EMBO/soda-nel huggingface dataset:
Ca2+/Calmodulin Binding to PSD-95 Mediates Homeostatic Synaptic Scaling Down (2017)
Figure 8

Figure 8. R126E mutation on CaM restores scaling down of AMPAR mEPSC amplitude that is impaired by PSD-95 E17R mutation.

Cultured hippocampal neurons were co-transfected at 10-11 DIV with RFP and the indicated CaM-IRES-ECFP plasmids and infected at 12-13 DIV with lentivirus expressing shRNA against PSD-95 (sh95) and sh-resistant wild-type (WT) or E17R PSD-95-EGFP. mEPSC recordings were performed at DIV 17-19, following 48 h of control or BIC (50 μM) treatment. (A) Representative traces of AMPAR mEPSC recordings (left) and averages of all mEPSC events from single representative neurons (right) drawn to scale (top) and normalized to peak (bottom). (B) Quantification of mEPSC amplitude revealed downscaling by BIC in neurons expressing PSD-95WT / CaMWT or PSD-95E17R / CaMR126E but not PSD-95WT / CaMR126E (left) over the full range of amplitude distributions (right). (C,D) Mini-EPSC frequency (C) and decay time (D) were comparable among all conditions (n=11-25 neurons per condition from 8-14 independent sets of cultures prepared from the embryos of 8-14 dams;* p<0.05; two-way ANOVA followed by Bonferroni's test).
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 1

Figure 1: RAB13 RNA and protein exhibit distinct subcellular distributions.

A. Representative FISH images showing RAB13 RNA distribution in MDA-MB-231 cells. Nuclei and cell outlines are shown in blue and green respectively. Arrows points to RAB13 RNA concentrated at protrusive regions. Boxed regions are magnified in the insets.

B. Representative immunofluorescence images of RAB13 protein in cells transfected with the indicated siRNAs. Reduction of intensity in RAB13 knockdown cells confirms the specificity of the signal. Arrows point to perinuclear RAB13 protein. Calibration bar shows intensity values.

C. Ratios of peripheral/perinuclear intensity calculated from images as shown in A and B. Bars: mean ± s.e.m..Values within each bar represent number of cells observed in 3 independent experiments.

D. Protrusions (Ps) and cell bodies (CB) of cells induced to migrate towards LPA were isolated and analyzed to detect the indicated proteins (by Western blot; left panels) or RNAs (by RT-ddPCR; right panel). Ps/CB enrichment ratios from 2 independent experiments are shown. Bars: mean ± s.e.m..The enrichment of pY397-FAK serves to verify the enrichment of protrusions containing newly formed adhesions in the Ps fraction.

Data information: P-values: **<0.01; ****<0.0001 by student's t-test (C) or analysis of variance with Dunnett's multiple comparisons test against GFP (D). Scale bars: 10 μm.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 2

Figure 2: A GA-rich motif in the mouse Rab13 3'UTR is necessary for localization at protrusions.

A. Schematic showing the %GA content along the mouse Rab13 3'UTR using a 30nt window size. Occurrences of the consensus GA-rich motif are indicated by a red rectangle. The exact sequence between nucleotides 153-216 is shown with GA motifs in red and deleted regions indicated by black bars. P-value by Fisher's exact test with Bonferroni correction.

B. FISH images of mouse fibroblasts expressing the β-globin coding sequence followed by the indicated UTRs. β-globin RNA is shown in yellow. Nuclei and cell outlines are shown in blue. Arrows points to β-globin RNA concentrated at protrusive regions. ∆1 and ∆1+2 indicate deletions of the regions shown in (A). Scale bars: 10 μm.

C. Distribution of β-globin RNA, or of Ddr2 RNA detected in the same cells, quantified by measuring a Peripheral Distribution Index (PDI). N=35-55 cells observed in 3 independent experiments. Bars: mean ± 95% CI. ****:p<0.0001 by analysis of variance with Dunnett's multiple comparisons test.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 3

Figure 3: Antisense oligonucleotides against the GA-rich region specifically interfere with localization of Rab13 RNA.

A. Schematic showing positions along the mouse Rab13 3'UTR targeted by the indicated PMOs. PMOs #1, 2 and 3 target the RGAAGRR motifs or the adjacent GA-rich region. PMOs #6 and #7 target the Rab13 3'UTR outside of the GA-rich region. The control PMO targets an intronic sequence of human β-globin. Red rectangles and text indicate the location of the GA-rich motifs.

B. FISH images and corresponding PDI measurements of mouse fibroblast cells treated with the indicated PMOs. Cyb5r3 is an APC-dependent RNA also enriched at protrusions. Arrows: peripheral Rab13 RNA. Arrowheads: perinuclear Rab13 RNA. Boxed regions are magnified in the insets. Note that Rab13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region. Scale bars: 10 μm. (4 μm in insets). ****: p<0.0001 by analysis of variance with Dunnett's multiple comparisons test. N=40-90 cells observed in 3-6 independent experiments. Bars: mean ± 95% CI.

C. Protrusion (Ps) and cell body (CB) fractions were isolated from cells treated with control-PMO or Rab13-PMO #2. The indicated RNAs were detected through nanoString analysis to calculate Ps/CB enrichment ratios (n=3; bars: mean ± s.e.m.). Note that only the distribution of Rab13 RNA is affected. **: p=0.01 by two-way ANOVA with Bonferroni's multiple comparisons test against the corresponding control.

D. Levels of the indicated RNAs were determined using nanoString analysis from control- or Rab13 PMO #2-treated cells. (n=4; bars: mean ± s.e.m.). No significant differences were detected by two-way ANOVA against the corresponding controls.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 4

Figure 4: The human RAB13 3'UTR contains a functionally conserved GA-rich region required for peripheral localization.

A. Schematic showing %GA content and positions along the human RAB13 3'UTR targeted by the indicated PMOs. Red rectangles indicate the location of GA-rich motifs. Graphs present PDI measurements of RAB13 RNA (upper panel) or NET1 RNA (another APC-dependent RNA; bottom panel) detected in MDA-MB-231 cells treated with the indicated PMOs. PDI=1 indicates a diffuse distribution. p-values: **<0.01, ***<0.001, ****<0.0001 by analysis of variance with Dunnett's multiple comparisons test. n.s.: non-significant. N=30-73 cells observed in 3-5 independent experiments. Bars: mean ± s.e.m..

B. Representative FISH images of cells treated with the indicated PMOs. Arrows point to peripheral RNA. Arrowheads point to perinuclear RNA. Boxed regions are magnified in the insets. Note that RAB13 RNA becomes perinuclear in cells treated with PMOs against the GA-rich region, while NET1 remains localized at protrusions. Scale bars: 10 μm (4 μm in insets).

C. RAB13 protein levels were measured by quantitative Western blot and normalized to total α-Tubulin or GAPDH levels (for representative blot, see Figure 5E). Relative levels in RAB13 PMO-treated compared to control are shown. No significant differences were detected by Kruskal-Wallis test with Dunn's multiple comparisons test. N=3-8. Bars: mean ± SD.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 5

Figure 5: Loss of peripheral RAB13 RNA localization disrupts cell migration and phenocopies acute RAB13 protein loss.

A. Transwell migration of MDA-MB-231 cells treated with control PMOs or RAB13 PMOs (191+230). Cells reaching the bottom surface after 4hrs were counted. n=25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m..

B. Cells expressing Cherry-NLS, and treated with the indicated PMOs, were tracked every 5min for 10hrs to derive average migration speed. n=65 cells. Bars: mean ± s.e.m..

C. PMO-treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n=4 independent experiments. Bars: mean ± s.e.m..

D. Lifeact-GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n=11-13 cells. Bars: mean ± s.e.m..See also movies EV2 and EV3.

E. Cells treated with the indicated PMOs or siRNAs were analyzed by western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n=55-78 cells (bottom graph). Bars: mean ± s.e.m..

F. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs was assessed as in (D). Graphs show average normalized values from n=10-13 cells imaged for 1 hr. Bars: mean ± s.e.m..

G. Migration speed of RAB13 knockdown cells re-expressing GFP-RAB13 or GFP-RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28-52 cells were analyzed. Bars: mean ± s.e.m.. Similar results were obtained in two additional independent experiments. Re-expressing cells were identified through GFP fluorescence and cells with similar, low GFP signal were tracked. All cells are expressing cherry-NLS for accurate tracking.

Data information: p-values: *<0.05; **<0.01, ***<0.001, ****<0.0001, by Student's t-test (A, B, C, D), analysis of variance with Dunnett's multiple comparisons test (E, F) or Kruskal Wallis test with Dunn's multiple comparisons test (G). n.s.: not significant.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 6

Figure 6: Peripheral RAB13 RNA translation is required for RAB13 protein activation but not steady state distribution or membrane association.

A. Widefield images of RAB13 immunofluorescence in MDA-MB-231 cells treated with control or RAB13 (191+230) PMOs and ratios of peripheral/perinuclear intensity. Scale bars: 10 μm. n=45-50 cells. Bars: mean ± s.e.m.. Similar results were observed in two additional independent experiments.

B. Fluorescence images (projections of confocal slices throughout the cell height) of cells expressing GFP-RAB13 with the indicated UTRs. Note that in both cases the protein assumes indistinguishable distribution. Scale bars: 10 μm.

C. Soluble/particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Graph shows quantitation from n=3 independent experiments. Bars: mean ± s.e.m..

D, E. Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from the indicated PMO-treated cells (D) or GFP-RAB13 expressing lines (E). The amount of endogenous or exogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..

Data information: p-values: *<0.05, **<0.01 by Kruskal-Wallis test.
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 7

Figure 7: Peripheral RAB13 RNA promotes the local association of RAB13 with the exchange factor RABIF.

A. Immunoprecipitation and Western blot analysis to detect proteins bound to GFP-RAB13 expressed from constructs carrying wt or ∆PMO RAB13 3'UTR. GFP-RAB13 (T22N) expresses a nucleotide-free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP-RAB13.

B. Quantification of RABIF binding to RAB13 from experiments as in (A). n=6. Bars: mean ± s.e.m..

C. Active RAB13 pulldown assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n=3. Bars: mean ± s.e.m..

D. Quantification of RABIF-RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m.. Similar results were observed in one additional independent experiment.

E. Representative RABIF-RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets.

F. Representative RABIF-GFP PLA images and quantitations from cells expressing GFP-RAB13(T22N) carrying wt or ∆PMO RAB13 3'UTR. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets.

Data information: p-values: *<0.05, **<0.01, ***<0.001, ****<0.0001 by Wilcoxon matched-pair signed rank test (B) or analysis of variance with Dunn's, Dunnett's or Tukey's multiple comparisons test (C-F).
RNA localization and co-translational interactions control RAB13 GTPase function and cell migration (2020)
Figure 8

Figure 8: RABIF associates with RAB13 co-translationally.

A. Schematic depicting experimental strategy for assessing co-translational association of RABIF with nascent RAB13.

B. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several-fold more to RAB13(T22N) (see Figure 7A, B) it binds similarly to the wild-type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP-loading of wild type RAB13, while it remains more stably bound to the nucleotide-free (T22N) form. N=6. Bars: mean ± s.e.m.. p-values: *<0.05 by analysis of variance with Dunn's multiple comparisons test.

C. Proposed model for regulation of RAB13 activity through local RNA translation. See text for details.

EXTENDED VIEW FIGURE LEGENDS
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 1

Figure 1 -TIMM50 and the import machinery

A Schematic structure of the human mitochondrial protein import machinery showing the components of the translocase of the outer (TOM) and inner membrane (TIM23-PAM and TIM22).

B Human TIMM50 isoforms (top), TIMM50L and TIMM50S, consist of three domains: a mitochondrial targeting sequence (MTS) that directs the protein to mitochondria, a trans membrane domain (TMD) and a presequence binding domain (PBD) that contains an active phosphatase FCP1-like domain. Isoform TIMM50L also contains a nuclear localization signal (NLS). Phylogenetic alignment of the human protein region containing the substitutions found in the affected individual is shown below. Altered residues in the affected subject are boxed in red and its position marked in relation to conserved domains. Abbreviations are as follows: Hs, H. sapiens; Pt, P. troglodytes; Bt, B. taurus; Mm, M. musculus; Rn, R. norvegicus; Xt, X. tropicalis; Dr, D. rerio; Dm, D. melanogaster; Ce, C. elegans; Sc, S. cerevisiae.

C TIMM50S levels in primary and immortalized skin fibroblast from control (C1-4) and mutant TIMM50 (Pt). SDHA and ETHE1 are shown as loading controls.

D qPCR quantification of TIMM50 transcript levels in control and TIMM50 mutant fibroblasts. Data are shown as mean ± SD, n = 4.
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 2

Figure 2 - Decreased TIM23 complex and membrane potential in TIMM50 mutant fibroblasts

A Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts grown in either glucose or galactose containing medium.

B Western-blot of whole cell extracts for different components of the protein import machinery in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium.

C Mitochondrial membrane potential in fibroblasts grown in glucose or galactose using JC-1 staining in untreated or FCCP treated conditions. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. Exact P-values are reported in Table EV1.

D TMRM live staining of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in either glucose or galactose containing medium. Scale bar corresponds to 10 μm. A digitally enhanced TMRM image from TIMM50 mutant fibroblasts is shown in Appendix Fig S4B.
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 3

Figure 3 - TIM23 but not TIM22 dependent import is impaired in TIMM50 mutant fibroblasts

A In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts in the absence or presence of the uncoupler FCCP. The radiolabeled precursors were incubated with isolated mitochondria for the indicated times followed by trypsin treatment only in the case of AAC1. Coomassie Blue (CB) staining was used as loading control and the radiolabeled protein (TNT) as reference for quantification. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown. Western blot for TIMM50 is shown as cell line control.

B Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. Exact P-values are reported in Table EV1.

C In organello import of the precursors of TFAM and AAC1 into isolated mitochondria from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 in the absence or presence of the uncoupler FCCP as described in A. The position of the precursor (empty arrowheads) and the mature protein (filled arrowhead) are also shown.

D Quantification of TFAM and AAC1 import from control and TIMM50 mutant fibroblasts transduced with the empty vector (EV) or with wild-type TIMM50 expressed as fraction of the input radiolabeled precursor converted into mature protein and normalized to Coomassie Blue signal. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05. Exact P-values are reported in Table EV1.
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 4

Figure 4 - Altered OxPhos protein level and respiration in TIMM50 mutant fibroblasts

A Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Two biological replicates are shown for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts compared to controls have been boxed in red. The quantification is shown in Appendix Fig S3A.

B Western blot for representatives of all five respiratory complexes in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Two biological replicates were obtained for each cell line and condition. Significant changes in protein levels in TIMM50 mutant fibroblasts complemented with TIMM50 compared to EV have been boxed in red. The quantification is shown in Appendix Fig S3B.

C Oxygen consumption measurements in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. Exact P-values are reported in Table EV1.

D Oxygen consumption measurements in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Values of basal and maximal respiration along with ATP-production dependent, proton leak respiration and spare capacity are shown. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. Exact P-values are reported in Table EV1.
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 5

Figure 5 - Increased ROS production and mitophagy in TIMM50 mutant fibroblasts

A ROS production as measured by intracellular oxidation of the cell-permeant probe DCF-DA in control and TIMM50 mutant fibroblasts grown in glucose or galactose. Measurements were made in untreated cells and after treatment with 250μM hydrogen peroxide for 30 min. Data are shown as mean ± SD, n = 3 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. Exact P-values are reported in Table EV1.

B Steady state levels of ROS related proteins SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control.

C Quantification of the steady state levels of SOD2 and ACO2 in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗p < 0.05, ∗∗p < 0.01 grouping both controls together. Exact P-values are reported in Table EV1.

D Steady state levels of mitophagy related proteins VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose were assessed by Western blot using GAPDH as internal control. GAPDH is from the same blot as panel B.

E Quantification of the steady state levels of VDAC1, p62 and LC3-II in control and TIMM50 mutant fibroblasts growing in either glucose or galactose. Data are shown as mean ± SD, n = 4 biological replicates; ∗∗p < 0.01, ∗*∗p < 0.001 grouping both controls together. Exact P-values are reported in Table EV1.
Mutations in TIMM50 compromise cell survival in OxPhos-dependent metabolic conditions (2018)
Figure 6

Figure 6 - Cell size, cell growth and apoptosis in TIMM50 mutant fibroblasts

A Cell size in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 biological replicates; ∗p < 0.05, ∗∗p < 0.01, ∗*∗p < 0.001. Exact P-values are reported in Table EV1.

B Growth curves of control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cell growth was monitored continuously by the Incucyte live cell imager (Essen Bioscience). One of the three independent experiments carried out is presented. Data are shown as mean of three technical replicates ± SD.

C Plot of Annexin V intensity against propidium iodine signal in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Cells in the late stage of apoptosis are boxed in the top right corner.

D Quantification of the number of late apoptotic cells in control and TIMM50 mutant fibroblasts, transduced with the empty vector (EV) or with wild-type TIMM50 grown in glucose or galactose. Data are shown as mean ± SD, n = 5 or 4 biological replicates in the case of transduced cells; ∗*∗p < 0.001. Exact P-values are reported in Table EV1.