Additional file 2: Figure S1. of Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

A- GST-Tau was incubated with p35/CDK5 or p25/CDK5 and [γ-32P]-ATP for the times indicated, then subjected to SDS-PAGE prior to autoradiography (upper section). Tau bands were digested with Lys-C and phosphopeptides isolated and identified as described in Methods. A major phosphopeptide eluted with mass/charge ratio of 802.4331 corresponding to the peptide containing Ser235 (lower section), and a second doubly phosphorylated peptide corresponding to a peptide including Ser202 and Thr205 was also identified but at much lower abundance. The same result was obtained from two different phosphorylation reactions. B- GST-tau was phosphorylated as above but using non-radioactive ATP. Proteins were transferred to nitrocellulose after SDS-PAGE and probed with the indicated antibodies. Data is representative of two experiments. Figure S2. Co-expression of CDK5 complexes with CRMP2 and CRMP4. Hela cells were co-transfected with equal amounts of expression constructs for CDK5 catalytic subunit, p35 or p25 and FLAG-tagged CRMP2 (A and B) or CRMP4 (C and D) as indicated. Cells were lysed and protein expression and phosphorylation assessed by Western blot analysis (A and C). Quantification was performed on a Licor Odyssey (B and D) with data shown as mean ± S.E.M of three experiments in duplicate. t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Figure S3. Co-expression of CDK5 complexes with tau. Hela cells were co-transfected with equal amounts of expression constructs for CDK5 catalytic subunit, p35 or p25 and tau. Cells were lysed and protein expression and phosphorylation assessed by (A) Western blot analysis. (B-D) Quantification was performed on a Licor Odyssey and the ratio between phospho-tau: total tau calculated for each phospho-tau antibody. Data shown as mean ± S.E.M. for three experiments performed in duplicate. t-test, *P < 0.05, **P < 0.01, ***P < 0.001. (PDF 2557 kb

At G1, NM1 is ubiquitinated in a GSK3β-dependent manner by the E3 ligase UBR5.

<p>(<b>A</b>) Lysates prepared from GSK3β^+/+ MEFs and GSK3β^−/− MEFs at G1 transiently expressing HA-tagged ubiquitin, treated with MG132 where indicated, were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. (<b>B</b>) Lysates from HeLa cells synchronized in G1 co-transfected with GSK3β RNAi oligonucleotides or scrambled scrRNAi oligonucleotides and transiently expressing HA-tagged ubiquitin. Where indicated lysates were obtained from HeLa cells treated with MG132. Immunoprecipitations were performed from all lysates with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin. (<b>C</b>) Lysates from G1-blocked GSK3β^−/− MEFs were subjected to RNAi-mediated gene silencing of the E3 ligases UBR5 and Fbxw8 or to scrRNAi oligonucleotides transiently expressing HA-tagged ubiquitin. The lysates were subjected to immunoprecipitations with the anti-NM1 antibody and the co-immunoprecipitated fractions were analyzed on immunoblots for HA-tagged ubiquitin.</p

GSK3β-dependent NM1 phosphorylation suppresses proteasome mediated degradation and mediates association with chromatin.

<p>(<b>A</b>) Cell cycle profile analyzed at the indicated time points, after release from a G1 arrest by serum starvation, on immunoblots of the corresponding lysates for NM1, cyclin A, cyclin E, p27 and β-actin. (<b>B</b>) Relative NM1 mRNA levels in GSK3β^+/+ MEFs and GSK3β^−/− MEFs monitored by RT-qPCR using β-tubulin mRNA as internal control. (<b>C</b>) rRNA synthesis in GSK3β^+/+ MEFs and GSK3β^−/− MEFs arrested in G1 by serum starvation. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT-qPCR using tubulin mRNA as internal control [p = 3.2e-09 (***)]. (<b>D</b>) Lysates from GSK3β^−/− MEFs untreated or treated with the proteasome inhibitor MG132, released from a G1 block were collected at the indicated time points and analyzed on immunoblots for NM1 and β-actin. (<b>E</b>) ChIP and qPCR analysis on chromatin isolated from GSK3β^+/+ MEFs and GSK3β^−/− MEFs synchronized in G1, untreated or treated with MG132, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). Significances p(−MG132) = 2.2e-05 (***) and p(+MG132) = 3.0e-05 (***) were respectively calculated against the NM1 values obtained in GSK3β^+/+ MEFs not treated with MG132. (<b>F</b>) Immunoblots of total lysates from GSK3β^+/+ MEFs untreated or treated with the kinase inhibitor BIO. Analysis was performed with antibodies to NM1, actin, and the GSK3β antibodies 27C10 and CGR11 as indicated. (<b>G</b>) ChIP and qPCR analysis on chromatin isolated from GSK3β^+/+ MEFs at G1, untreated or treated with BIO, at the rRNA gene promoter with antibodies against NM1 and GSK3β (CGR11). The significance p = 0.009 (**) was calculated against the NM1 values obtained in GSK3β^+/+ MEFs not treated with BIO.</p

GSK3β phosphorylates NM1.

<p>(<b>A</b>) GSK3β, NM1 and actin are co-precipitated from nuclear protein extracts prepared from growing GSK3β^+/+ MEFs. Bound proteins were detected on immunoblots with antibodies against WSTF, SNF2h, NM1, PCAF, GSK3β (CGR11) and actin. 10% of the input is shown in Lane 1. IP, immunoprecipitation. (<b>B</b>) Schematic representation of V5-tagged wt and mutated NM1 constructs stably expressed in HEK293T cell lines. (<b>C</b>) Co-precipitations of GSK3β from total lysates obtained from HEK293T cells stably expressing wt and mutated V5-tagged NM1 constructs as indicated. 10% of the input is shown. IP, immunoprecipitation. (<b>D</b>) Lysates were prepared from growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs or from GSK3β^+/+ MEFs and GSK3β^−/− MEFs arrested in G1 by serum starvation. Where indicated extracts were subjected to alkaline phosphatase (AP) treatment. Lysates were analyzed on immunoblots for NM1 and actin. (<b>E</b>) Kinase assays were performed on lysates from G1-arrested GSK3β^−/− MEFs untreated or treated with the proteasome inhibitor MG132, supplemented with γ-³³P-ATP. Where indicated the lysates were incubated with recombinant GSK3β. To monitor NM1 phosphorylation, the lysates were subjected to immunoprecipitations with anti-NM1 antibodies. Phosphorylated NM1 was detected by phosphorimaging against the levels of unphosphorylated NM1 detected on immunoblots. (<b>F</b>) Kinase assays were performed on endogenous NM1 or actin immunoprecipitated from lysates of G1-arrested GSK3β^−/− MEFs treated with MG132; after immunoprecipitations the beads were washed and incubated with γ-³³P-ATP and recombinant GSK3β. Phosphorylated NM1 (NM1*) and autophosphorylated GSK3β (GSK3β*) were detected by phosphorimaging. The immunoprecipitated endogenous NM1 and actin were detected on immunoblots. Non-specific IgGs were used as negative control for the immunoprecipitations. (<b>G–H</b>) Tandem MS spectra of phosphoprylated and non-phosphorylated peptide DGIIDFTSGSELLITK identified within the primary NM1 sequence immunoprecipitated from G1-arrested lysates of GSK3β^+/+ MEFs and GSK3β^−/− MEFs, respectively.</p

GSK3β regulates pol I transcription activation.

<p>(<b>A</b>) rRNA synthesis in GSK3β^+/+ MEFs and GSK3β^−/− MEFs. For the analysis, relative 45S pre-rRNA levels were monitored from total RNA preparations by RT–qPCR using actin mRNA as internal control. Error bars represent the standard deviation of three independent experiments [p = 3.39e-05 (***)]. (<b>B</b>) FUrD incorporation assays on living GSK3β^−/− and GSK3β^+/+ MEFs subjected to DRB treatment. Transcription was monitored by a short FUrd pulse to monitor incorporation into nascent nucleolar transcripts. After fixation, cells were co-stained with a fluorochrome conjugated anti-BrdU antibody to detect the incorporated FUrd and with a human auto-immune serum against pol I (S57299). Detection was by confocal microscopy. Scale bar, 5 ìm. (<b>C</b>) MeDIP and qPCR analysis on growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs performed with an antibody for 5-methylcytidine. qPCR analysis on the precipitated DNA was performed with primers amplifying rRNA gene promoter and reference genes TSH2B and GAPDH. (<b>D</b>) ChIP and qPCR on growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs at the rRNA gene promoter and 18S with the pol I specific autoimmune serum S57299 and an anti-UBF antibody. (<b>E</b>) ChIP and qPCR on growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs at the rRNA gene promoter, 18S and IGS with the anti-GSK3β antibody CGR11 and antibodies against WSTF, SNF2h, NM1, actin and non-specific rabbit IgGs. (<b>F</b>) ChIP and qPCR on growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs at the rRNA gene promoter with antibodies against H3K9Ac, H3K4me3 and PCAF.</p

GSK3β distributes through the entire rDNA transcription unit, occupying the rRNA gene promoter and transcribed sequences.

<p>(<b>A</b>) Schematic representation of the primary structure of human GSK3β, including the N-terminal stretch of amino acids used as epitope for the GSK3β antibody CGR11. (<b>B</b>) Immunoblots of total lysates obtained from GSK3β^+/+ MEFs, GSK3β^−/− MEFs and HeLa cells analyzed with the anti-GSK3β antibodies CGR11 and 27C10 and with an anti-actin antibody. (<b>C</b>) ChIP and qPCR on growing GSK3β^+/+ MEFs and GSK3β^−/− MEFs at the rRNA gene promoter, 18S, 5.8S, 28S rDNA and IGS with the anti- GSK3β antibodies CGR11 and 27C10. Positions of all primers are indicated in bracket. The structure of individual mouse ribosomal rDNA repeat is shown to show the location of the different rDNA fragments analyzed. (<b>D</b>) ChIP-Seq performed on GSK3β^+/+ MEFs. The previously sequenced mouse rDNA repeat BK000964 was utilized in our analysis procedure. The frequency of hits by sequences matching the region spanning the rDNA repeat sequence and IGS is shown by the resulting graph.</p

The effects of GSK3β knockout on the morphology of the nucleolus.

<p>(<b>A</b>) The size and number of nucleoli in GSK3β^+/+ and GSK3β^−/− MEFs were analyzed in double-stained preparations using anti-UBF (red) and anti-nucleolin (green) antibodies as nucleolar markers. (<b>B</b>) Quantitative evaluation of the number of nucleoli per cell. The histogram shows the average number of nucleoli per cell based on the analysis of 212 GSK3β^+/+ MEFs and 211 GSK3β^−/− MEFs, from two independent experiments. (<b>C</b>) The ultrastructure of the nucleolus in GSK3β^+/+ and GSK3β^−/− MEFs analyzed by transmission electron microcopy. GSK3β^+/+ nucleolus. DFC: dense fibrillar component; FC: fibrillar center; GC: granular component; chrom: dense chromatin. The magnification bars represent 0.5 µm. (<b>D</b>) GSK3β^+/+ and GSK3β^−/− MEFs stained with an antibody against nucleolin (green) and counterstained with DAPI (blue) to visualize patterns of chromatin condensation. Dense chromatin of GSK3β^−/− MEFs is found in small patches that are often located at the nuclear periphery, whereas in GSK3β^+/+ cells they are often larger and more centrally located. (<b>E</b>) Transmission electron microscopy images showing the accumulation of dense chromatin near the nuclear envelope in GSK3β^−/− MEFs. Nuc: nucleus; Cyt: cytoplasm. The bar represents 200 nm.</p

Additional file 1: Table S1. of Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

All proteins identified from synaptic and non-synaptic mitochondrial proteomic analyses. (XLSX 273 kb

Additional file 2: Table S2. of Proteomic profiling of neuronal mitochondria reveals modulators of synaptic architecture

All proteins identified from synaptic and non-synaptic mitochondrial proteomic analyses with >2 fold-change, p≥0.05, ≥2 unique peptides. (XLSX 106 kb