriptional activity. Deleting the APP-binding PTB2 domain inhibited AICD spot localization, but allowed FT spot formation and presented an even higher transcriptional activity than full-length Fe65.

On first sight, this implies that AICD is not necessary for Fe65-mediated transcription. However, transcriptional activity in the assay relies on the Gal4-DBD that binds introduced UAS promoter elements and the fused protein just needs to have transcriptional activity or attract transcription factors, not DNA-binding activity. We have shown that the Gal4-DBD does not colocalize with AFT spots but rather locates to the genomic integration sites of the UAS elements. The dissociation of transcriptional activity from the capacity to form AFT spots relates to this. It is well possible that only with bound AICD, is Fe65 is able to adopt a conformation to target endogenous promoters. We clearly see AICD localizing to nuclear AFT complexes in our experiments and have shown that AFT spots localize to APP and KAI1 promoters (Konietzko et al., 2010), two proposed target genes of AICD and Fe65 (Baek et al., 2002; von Rotz et al., 2004). Furthermore, AICD has been shown to bind mediator subunits (Xu et al., 2011), which also form transcription-dependent condensates that resemble AFT spots (Cho et al., 2018). Dissection of Fe65 by deletion and fusion constructs revealed that, in addition to the WW and PTB1 domains, the C-terminal L3-PTB2-Ct region is absolutely required for AFT spot formation. It even enabled a N-terminally truncated Fe65L1 to localize to spots when replacing the Fe65L1 sequence. Besides AICD, Fe65 and Tip60, more proteins are likely localized to spots that could be scaffolded by the different Fe65 domains, such as SET (Telese et al., 2005), akin to the cytosolic scaffolding function of Fe65 (Chow et al., 2015a).

Fe65 is a long-lived protein with a half-life of ∼25 h, and control of its transcriptional activity is unlikely to be mediated by the regulation of Fe65 levels. Although a cytosolic protein, in fractionations it mostly distributes to the membrane fraction where it binds to APP and other transmembrane proteins such as the ApoE receptor family and calsyntenin/alcadein proteins (Araki et al., 2004; Hoe et al., 2006; Kinoshita et al., 2001; Trommsdorff et al., 1998). Nevertheless, in contrast to the APP family, which binds to the PTB2 domain, these transmembrane proteins bind the PTB1 domain of Fe65 and it is not known whether that enables the simultaneous binding of Tip60 as for APP–Fe65 complexes. All currently described transmembrane interaction partners of Fe65 are substrates for γ-secretase-mediated RIP. We revealed that manipulation of γ-secretase cleavage of APP directly controlled Fe65 nuclear translocation and transcriptional activity. Deletion of the PTB2 domain prevented sequestration of Fe65 at the membrane after inhibition of γ-secretase cleavage. Therefore, APP is a break for Fe65 signaling, and the rate of APP cleavage determines the strength of the transcriptional response as reported recently (Konietzko et al., 2019). In contrast to Fe65, APP is turned over rapidly with a half-life of less than an hour (Allinquant et al., 1995; Gersbacher et al., 2013; Weidemann et al., 1989). Proteomic analysis of synaptosome protein turnover identified APP as a protein with one of the fastest turnover rates (Heo et al., 2018). In addition, the APP–Fe65 interaction is regulated by phosphorylation of interacting residues in both proteins. APP Y682 phosphorylation inhibits Fe65 binding (Tamayev et al., 2009) and Fe65 S610 phosphorylation similarly blocks APP binding (Chow et al., 2015b). Thus, the turnover of APP and modification of its binding to Fe65 determine transcriptional signaling by Fe65.

The PTB2 deletion enhanced the transcriptional activity of Fe65, but a C654F mutation that also disrupts APP binding (Borg et al., 1996; Minopoli et al., 2001) had wild-type levels of activity. Fe65 has been shown to form dimers via the PTB2 domain (Feilen et al., 2017). We now show that the C654F mutant Fe65 can still form dimers. C654 is located in α-helix 3 of the Fe65 PTB2 in Fe65–AICD complexes and its mutation creates a steric overlap with the YENPTY motif of AICD, thus disrupting APP binding (Radzimanowski et al., 2008). Fe65 dimerization occurs via β-completion–C654 is part of a β-sheet that is far less vulnerable to accommodating a phenylalanine (Feilen et al., 2017). On the other hand, the PTB2 domain is not necessary for spot localization, although it disrupts Fe65 dimers. We conclude that Fe65 dimers are located in the cytosol. Whereas PTB2 deletion disrupts both dimers and APP binding, leading to increased nuclear translocation and transcriptional activity of Fe65, the C654F mutation keeps Fe65 dimers intact and thus some Fe65 is retained in the cytosol. Stimulation of amyloidogenic APP cleavage by Fe65 has previously been shown to be regulated by T579 phosphorylation in the PTB2 domain. This suppressed Fe65 PTB2 intermolecular dimerization and enhanced FE65–APP complex formation (Lee et al., 2017). The picture that emerges is a reservoir of cytosolic Fe65 dimers, whose dissociation and APP association is regulated, whereby the binding of Fe65 PTB2 to AICD opens up Fe65 for additional binding of Tip60. Upon γ-secretase-mediated cleavage of APP, the AFT complex can then translocate to the nucleus with the help of the NLSs from Tip60, to form AFT spots.

Fe65/Fe65L1 double KO mice show a much more severe phenotype than the single KOs (Guénette et al., 2006). Because only Fe65 has transcriptional activity, the severe phenotype cannot be explained by the loss of transcriptional function of Fe65, but probably the role of the Fe65 family as cytosolic scaffold (Chow et al., 2015a). Nevertheless, in contrast to Fe65L1 single KO, Fe65-KO animals have reduced early long-term potentiation, pointing towards defects in synaptic plasticity (Strecker et al., 2016). In support of this, AICD has been shown to regulate transcription of NMDAR subunits and modify electrical properties of neuronal signaling (Pousinha et al., 2019, 2017). Furthermore, APP also regulates activity-dependent genes to influence inhibitory neurotransmission (Opsomer et al., 2020). APP turnover is regulated by synaptic activity (Cirrito et al., 2005; Kamenetz et al., 2003). Thus, activity-dependent synapse to nuclear signaling by AICD–Fe65–Tip60 complexes might be involved in regulating synapse function.

DNA constructs

The CFP-Tip60 (human, isoform 2), Citrine-AICD (Cit-AICD), human APP695-Citrine (APP-Cit) and human Fe65 expression constructs were previously described (von Rotz et al., 2004). APP695-Cerulean (APP-Cer) (Kohli et al., 2012) and Myc-Tip60 (Konietzko et al., 2010) are as described previously. The Myc tag in Tip60 was further exchanged with a V5 tag. The 3myc-APP695-3HA construct was previously described (Gersbacher et al., 2013) and AICD-3HA was derived from this construct by PCR-based cloning. Mouse Fe65L1 (courtesy of Suzanne Guénette and Stefan Kins, TU Kaiserslautern, Germany) and human Fe65L2 (variant 4, OHu05198, Genescript) were cloned into the pUKBK vector system (Kohli et al., 2012). We used PCR-based cloning methods to extend the pUKBK vectors with N-terminal tags – streptavidin-binding protein (SBP) was derived from a construct described previously (Kohli et al., 2012), and the Gal4 DNA-binding domain (DBD) was derived from APP-Gal4 (courtesy of Thomas Südhof, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, USA). SBP and Gal4-DBD were followed by a Myc tag and a unique AscI cleavage site designed to code for glycine-alanine-proline. Fe65 family proteins and mutants thereof were inserted after the AscI site. Deletion, point mutation, and fusion protein constructs of Fe65 family proteins were created using standard PCR-based cloning methods. The SBP-myc tag was further exchanged with mCherry (mChe) via the AscI cleavage site. As a control construct we also fused the Gal4-DBD via a Myc tag to Cerulean with a triple NLS sequence (Gal4-myc-Cer-NLS). Table S1 provides an overview of the amino acid sequences of the different Fe65 family protein constructs. A CMV promoter was used to express all constructs, except for 3Myc-APP-3HA, which was expressed via a GAPDH promoter. The lentiviral reporter construct UAS-Cit-NLS has been recently described (Konietzko et al., 2019).

Cell culture, lipofection and drug treatment

Human embryonic kidney cells (HEK293; DSMZ, Braunschweig, Germany), the Lenti-X™ 293T cell line (#632180, Clonentech, Takara, Berkely, USA) and N2a (DSMZ, Braunschweig, Germany) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, #41965039, ThermoFisher Scientific) containing 10% fetal bovine serum (FBS; #10270106, ThermoFisher Scientific) using standard conditions (37°C, 5% CO2, humified atmosphere). Cells were free of mycoplasma contamination. For microscopy experiments, cells were grown on four-well-chamber glass slides (Falcon), successively coated with poly-L-ornithine (50 μg/ml; Sigma-Aldrich) and fibronectin (5 μg/ml; Sigma-Aldrich). Cells were transfected with expression constructs using Lipofectamine™ 2000 Transfection Reagent (#11668019, ThermoFisher Scientific) according to the manufacturer's instructions, and fixed or lysed after 20 to 26 h. Plasmid DNA concentration was measured on a Nanodrop 2000 (ThermoScientific) and 500 ng of undigested DNA was routinely analyzed on agarose gels to ensure identical quality (i.e. supercoiled status, which affects lipofection efficiency). Drugs were added with medium replacement 2.5 h after transfection. The nuclear export inhibitor leptomycin B (LMB, #431050, Calbiochem, in ethanol) was used at 10 ng/ml for 18 h and the γ-secretase inhibitor DAPT (#D5942, Sigma, in DMSO) was used at 1 or 3 µM for 20 h. To inhibit protein synthesis, cells were treated with 100 μg/ml cycloheximide (CHX, #66819, Sigma) 22 h after transfection for 0.5 h to 4 h.

Generation of Fe65-KO cells

HEK293 Fe65-KO cells were generated using Alt-R® CRISPR-Cas9 (IDT, Coralville, Iowa, US). The crRNA/tracrRNA/Cas9 complexes were created using 0.2 nmol Alt-R® CRISPR-Cas9 crRNA against Fe65 (HEK293 Fe65ko clone 1: #Hs.Cas9.APBB1.1.AA, 5′-ATTGCGATTCTGGTCACGGT-3′, starting at amino acid 84; HEK293 Fe65ko clone 3: #CD.Cas9.TPCK3618.AB, 5′-CCCCACGGAATACCAACCCA-3′, starting at amino acid 359; HEK293 Fe65ko clone 4: #CD.Cas9.ZGJY0202.AH, 5′-ACCCAGTGATGAGGCCCCAA-3′, starting at amino acid 320), 0.5 nmol Alt-R® CRISPR-Cas9 tracrRNA (#1072532) and 61 pmol Alt-R® S.p. Cas9 nuclease V3 (#1081058). The ribonucleoprotein complexes were electroporated into 1.5×106 HEK293 cells using the Neon™ Transfection System 100 µl Kit (#MPK10096, ThermoFisher Scientific), and applying one 30 ms pulse of 1150 V (Yu et al., 2016). Single clones were generated using limited dilution on 96-well plates. Deletion of Fe65 was verified by immunoblotting.

Virus production and cell line generation

Lentiviral vector production and generation of HEK:UAS-Cit, HEK1:UAS-Cit-NLS and HEK2:UAS-Cit-NLS cells was as recently described (Konietzko et al., 2019). To generate the N2a:UAS-Cit-NLS reporter cell line, 1.25×105 N2a cells were plated and infected the next day with concentrated UAS-Cit-NLS reporter virus (5×108 particles). Cell lines were isolated by limited dilution on 96-well plates.

Streptavidin purification

Pulldown assays were performed to purify the SBP-tagged constructs and to co-precipitate bound proteins. At ∼22 h following transfection, HEK293 cells were lysed in homogenization buffer [HB; 140 mM KCl, 20 mM HEPES pH 7.2, 10 mM NaCl, 5% (v/v) glycerol, 2 mM MgSO4, 1% (v/v) Triton-X100, 2 mM DTT, EDTA-free Protease-Inhibitor Cocktail (#11873580001, Roche) and 2 mM Phenantrolen]. Lysates were pushed 10× through a 26 G needle, incubated on a rotating wheel for 15 min at 4°C, and cleared by centrifugation at 800 g for 10 min at 4°C. 100 μl of the supernatant was used as lysate sample; the remaining 700 μl was incubated with Dynabeads® M-280 Streptavidin (#11205D, ThermoFisher Scientific) for 4 h at 4°C. Next, the beads were washed, and the bound proteins were eluted with 14 mM biotin.

Subcellular fractionation

Cells were lysed with the same buffer as for streptavidin purification and transferred to Eppendorf tubes. Tubes were vortexed with maximum speed for 20 s, with the finger placed on top of the tube to achieve reproducible conditions. After 15 min on a turning wheel at 4°C, cell were vortexed again for 20 s and centrifuged for 15 min at 800 g. The supernatant was kept as cytosol/membrane (C/M) fraction and the pellet resuspended in streptavidin purification buffer and centrifuged for 15 min at 800 g. The nuclear pellet was resuspended in RIPA buffer, 125 Units benzonase added, incubated at 37°C for 5 min, and resuspended by vortexing and trituration with 18G and 22G canula.

Western blotting

Medium was collected, and cells were lysed using RIPA buffer [50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 1 tablet EDTA-free protease inhibitor cocktail/50 ml (#04693159001, Sigma)]. After centrifugation for 5 min at 20,817 g, 4°C, equal amounts of lysate supernatant and collected medium were separated on Novex™ 10%–20% Tricine Protein Gels (#EC6625BOX, ThermoFisher Scientific), and transferred on a 0.1 µm Amersham™ Protran™ nitrocellulose blotting membrane (#GE10600000, GE Healthcare). Blots were blocked with PBS, 0.05% Tween-20, 5% milk and incubated in blocking solution with following primary antibodies: HA high affinity (rat IgG1, 3F10, monoclonal, 1:1000, #11867431001, lot# 15645900, Roche), APP C-term (rabbit IgG, Y188, monoclonal, 1:250–1:1000, #ab32136, lot# GR211501-24, Abcam), β-amyloid 1-16 (mouse IgG1, 6E10, monoclonal, 1:1000, #SIG-39320, lot# B228658, Biolegend), c-myc (mouse IgG1K, 9E10, monoclonal, 1:1000, #11667149001, lot# 11776800, Roche), mCherry (rat IgG2a, 16D7, monoclonal, 1:2000, #M11217, lot# UD284615, ThermoFisher), Tip60 (rabbit, polyclonal, 1:500, #ab137518, lot# GR315199-11, Abcam), acetylated histone H3 (rabbit, polyclonal, 1:4000, #06-599, lot# DAM1588236, Millipore), Fe65 (mouse, OTI3H9 monoclonal, 1:1000, #MA5-27408, lot# UE2761282, ThermoFisher) and GAPDH (mouse IgG1, monoclonal, 1:5000, #H86504M, lot# H86504M, Meridian). Secondary antibodies were peroxidase-linked goat anti-mouse-IgG (1:2500, #115-035-146, lot# 142637, Jackson ImmunoResearch), peroxidase-linked donkey anti-rabbit-IgG (1:2000, #NA9340, lot# 9672941, GE Healthcare) and peroxidase-linked goat anti-rat-IgG (1:2500, #NA935, lot# 354130, GE Healthcare). For improved APP and AICD detection, the membranes were air-dried and incubated with boiling PBS after transfer (Pimplikar and Suryanarayana, 2011), followed by blocking with Tris-buffered saline (TBS) with 0.1% Tween-20 and 10% FBS. Protein bands were detected with the ImageQuant LAS 4000 (GE Healthcare Life Sciences), using Pierce™ ECL Western Blotting Substrate (#32106, ThermoFisher Scientific) or with ECL Prime Western Blotting Detection Reagent (#RPN2232, GE Healthcare).

The ImageQuant TL software was used to quantify the latest exposure before the brightest band was saturated on the blot. APP, Fe65, Fe65L1 and Fe65L2 protein levels were normalized to GAPDH. For the CHX chase experiments, the normalized protein levels were fitted to the one-term exponential model f(x)=a×e(bx) using MATLAB R2014b. The obtained coefficient b was further used to calculate the half-life using the formula: ln(2)/−b.

Immunocytochemistry

HEK293 cells were fixed with 4% (w/v) paraformaldehyde, blocked with TBS supplemented with 0.2% Triton X-100, 5% goat serum, 5% horse serum, and incubated with primary antibodies (same as for western blots, 5-fold less diluted) in blocking solution. Highly cross-purified secondary antibodies from Jackson ImmunoResearch were Cy™3-conjugated donkey anti-mouse-IgG (#715-165-151), Alexa Fluor® 647-conjugated donkey anti-mouse-IgG (#715-606-151) and Alexa Fluor® 647-conjugated donkey anti-rat-IgG (#712-605-153). Cell nuclei were stained with DAPI, and cells were mounted with Mowiol containing 2.5% 1,4-diazabicyclo(2.2.2)octane (DABCO) (Valnes and Brandtzaeg, 1985).

Confocal microscopy

Images were acquired on a TCS/SP8 confocal microscope (Leica) with a 63× glycerol objective and a pinhole of 0.5 airy. Hybrid detectors (HyDs) were operated in standard mode with 8-bit intensity resolution and detection windows adjusted to fluorophore emission spectra. DAPI was excited with a 405 nm diode laser and detected with a HyD window of 407–507 nm. Cit was excited with the 514 nm line of the argon laser and detected with a HyD window of 516–560 nm with Cy3 co-staining or 516–610 nm when no Cy3 was present. CFP was excited with the 458 nm line of the argon laser and detected with a HyD window of 460–512 nm. In this case, the HyD window for DAPI was set from 407–456 nm to prevent signal pickup from Cer. Cy3 was excited with a 561 nm laser and detected with a HyD window of 563–631 nm. Alexa Fluor 647 was excited with a 633 nm helium-neon laser and detected with a HyD window of 635–750 nm. Images shown are mostly maximum projections of 5–8 z-stacks encompassing the nucleus (unless noted). Single sections imaged with a 20× glycerol objective and a pinhole of 1 airy, were used for UAS-Cit assay measurements and determination of the nuc/cyt ratio. For quantification of the nuc/cyt ratio, we measured the total fluorescence and subtracted the fluorescence signal in the DAPI-demarcated nuclei to derive the cytoplasmic signal. Background fluorescence was determined with non-transfected and stained cells to calculate the Limit of Blank (Armbruster and Pry, 2008), which was subtracted from fluorescence measurements. All fluorophores used are consistently color-coded in all images: CFP and Cer (cyan), Cit (yellow), Cy3 (red), Alexa Fluor 647 (blue) and DAPI (white).

UAS-Cit transactivation assay

Image acquisition and quantification for UAS-Cit assay experiments was performed as recently described (Konietzko et al., 2019). Briefly, we used a 20× glycerol objective and HyDs were operated in count mode with 12-bit intensity resolution to image Cit, DAPI and Alexa Fluor 647. After adjusting laser intensity and accumulation number to not saturate the HyDs, we selected frames to image via the DAPI signal to select areas of similar cell density, adjusted the z-position to achieve the brightest DAPI signal and acquired 7 images per condition and passage (frame 775×775 µm, ∼1000–2000 cells per frame). This way, the Cit channel is imaged for the first time during actual acquisition, preventing any bias or bleaching. All image analysis was performed using Fiji/ImageJ and described in detail in Konietzko et al. (2019). We measured total Cit fluorescence in the case of UAS-Cit reporters or nuclear Cit fluorescence in the case of UAS-Cit-NLS reporters. DAPI was used to determine the nuclear outline and cell number. Data from Fiji were transferred to Excel or Prism for further analysis.

Statistical analysis

Statistics were calculated with Prism 8 (GraphPad). Data were controlled for normal distribution by Shapiro–Wilk testing. Normal distributed data were analyzed by Student's t-test or one-way ANOVA with multiple comparisons against control or wild type. Non-normal distributed data were analyzed by Kruskal–Wallis or Mann–Whitney U-test. Asterisks above the horizontal significance line refer to all bars with tick marks except when the ticks are marked individually (*P<0.05, **P<0.01 and ***P<0.001. Bars not indicated by a tick mark are not significantly different. Data are presented as mean±s.d. for UAS-Cit assays and determination of nuc/cyt ratios. Data are presented with mean±s.e.m. for western blots.

Author contributions

Conceptualization: S.P., U.K.; Methodology: S.P., M.K., L.K., S.T., D.S., U.K.; Software: M.K.; Validation: L.K., S.T.; Formal analysis: S.P., M.K., U.K.; Investigation: S.P., M.K., L.K., U.K.; Resources: M.K., L.K., S.T., D.S.; Writing - original draft: U.K.; Writing - review & editing: S.P., R.M.N., U.K.; Visualization: S.P., U.K.; Supervision: R.M.N., U.K.; Project administration: R.M.N., U.K.; Funding acquisition: U.K.

Funding

This work was supported by the Swiss National Science Foundation SNF (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung) 31003A_166177 and the resources of the IREM.

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Competing interests

The authors declare no competing or financial interests.

Supplementary information