EMBO/soda-nel huggingface dataset:
Myotubularin functions through actomyosin to interact with the Hippo pathway (2022)
Figure 1

Figure 1. Identification of Mtm as a novel growth regulator.

(A-D) Images of a representative wild-type adult eye (A) or adult eyes containing mtm mutant clones (B and C). Quantification of relative eye sizes (n=10) (D).

(E) Schematic diagram of Drosophila Mtm protein (top; Dm) and its human homologue MTMR2 (bottom; Hs). mtm mutant alleles from this study are indicated.

(F-G) A pupal eye disc containing mtm mutant clones (marked by the absence of GFP) was stained for Dlg. Note the increased interommatidial cells in the mtm mutant clone. Quantification of numbers of interommatidial cells per ommatidium (n=10) (G). Scale bar = 10 µm.

(H-I) Third instar larval eye discs containing mtm mutant clones (marked by the absence of GFP) were stained for the Hippo pathway target genes diap1 and ex. Note the increased expressions of Diap1 and Ex in mtm mutant clones indicated (arrows). Scale bars = 10 µm.

(J) A third instar larval wing disc containing mtm mutant clones (marked by the absence of GFP) was stained for Yki and DAPI. Note increased Yki nuclear localization in mtm mutant clone. Scale bar = 10 µm.

(K) RNAi of Mtm reduces Yki phosphorylation. Kc167 cells were incubated with dsRNA of gfp or mtm for three days before western blot analysis. Total cell lysates were probed with anti-phospho-Yki (p-Yki) antibody. Note that RNAi of mtm decreased Yki phosphorylation.

Data information: Data are shown as mean ± SEM, ***P < 0.001, ****P < 0.0001, Student's t-test. "n" indicates the numbers of samples used for the statistical analysis. D and G represent results from one of three biological replicates.
Myotubularin functions through actomyosin to interact with the Hippo pathway (2022)
Figure 4

Figure 4. Mtm-mediated PI(3)P dynamics is critical for its function in Growth Suppression

(A-C) PI(3)P expression levels detected by a 2xFYVE-GFP reporter in a third instar larval wing disc (A) and eye disc (B) containing mtm mutant clones (marked by the absence of RFP). Quantification of the mean FYVE-GFP fluorescence intensity of mtm mutant clones and surrounding wild-type (WT) cells are shown (C). Ten different mtm clones and surrounding regions in three biological replicates were used for the analysis. Note the strong increases of PI(3)P levels in the mtm mutant clones. Data are shown as mean ± SEM, ****P < 0.0001, Student t-test. Scale bars = 10 µm.

(D-F) Pupal eye discs containing clones (GFP positive) of indicated genotypes were stained for Dlg. The average numbers of extra interommatidial cells per ommatidium in each genotype were shown in the lower right corners. 10 ommatidia from different clone regions in each genotype were used for the quantification. Note pi3k68d RNAi almost completely suppressed loss-of-mtm-induced extra interommatidial cells. Scale bars = 10 µm.

(G-L) Pupal eye discs of indicated genotypes were stained for Dlg. The average numbers of extra interommatidial cells per ommatidium in each genotype were shown in the upper right corners of each panel. 10 ommatidia from different clone regions in each genotype were used for the quantification. Note pi3k68d RNAi dramatically suppressed the interommatidial cell increases in the mtm,ex and mtm,ft double mutant flies. Scale bars = 10 µm.

(M-P) Images of representative adult fly nota containing indicated mutant clones. Note that the massive notum tissue overgrowth was largely suppressed by pi3k68d RNAi knockdown in mtm,ex or mtm,ft double mutant flies.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 1

Figure 1: Molecular composition of reconstituted K. lactis COMA-Nkp1-Nkp2 or reconstituted K. lactis COMA

A) Representative size-exclusion chromatography (SEC) chromatogram with absorbance measured at nanometers (nm) 260 or 280 (for this chromatogram and the following ones, absorbances are in units of 1000-1 (mAU)) of reconstituted K. lactis COMA-Nkp1-Nkp2, and image of Coomassie Blue-stained SDS-PAGE gel with fractions from principal SEC peaks. Molecular masses of protein standards (Mm. S.) are in kiloDalton (kDa); L: sample loaded on column. †: Escherichia Coli DnaK; ╪: proteolysed Nkp1-Nkp2; Nkp1-Nkp2 is less prone to spontaneous proteolysis when associated with COMA. B,C) Nanoflow mass-spectra of COMA-Nkp1-Nkp2 (B) or COMA (C), acquired with different injected concentrations, showing an increase in COMA-Nkp1-Nkp2 dimers or COMA dimers with increasing injectant concentration. D) Graph with measured differential refractive index (relative scale) and molar mass calculated with multi-angle light scattering (MALS) data of COMA eluting from SEC (SEC-MALS). We injected COMA at a concentration of ~ 43 μM (COMA dimer) on the column. Dashed lines indicate expected molar masses for monomeric COMA or dimeric COMA: 142337 g mol−1 or 284674 g mol−1; our experimentally determined molar mass from the principal SEC elution peak: 299100 g mol−1 ± 5683 g mol−1 (mean ± standard deviation from a single SEC-MALS experiment); max. dimeric COMA concentration, from refractive index measurement, in principal peak: 2.4 μM.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 2

Figure 2: Subunit topology of COMA

A) Excerpt from a representative nanoflow mass-spectrum of COMA (full spectrum and enlarged spectrum areas are in Appendix Fig S2A); B) mass-spectrum from tandem mass-spectrometry of the isolated 28+ charge state of COMA (from spectrum shown in A), after its collision-induced dissociation in the gas phase. Charge states of assigned masses are indicated above peaks. For this spectrum and our other spectra of this type, charge states of assigned masses are indicated above peaks. Inset table shows identified proteins or protein assemblies, their measured mass values as mean ± standard deviation from multiple peaks in a peak series assigned to the same molecular species; and their respective expected mass.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 3

Figure 3: Comparative deuterium-exchange experiments identify structured segments and flexible elements of Okp1 or Ame1 in COMA-Nkp1-Nkp2

A,B) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Okp1 (A) or Ame1 (B), each in COMA or COMA-Nkp1-Nkp2. For this representation, and all following of this type, detected peptides are colour-coded according to their measured deuterium-exchange in % of the maximum measured exchange (1-25 %: dark blue; 26-50 %: light blue; 51-75 %: yellow; 76-100 %: red). Peptides are represented as bars. Each bar is plotted row-wise corresponding to its position in the amino-acid sequence of the protein it derives from. Amino-acid sequence scheme is illustrated at the top with thin or thick lines; thin line: flexible element: thick line: structured segment. Positions of predicted coiled coils are shown as helices. Plots for full time courses are in Appendix Fig S3C,E.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 4

Figure 4: Molecular requirements for COMA assembly

A) Schematics of our designed Ame1 and Okp1 constructs; our identified structured segments and coiled coils are as we show in Fig 3. B,C) Representative images of Coomassie Blue-stained SDS-PAGE gels with Ni2+-affinity purification eluates. B) of full-length COMA (lane 11) with full-length polyhistidine-tagged Mcm21 (His-Mcm21) and Ctf19, or COMA variants with polyhistidine-tagged Mcm21 D-RWD domain (His-Mcm21_D-RWD; K. lactis Mcm21 residues 108-293) and Ctf19 D-RWD domain (Ctf19_D-RWD; K. lactis Ctf19 residues 107-270) C) of Ame1-Okp1 variants with polyhistidine-tagged Okp1 variants (His-Okp1 variants). We show one-letter identifiers for protein versions in A. Lanes between lanes 5 and 6 are not relevant. D) Image of SDS-PAGE gel with fractions of two purified Ctf19-Mcm21-Okp1 variants (after SEC); expected migrating position of non-proteolysed Okp1 variant (compare with its position in SDS-PAGE images of COMA or Ame1-Okp1 variants in B or C) is marked; 2 and 3: SEC-purified fractions of samples from different elution fractions from ion-exchange chromatography. When we co-expressed full-length Okp1 with Ctf19 and Mcm21, which has a coding regions for an N-terminal polyhistidine-tag, we did not co-purify substantial amounts of full-length Okp1 with Ctf19-Mcm21, probably because Okp1 was prone to proteolysis (in the absence of Ame1).
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 5

Figure 5: Interactions of Okp1 or Ame1 with Nkp1-Nkp2

A,B) Overlayed chromatograms showing absorbance at 280 nm from SEC of Ctf19-Mcm21-Okp1 variants without Okp1 segment 3 (A) or with Okp1 segment 3 (B); Ctf19-Mcm21-Okp1 variants: CMO, Nkp1-Nkp2: NN, Ctf19-Mcm21-Okp1 variants combined with Nkp1-Nkp2: CMO-NN; and images of SDS-PAGE gels with equivalent elution fractions from five SEC experiments; L: loaded on column. The elution-volume range that we analysed fractions of on SDS-PAGE is indicated below the chromatograms. C) Representative microscopy-images of living haploid S. cerevisiae cells with Okp1_fl (integrated full-length Okp1) or our Okp1 version that lacks the coding region for segment 3-Okp1_nnΔ, with Nkp1-GFP or Nkp2-GFP. Fluorescence images are maximum intensity projected, and we show those from Okp1_fl or Okp1_nnΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale; scale bar: 2 μm. D,E) Plots showing hydrogen-deuterium exchanged peptides, after 10 sec of deuterium exchange of Nkp1 (D) or Nkp2 (E), each in Nkp1-Nkp2 or COMA-Nkp1-Nkp2. Nkp1 has an N-terminal SNA residual. Plots for full time courses are in Appendix Fig S5A,B.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 6

Figure 6: Molecular structural characteristics of the Ctf19-Mcm21 binding motif of Okp1

A) Plots showing deuterium-exchanged peptides, after deuterium exchange of 10 sec of Okp1 in Ctf19D-RWD-Mcm21D-RWD-Okp1229-336, in full-length COMA, or in Ame11-260-Okp1123-336. Plots for full time course are in Appendix Fig S6C. B) Deuterium-exchange of peptides, from deuterium exchange data after 1200 sec of deteurium-exchange of Ctf19 and Mcm21, mapped onto the surface of the structure of the Ctf19-Mcm21 D-RWD domains (PDB code: 3ZXU); left: Ctf19-Mcm21 in COMA; right: Ctf19-Mcm21 alone. Residues without deuterium-exchange data are coloured dark grey. Plots for full time courses are in Appendix Fig S6D,E. C) Semi-transparent surface representation of our Ctf19D-RWD-Mcm21D-RWD-Okp1295-360 crystal structure, with secondary structure cartoon-diagram underneath; left: in a similar orientation as Ctf19-Mcm21 in B. D) Close-up view of the Okp1 binding site of Ctf19-Mcm21 with Ctf19-Mcm21 binding motif of Okp1; Okp1 residues, and some Ctf19 or Mcm21 sidechains, are shown as sticks (nitrogen: blue, oxygen: red); Ctf19-Mcm21 shown as secondary structure cartoon-diagram. E) Sequence alignment of budding yeasts' Ctf19-Mcm21 binding motif of Okp1. Similar residues are coloured red; conserved residues are on red background. We show full alignment in Fig EV5. F) Representative baseline-subtracted raw isothermal calorimetry data and derived binding isotherm from titration of Okp1-derived peptide, with the Ctf19-Mcm21 binding motif, to full-length Ctf19-Mcm21. Mean value for dissociation constant (Kd) and its standard error of regression are from three independent experiments. G) Plots from microscale-thermophoresis measurements with fluorescently labeled full-length Ctf19-Mcm21 and with Okp1-derived synthetic peptide (same as in F), or with Okp1-peptide variants that differ in one or two residue positions from the native Okp1 sequence (e.g. N330A: N330A). Normalized fluorescence, from which the fluorescence of unbound Ctf19-Mcm21 had been subtracted (ΔFnorm), is plotted against peptide concentration (logarithmic scale). For dissociation constants (Kd), we calculated mean value and confidence interval (68 % probability that Kd is within given range) with data from three independent experiments. Variant N330A-K332P had no detectable binding in the peptide concentration-range that we measured.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 7

Figure 7: Effect of absence of the Ctf19-Mcm21 binding motif on kinetochore organization

A) Schematic of K. lactis Okp1 or K. lactis Okp1 without the Ctf19-Mcm21 binding motif (Okp1_cmΔ); annotation is as shown in Fig 3A. B) Representative images of Western-blots of immunoprecipitated fractions from S. cerevisiae extracts with Okp1-6×flag and Ctf19-4×myc; fl: Okp1_fl; cmΔ: Okp1 variant without the Ctf19-Mcm21 binding motif (Okp1_cmΔ); IP: immunoprecipitated samples. Protein G: beads coated with Protein G only (used as control); positions of molecular masses of standards are indicated; uncropped images are in Appendix Fig S8B,C. C) Representative microscopy images of living haploid S. cerevisiae cells with Nuf2-mCherry, and Ctf19-GFP or Mcm21-GFP, either with Okp1_fl or with Okp1_cmΔ. We show merged green fluorescence signals and red fluorescence signals as pseudo-colours. We show fluorescence images of cells with Okp1_fl or Okp1_cmΔ with the same fluorophor-fusion kinetochore protein on the same intensity scale (also applies to images in D). Fluorescence images and those in D are deconvoluted and maximum intensity projected; scale bar: 2 μm. D) Representative microscopy images of living haploid S. cerevisiae cells with Okp1_fl or Okp1_cmΔ, with GFP-tagged kinetochore subunits. Image pixels for images of cells with GFP-fusion proteins of Chl4, Iml3, Mif2, Ctf3, Cnn1, Nkp1, or Nkp2 are 2×2 binned; scale bar: 2 μm. The low GFP signal-intensity that we observe for Chl4-GFP and Iml3-GFP in Okp1_fl cells is consistent with the low GFP signal for Chl4-GFP and Iml3-GFP at centromeres that was previously reported (Joglekar et al, 2008; Lawrimore et al, 2011). E) Scheme summarizing our in vivo observed centromere localization dependencies. Solid arrows: localization dependencies found by our study or by studies of others; dashed arrows: protein-protein interactions.
Molecular basis for inner kinetochore configuration through RWD-domain interactions (2017)
Figure 8

Figure 8: Relevance of the Ctf19-Mcm21 binding motif for cell viability

A) Representative images of dilution-series growth assay of haploid S. cerevisiae clones with cnn1Δ, Okp1_fl, Okp1_cmΔ, Okp1_fl cnn1Δ, or Okp1_cmΔ cnn1Δ, grown for two or three days on solid YPD agar or on solid YPD agar with 20 μg ml−1 benomyl. We show two unique clones each for Okp1_fl cnn1Δ or Okp1_cmΔ cnn1Δ. Arrow indicates decreasing cell-density. Spots are from cell samples that we sequentially diluted four fold. B) Representative images of haploid S. cerevisiae spores from dissection of three tetrads (we placed the four spores from each tetrad row-wise) from heterozygous diploid cells with Okp1_fl mad1Δ or Okp1_cmΔ mad1Δ grown for four and three days, respectively on solid YPD agar at 25 °C. Coloured circles indicate spore genotypes (see bottom). C) Representative images of haploid S. cerevisiae with mad1Δ, Okp1_fl mad1Δ, or Okp1_cmΔ mad1Δ; native: S. cerevisiae S288C type (identifier DDY904 in Table EV6). Dilution is as we describe for A. We show two unique clones for Okp1_fl mad1Δ. D) Quantification of spore viability after meiosis, of spores of dissected tetrads from homozygous diploid S. cerevisiae clones with either Ctf19_1-954Δ (without Ctf19 basepairs 1-954; leaving the 3' end of Ctf19, and, on the complementary DNA strand, the IRC15 gene, which encodes a microtubule binding protein (Keyes & Burke, 2009) intact), Ctf19Δ (that lacks the entire Ctf19), Okp1_fl, Okp1_cmΔ, or Okp1_cmΔnnΔ. Quantification is from spores from multiple tetrad dissections.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 1

LAMP‐2A localizes in detergent‐resistant lipid microdomains of the lysosomal membrane. (A) Fed rat liver lysosomes were extracted with 1% of the indicated detergents and then subjected to sucrose gradient centrifugation. Aliquots collected from top to bottom were subjected to SDS–PAGE and immunoblotted for LAMP‐2A and flotillin‐1. Stars indicate the aliquots of the gradient of translucent appearance (highly enriched in lipids). (B) The distribution of LAMP‐2A through the sucrose gradient after extraction with 1% Triton X‐114 was calculated from the densitometric quantification of immunoblots as the one shown here. Values are expressed as percentage of the total lysosomal levels and are mean+s.e. of four experiments.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 2

Properties of the LAMP‐2A‐containing lipid microdomains at the lysosomal membrane. (A) Lysosomes from livers of fed (Fed) or 24 h starved rats (Stv) were extracted with 1% Triton X‐114 and processed as in Figure 1 or the aliquots in the translucent region were pooled together as the detergent‐resistant (DR) fraction, and anything below this fraction was pooled, as the solubilized fraction (Sol) (B). Lanes 5–6 are one‐tenth of the amount of lysosomes loaded in the gradient. (C) Distribution of LAMP‐2A (L‐2A) and flotillin‐1 (Flot‐1) in fed rats liver lysosomes extracted with 1% Triton X‐114 at 4 or 37°C. DR fractions were pooled as in (B), and the aliquots below this region were divided in three fractions (a soluble (Sol; aliquot 15) and two intermediate fractions (light: aliquots 11–12; heavy: aliquots 13–14). (D) Fed rat liver lysosomes were incubated with MBCD or filipin, as described under Material and methods, and then processed as in (C). The mean value of the densitometric quantification of the LAMP‐2A immunoblots from two experiments is shown at the bottom in (C) and (D).
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 4

LAMP‐2A localizes in cholesterol‐ and GM‐1‐enriched intracellular membrane domains. Mouse fibroblasts grown on coverslips in the presence (+) or absence (−) of serum were fixed and labeled with filipin (red) (A) or incubated with Texas‐Red‐cholera toxin B subunit to label GM‐1‐enriched membrane regions (red) and then fixed. (B) Both groups of cells were then subjected to indirect immunofluorescence for LAMP‐2A (green). Where indicated, cells were treated with MBCD (25 mM) for 30 min before fixation. The merged images of both fluorophores are shown on the right. Insets show vesicles at higher magnification. Bar: 10 μm. The quantification of the colocalization of the two fluorophores is shown in (C). Values are expressed as the mean+s.e. of percentage of colocalization in 20–40 cells.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 5

Distribution of LAMP‐2A in discrete lysosomal membrane regions. (A, B) Regions of the lysosomal membrane resistant to 1% Triton X‐114 recovered from the sucrose gradients were fixed and processed for electron microscopy (A) or visualized without fixation by electron microscopy (B). Arrows point to the membrane bilayer. (C) Intact rat liver lysosomes (top) and DR regions of the lysosomal membranes (bottom) were fixed and subjected to immunogold labeling for LAMP‐2A and LAMP‐2B, and a gold‐conjugated secondary antibody as described under Material and methods. Top right panels show ‘clusters’ of gold particles (black arrows) in individual lysosomes at higher magnification. Graph shows the labeling density (gold particles per μm) in the indicated fractions for both proteins. Values are mean+s.e. from the quantification of 15 different micrographs. (D) Intact rat liver lysosomes were fixed and subjected to double immunogold labeling for LAMP‐2A and flotillin‐1. Representative lysosomes are shown. Far right panel: zoomed‐in image of the distribution of LAMP‐2A in regions containing (black arrows) and not containing (white arrows) flotillin‐1. Bars: 0.2 μm.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 6

The distribution of LAMP‐2A in discrete lysosomal membrane regions changes with changes in CMA. (A) Fed rat liver lysosomes were incubated in an isotonic buffer without additions, or in the presence of rat liver cytosol (25 μg), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (25 μg), GST‐hsc70 (10 μg/ml), ATP (5 mM) or the indicated combinations for 15 min at 37°C. Lysosomes incubated directly with apyrase or with cytosol treated with apyrase are shown in the right bottom panel. At the end of the incubation, lysosomes were subjected to Triton X‐114 extraction, sucrose density gradient centrifugation and immunoblot as in Figure 2C. Graph shows changes in LAMP‐2A distribution expressed as times the value in untreated lysosomes and are the mean of the densitometric quantification of 3–6 experiments as the one shown here. Because all the incubations were performed in the presence of protease inhibitors, total levels of LAMP‐2A in lysosomes remained constant throughout the incubation. (B) Lysosomes from livers of fed rats, untreated or treated with paraquat (+PQ), were processed as in (A). Graph shows changes in LAMP‐2A distribution induced by the PQ treatment, expressed as times the value in untreated animals and are the mean of three experiments. (C) Lysosomes with high (CMA+) and low (CMA−) ability for CMA isolated from livers of fed or 48 h starved rats were processed as in (A). Top left: Levels of two CMA components (LAMP‐2A and hsc70) and of an unrelated lysosomal membrane protein (LAMP‐1) in each of the four groups of lysosomes. Bottom left: Percentage of total lysosomal LAMP‐2A present in each fraction, determined by densitometric quantification of the immunoblots.
Lysosome membrane lipid microdomains: novel regulators of chaperone‐mediated autophagy (2006)
Figure 7

Consequences of changes in the lysosomal cholesterol content on CMA activity. (A) Degradation of long‐lived proteins in mouse fibroblasts (NIH3T3 cells) treated or not with 25 mM MBCD. Where indicated, 15 mM NH4Cl/100 μM leupeptin (NH4Cl+Leup) were added into the medium. Values are the mean+s.e. of three experiments with triplicate samples. (B) Effect of MBCD on total rates of protein degradation, measured as in (A), in wild‐type mouse fibroblasts and in fibroblasts stably RNA interfered for LAMP‐2A (LAMP‐2A(−)). The ratio of degradation in MBCD‐treated and untreated cells is shown for cells maintained with or without 10 mM 3‐methyladenine (3MA). (C) Proteolysis of [14C]GAPDH or a pool of [3H]labeled cytosolic proteins (pool proteins) by isolated rat liver lysosomes, treated or not with MBCD. Broken: lysosomes disrupted by a hypotonic shock before the incubation with the radiolabeled proteins. Values are expressed as percentage of the degradation by untreated lysosomes and are the mean+s.e. of three experiments with triplicate samples. (D) Lysosomal–mitochondrial fractions were isolated from mouse fibroblasts untreated or loaded with cholesterol (50 μM). Top: Distribution of LAMP‐2A after Triton X‐114 extraction and sucrose density gradient centrifugation. Bottom: Ability of intact or disrupted lysosomal–mitochondrial fractions to proteolize [14C]GAPDH. Values are expressed as percentage of the degradation in fractions from untreated cells and are the mean±s.e. of triplicate samples.