EMBO/soda-nel huggingface dataset:
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 6

Figure 6. Ift74 regulates apical centriole migration and F-actin enrichment during multiciliation.

A, B. ift74 CRISPR mutants display defective cilia formation. Embryos at one cell stage were injected and fixed at stage 27 with two independent sgRNA targeting ift74. Representative confocal images (A) and 3D-SIM images (B) of the Xenopus epidermis, and plots display defective ciliation. (A) >60 MCCs from 6 embryos and (B) >70 MCCs from 6 embryos for each condition. Cell membranes (mGFP), cilia (Ac-tub, green) and basal bodies (Cep164) were labeled with indicated antibodies.

C. Ift74 depletion affects centriole apical migration in MCCs. Representative 3D-SIM images (x-y) and 3D-reconstructions (x-z) of MCCs show failure of apical trafficking of basal bodies. Basal bodies (Cetn1, green) and distal appendages (Cep164, purple) were labeled with indicated antibodies. Quantitative plot shows a slight significant reduction in basal bodies by the depletion of Ift74. >70 MCCs from 6 embryos for each condition.

D. Representative 3D-SIM images and plots reveal that apical expansion of MCCs is slightly affected by Ift74 depletion. >70 MCCs from 6 embryos for each condition. Embryos were fixed and stained with phalloidin (purple). Cilia (Ac-tub, gray) were also presented.

E. Representative confocal images and plot show a significant reduction in apical F-actin enrichment upon Ift74 depletion. >70 MCCs from 6 embryos for each condition. Cell boundaries and apical actin network were marked with mGFP (green) and phalloidin (purple).

Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 7

Figure 7. Ccdc108 governs the centriolar localization of IFT-B complex proteins during centriole migration.

A. Live imaging of MCCs shows the ability of wild type Ccdc108 and mutant proteins lacking the seven amino acid IFT interaction domain to localize to migrating centrioles. Embryos expressing GFP tagged Ccdc108 proteins (green) and Cetn4-RFP (purple) were imaged at stage 18.

B. Live imaging of MCCs shows the ability of IFT-B complex proteins to localize to migrating centrioles. Embryos expressing GFP tagged IFT-B proteins (green) and Cetn4-RFP (purple) were imaged at stage 18.

C-E. Ccdc108 depletion affects centriolar distribution of IFT-B proteins during centriole migration in MCCs. Embryos were co-injected with GFP-IFTs (C: Ift74; D: Ift80; E: Ift88; green), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of IFT-B proteins (Ift74, Ift80 and Ift88), and Ccdc108-IFT interaction deficient mutants are unable to rescue the decreased centriolar accumulation of IFT-B proteins. >40 MCCs from 6 embryos for each condition.

Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001). Mean ± s.d. values are also presented.
Male infertility associated Ccdc108 Regulates Multiciliogenesis via the Intraflagellar Transport Machinery (2022)
Figure 8

Figure 8. Ccdc108 and its interaction with the IFT-B complex is required for actin cytoskeleton regulator recruitment to centrioles for their apical membrane migration in MCCs.

A, B. Centriolar distribution of ciliary adhesion complex related proteins Fak and Cp110 are not reduced following Ccdc108 depletion. Embryos were co-injected with GFP-Cp110 (A) GFP-Fak (B), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. >30 MCCs from 6 embryos for each condition.

C, D. Ccdc108 depletion affects centriolar distribution of PCP associated actin cytoskeleton regulators important for centriole migration in MCCs. Embryos were co-injected with GFP-Drg1 (C) GFP-RBD (D), Cetn4-RFP (purple) and each morpholino, and imaged at stage 18. HA tagged Ccdc108 proteins were co-expressed to test for the rescue of phenotypic defects. Xenopus embryos were subjected to immunoblotting to evaluate the protein expression levels. α-tubulin (α-Tub) served as loading control. Quantitative plots show that Ccdc108-depleted MCCs showed a significant decrease of centriolar distribution of Drg1 and RBD, and neither of Ccdc108-IFT interaction deficient mutants can rescue the centriolar phenotypic defects. >30 MCCs from 6 embryos for each condition.

Data information: Quantitative data from three independent experiments were scored. Unpaired two-tailed t-test was performed (*** p<0.001; ** p<0.01). Mean ± s.d. values are also presented.

Expanded View Figure Legends
A new family of CRISPR-type V nucleases with C-rich PAM recognition (2022)
Figure 3

Figure 3 - Identification and confirmation of Cas12l guide RNA(s).

(A) Alignment of the sequences between the genes encoding the Cas12l effector and Cas1 exhibit features of a trans-activating CRISPR RNA (tracrRNA). Percent identity is shown in teal. These include a sequence encoding a 5' hairpin (Hairpin 1 - gray), a conserved sequence encoding a nexus-like stemloop (Hairpin 2 - blue), a region capable of base-pairing with the CRISPR repeat (Anti-repeat - orange) and a GC-rich sequence capable of forming a terminator-like hairpin (Hairpin 3 - red).

(B) Four single guide RNA (sgRNA) designs were engineered for Asp2Cas12l and Asp3Cas12l based on the secondary structure prediction of tracrRNAs in (A). They differed by the presence or omission of a 5' region of the tracrRNA predicted to be unstructured and by the inclusion or exclusion of the terminator-like Hairpin 3. tracrRNA features are colored as described in (A).

(C) Cleavage of supercoiled (SC) plasmid DNA substrates using purified Cas12l nuclease and respective sgRNA variants. Efficient full length linearization (FLL) of the substrate resulting from a complete double-strand break was only observed when using sgRNAs bearing the 5' most end of the tracrRNA (variants 1 and 3). Moreover, the terminator-like Hairpin 3 is not required for target cleavage (variant 1). OC - open circular; FLL - full length linear; SC- supercoiled.
A new family of CRISPR-type V nucleases with C-rich PAM recognition (2022)
Figure 4

Figure 4 - Biochemical characterization of Cas12l dsDNA cleavage.

(A) Positions of Asp2Cas12l and Asp3Cas12l target and non-target strand cleavage. Frequency of adater ligated reads from PAM library cleavage experiments for both target and non-target strands. The target strand is cleaved 23-24 nt 3' of the PAM and the non-target strand is cleaved 15-18 nt downstream of PAM recognition.

(B) Substitution of alanine residues disrupts linear dsDNA cleavage confirming key catalytic positions within the RuvC nuclease domain of Asp2Cas12l and Asp3Cas12l. wt - wildtype.

(C) Cleavage of oligoduplex dsDNA substrates with purified RNP complexes confirm PAM recognition. The molar ratio of RNP to substrate was kept low (5:1) to increase reaction stringency. Asp2Cas12l predominantly recognizes a 5'-CCY-3' PAM and Asp3Cas12l a 5'-CCB-3' PAM. N=3.

Data information: In (C), individual data points are plotted, where n = 4 replicates from independent experiments (Asp2Cas12l) and n = 3 replicates from independent experiments (Asp3Cas12l). The data points were fitted to a single exponential association curve (solid lines).
A new family of CRISPR-type V nucleases with C-rich PAM recognition (2022)
Figure 5

Figure 5 - Cas12l collateral nuclease activity.

(A) Asp2 and Asp3Cas12l RNP complexes degrade M13 ssDNA in the presence of single-stranded (ss) DNA or dsDNA (including PAM) activators with target sequences complementary to the gRNA spacer. NS activator - non specific activator (ssDNA oligonucleotide or dsDNA duplex) with no sequence complementarity to the spacer of the gRNA.

(B) Asp2Cas12l RNP complexes activated with ss or dsDNA degrade quenched fluorescent ssDNA or ssRNA probes. Background-subtracted traces from fluorescent reporter assays with favored ssDNA (5'-CCCCCCCC-3') or ssRNA (5'-CCCCCCCC-3') probes. RFU - relative fluorescence units.

(C) Background-subtracted traces of fluorescent reporter cleavage using ssDNA or ssRNA probes.

(D) Rates of trans-degradation with ssRNA or ssDNA reporters activated with either ssDNA or dsDNA targets. Collateral ssDNAse activity is about 3-fold higher than the rate of ssRNA degradation.

Fluorescence intensities in B-C were normalized against the fluorescence of a reaction containing only the probe to account for imperfect quenching or degradation of reporters.

Data information: In (B-D), individual data points from n = 3 replicates from independent experiments are plotted. In (D), bars represent the mean.
Heterotypic Amyloid β interactions facilitate amyloid assembly and modify amyloid structure (2021)
Figure 4

FIG 4. Presence of proteins with homologous regions to Aβ APRs in human amyloid plaques

A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.

B. Log-odd ratio of random sampling from mass spectrometry background. In blue is the distribution upon random sampling (x1000) from tissue proteins. Red dot indicated the true values of analysis. In APR regions the actual value (red) resides either in the edges or outside of the random distribution. (statistics: Z-test). * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001

C. Log-odd ratio of random sampling from human proteome. In blue is the distribution upon random sampling (x1000) from proteome. Red dot indicated the true values of analysis. (statistics: Z-test) * P ≤0.05, ** P≤0.01

D. TANGO scores of homologue APRs in amyloid plaques and non-amyloid plaques proteins. (statistics: Kolmogorov-Smirnov test).

E. Biological pathways enrichment of Aβ APR homologue related and non-related proteins derived from AD amyloid plaques

F. APR homologue proteins identified in other MS studies

G. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.

H. Log-odd ratio of random sampling from mass spectrometry background. In blue is the distribution upon random sampling (x1000) from tissue proteins. Red dot indicated the true values of analysis. (statistics: Z-test) * P ≤0.05, ** P≤0.01

I. Log-odd ratio of random sampling from human proteome. In blue is the distribution upon random sampling (x1000) from proteome. Red dot indicated the true values of analysis. (statistics: Z-test) * P ≤0.05, ** P≤0.01

J. Biological pathways enrichment in of Aβ APR homologue related and non-related proteins derived from nonAD amyloid plaques.
Heterotypic Amyloid β interactions facilitate amyloid assembly and modify amyloid structure (2021)
Figure 5

FIG 5. No overrepresentation is observed in amyloid plaques from APP/PS1 mouse or in Glial cytoplasmic inclusions from

A. Overrepresentation of Aβ APRs in amyloid plaques of APP/PS1 mouse brains (mean ±SD) from two biological replicates. (statistics: hypergeometric test with Bonferroni correction)

B. Log-odd ratio of random sampling from mass spectrometry background for both replicates. Red dot indicated the true values of analysis. (statistics: Z-test) * P ≤0.05

C. Log-odd ratio of random sampling from mouse proteome for both replicates. Red dot indicated the true values of analysis. (statistics: Z-test) * P ≤0.05

D. No overrepresentation of Aβ APRs was observed in proteins from Glial cytoplasmic inclusions (α-synuclein aggregates). (statistics: hypergeometric test with Bonferroni correction)
Heterotypic Amyloid β interactions facilitate amyloid assembly and modify amyloid structure (2021)
Figure 6

FIG 6. Proteins with homologues to Aβ regions can induce the aggregation of Aβ1-42 in HEK293T cells Aβ biosensor.

A. Experimental setup of inducing aggregation in Aβ biosensor cell line.

B. Representative images of Aβ biosensor seeding with 3 different seed concentration (10, 50, 100nM) Scale bars: 100μm

C. Treating Aβ biosensor with different concentration of rAβ1-42 seeds induces the aggregation of mCherry-Aβ1-42 in a dose dependent matter (n=3 independent experiments, graph: mean and 95%CI). FRAP of Aβ spots shows limited recovery confirming that are aggregates (in detail at Appendix figure S10b,c). Scale bar: 2.5μm

D. Representative images of 3 proteins that can induce aggregation of Aβ1-42 in biosensor cell. Increase aggregation is observed in cells expressing the construct but not GFP alone (left panels). Scale bar: 100μm. FRAP of the resulting aggregates shows no recovery (in detail at Appendix Figure S11). Scale bar: 2.5μm Removal of the homologue regions resulting in reduced aggregation (right panels). (n=3 independent experiments).

E. Quantification of number of spots per cell in cells expressing/not expressing the construct (identified by GFP, transfection reporter) (n=3 independent experiments, statistics: ordinary one-way Anova with Dunnett T3 multiple comparison correction, unpaired t-test for transfected/non-transfected cells). Bar plot: mean with 95%CI. * P ≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001

F-G. Quantification of Spots per cell for construct6 and construct6 control (6Ctrl, removal of homologue region) (F) and construct10 and construct10 control (10ctrl, removal of homologue region) (G). (n=4 independent experiments, statistics: ordinary one-way Anova). Bar plot: mean with 95%CI. **** P≤0.0001

H-I) Confocal images of colocalization of protein of interest with Aβ aggregates. (Contrast of images were enhanced to 0.1% saturated pixels using Fiji). Scale bars: 22μm

Expanded View Figure Legends
Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction. (2011)
Figure 1

The viability of <i>atg</i> mutants in buffered starvation medium.

(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N or SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours, and dead cells stained by phloxine B were observed by fluorescent microscopy. Scale bar, 25 µm. (B–C) WT and atg1Δ cells grown nitrogen starved as (A) for the indicated times. Cell viability (B) and medium pH (C) were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction. (2011)
Figure 2

Loss of respiratory function in <i>atg</i> mutants during nitrogen starvation.

(A) WT and atg1Δ cells grown in YEPD medium were transferred to SD-N +50 mM MES-KOH (pH 6.2). After 120 hours, cultures were diluted 5.0×105 fold and plated onto YEPD agar. The plates were incubated at 30°C for four days. Insets indicate the plate overlaid with TTC agar to examine the respiratory competency of formed colonies. (B) WT, and atg1Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. (C) WT, atg1Δ, atg2Δ, atg7Δ, atg11Δ, atg15Δ, and atg32Δ cells were nitrogen-starved as (A). Cell viability and respiratory competency was determined by phloxine B staining and TTC overlay technique as (A), respectively. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction. (2011)
Figure 3

Mitochondrial defect in <i>atg</i> mutants.

(A) WT and atg1Δ cells expressing mitochondria targeted mCherry cultured in SD-N +50 mM MES-KOH (pH 6.2) medium for 120 hours were observed by fluorescent microscopy. Mitochondrial DNA was stained with SYBR green I, and mitochondria were visualized by mitochondria targeted mCherry. Scale bar, 2 µm. (B–C) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. ROS accumulation was detected by DHE staining (B). Each photo contains about 200 cells. Scale bar, 20 µm. (C) shows quantification of ROS accumulated cells (n>200 cells). (D) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) with 10 mM NAC for the indicated time. Cell viability was determined by phloxine B staining. Cells from these cultures were plated on YEPD agar and overlaid with TTC agar to examine respiratory competency. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction. (2011)
Figure 4

Altered expression of respiratory components and ROS scavengers by <i>atg</i> mutant cells.

(A) WT and atg1Δ cells were transferred to SD-N +50 mM MES-KOH (pH 6.2) medium for the indicated time. Lysates were prepared using the alkaline-trichloroacetic acid method and subjected to immunoprecipitation with anti-Cox2, anti-Cox4, anti-Tim17 and anti-Pgk1. Pgk1 was used as loading control. (B) WT and atg1Δ cells expressing Cta1-3×FLAG or Ctt1-3×FLAG were nitrogen-starved as in (A) for the indicated time. Cell lysates were prepared as (A) and subjected to immune precipitation with anti-FLAG and anti-Pgk1. Pgk1 was used as loading control.
Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction. (2011)
Figure 5

Viability of respiratory deficient cells during nitrogen starvation.

(A) WT and atg1Δ cells were transferred to non-buffered SD-N medium for the indicated time. Cell viability was determined by phloxine B staining. Nitrogen-starved cells were plated onto YEPD agar and overlaid with TTC agar to examine the respiratory competency of formed colonies. The black and gray areas indicate the percentage of viable cells that are respiratory competent or respiratory deficient, respectively. (B) WT, atg1Δ, rho0, and rho0 atg1Δ cells grown in YEPD medium were transferred to SD-N with or without 10 mM NAC for the indicated time. In the presence of NAC, medium pH was adjusted by using KOH. Cell viability and medium pH were examined by phloxine B staining and pH meter, respectively. These data represent the average of three independent experiments and bars indicate standard deviations.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 1

Figure 1. COA7 interacts with MIA40 by disulfide bonding.

(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.

(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.

(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.

(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 2

Figure 2. COA7 is an oxidized protein in the intermembrane space of human mitochondria.

(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.

(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.

(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.

(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.
Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7 (2019)
Figure 3

Figure 3. COA7 does not influence the MIA pathway.

(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.

(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.

(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.

(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.

(E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.