EMBO/soda-nel huggingface dataset:
Diverse roles of TssA-like proteins in the assembly of bacterial Type VI secretion systems. (2019)
Figure 4

Figure 4. TagAVC localization and influence on sheath stability

A. The ∆tagA mutant displays long and bent but still dynamic structures. Scale bar: 2 µm.

B+C. Frames from time lapse movies showing sheath dynamics in the ∆tagA mutant. Sheath polymerization does not stop when the distal end reaches opposing membrane (white arrow). Continued polymerization causes the sheath to tilt and distal end to move towards the bacterial cell pole, ultimately effecting bending of the whole structure. C. Sheath detaches from its membrane anchor (orange arrow), keeps extending to maximum cell length and then contracts. Scale bars: 1 µm.

D. Time lapse series of T6SS activity in tagged strain (VipA-mCherry2 TagAVC-mNeonGreen). Orange arrow indicates sheath distal end, white arrow indicates initiation site of another T6SS sheath. Scale bar: 1 µm.

E-H. Kymographs of dynamics observed for TagAVC. E. TagAVC localizes at the distal end when sheath reaches the cell periphery. Signal of TagAVC-mNeonGreen disappeared and reappeared on this site repeatedly. F. TagAVC localizes at the distal end when sheath reaches the cell periphery and stays associated to this site until contraction. G. TagAVC briefly localizes at T6SS initiation site. H. TagAVC briefly localizes at T6SS initiation site and at distal end, where it stays attached after contraction.
Diverse roles of TssA-like proteins in the assembly of bacterial Type VI secretion systems. (2019)
Figure 5

Figure 5. TssA1PA localizes at T6SS initiation sites in P. aeruginosa PAO1

A. H1-T6SS dynamics in tagged parental strain (∆retS TssB1-mCherry2, referred to as WT H1) and ΔtssA1PA strain. Blue arrow indicates extending and contracting T6SS sheath structures, white arrow points to dynamic sheath spots. Scale bars: 2 µm.

B. Fluorescence microscopy of T6SS dynamics in ∆retS TssB1-mCherry2 strain harboring pPSV35 plasmid with TssA1PA-mNeonGreen fusion. Scale bar: 2 µm.

C-E. Kymographs of TssB1 and TssA1PA dynamics. TssA1PA localizes exclusively at sheath initiation site (B-E), disappears after contraction (C,D) or remains in place for longer timeframe (C,E). TssA1PA signal appears in neighboring cell shortly after T6SS attack (B,C).
Capturing protein communities by structural proteomics in a thermophilic eukaryote (2017)
Figure 4

Fig. 4 Higher-order assemblies identified by proteome-wide cross-linking mass spectrometry, biomolecular modeling and negative-stain electron microscopy. (A) Distance distributions of identified cross-links on top of the modeled protein complexes and identification of novel interactions. Satisfied distances are shown in blue and over-length cross-links are shown in red. (B) Negatively stained electron micrographs of fractions 3-7 and 27 directly derived from size exclusion chromatography showing the structural signatures and their structural integrity within the fractions. Decreasing molecular weight correlates with increased protein concentration as a function of protein complex elution is highlighted. (C) Abundance profiles as determined by quantitative mass spectrometry correlate with the number of observed single particles of the corresponding structural signature within the negative-staining electron micrographs; shown for fatty acid synthase, 20S proteasome, 60S and 40S ribosome (the number of particles per image per fraction is indicated below the class averages). (D) Simplification of lysate (collecting only the flow-through from anion exchange chromatography) prior to SEC separation allows class-averaging of structural signatures from complex fractions that were previously too low abundant. Proteins identified in this simplified fraction are found in Table S4.
Capturing protein communities by structural proteomics in a thermophilic eukaryote (2017)
Figure 5

Fig. 5 Visualization of transient interactions in fatty acid synthesis. (A) Communities in fatty acid metabolism and the quantification of intra-community distances within cryo-electron micrographs. Fatty acid synthase (FAS) frequently interacts with other sizeable protein complexes in a linear 'pearl-string-like' arrangement and usually localizes at the edges of the community. Scale bars correspond to 25 nm. FAS particles (circles) and their nearest neighbors (arrow heads) are indicated. (B) Additional density outside of the ctFAS dome is observed in ~10% of the single particles; 2D class averages shown at the bottom. (C) Related to (A). Calculation of minimum distances between pairs of FAS molecules as well as FAS molecules and their closest non-FAS neighbors in comparison to random distributions. Whereas FAS molecules are randomly distributed, their binders are not, confirming physical interactions. Supervised picking means that all single-particles were manually picked from the images. Randomized distance means that these manually picked particles were assigned random coordinates in each image (randomization of x,y coordinates considering image borders) and then their distance is calculated. (D) Cross-linking mass spectrometry data show that the binder is a carboxylase that is bound to the malonyl transacylase domain and acyl carrier protein (ACP) is in the vicinity, considering cross-link length and the positions of the lysine on the ctFAS structure. Cross-links come from both affinity-purified and fractionated cell extracts. (E) The molecular mechanisms in fatty acid synthesis (Wakil et al, 1983), and the relevance of the position of the ACP (see Fig. 6 for details) and carboxylase to the catalytic cycle is indicated (see text). ACP, acyl carrier protein; CoA, acetyl-Coenzyme A; MPT, malonyl/palmitoyl transferase; KS, ketoacyl synthase; KR, ketoacyl reductase; DH, dehydratase; ER, enoyl reductase; AT, acetyltransferase.
Capturing protein communities by structural proteomics in a thermophilic eukaryote (2017)
Figure 6

Fig. 6 CryoEM structure of fatty acid synthase resolved to 4.7 Å as obtained from cryo electron micrographs of fraction numbers 7-9. (A) The cryoEM of C. thermophilum fatty acid synthase (ctFAS) map is shown isosurface rendered and superimposed with the fitted X-ray structure of yeast FAS (Jenni et al, 2007). The domes and the cap show the unambiguous fit of α-helices and β-strands. (B) A slice through the central wheel of fungal FAS. The pitch of α-helices is resolved. (C) As for (B) but sliced through the dome structure. (D) Location of acyl carrier protein (ACP) within the cryoEM map of ctFAS and the position of additional density outside the dome. (E) Fit of ACP in the cryoEM map of ctFAS and comparison with the crystallographically determined location in yeast FAS; additional density within the active site, possibly resembling the acyl chain bound on the ACP is observed. (F) Molecular model of the interaction between the ACP and the ER domains of ctFAS in cartoon representation. The model was derived from a rigid fit from (B) and subsequently flexibly refined for clash removal and interface energetics optimization.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 1

Figure 1. CARD14 and CARD14sh induce NF-B signaling, IL-8 secretion and JNK/p38 MAP kinase activation.

HEK293T were transfected with NF-B luciferase reporter plasmid and FLAG-tagged full-length CARD14 (FL) or CARD14sh (short) at the indicated concentrations and analyzed 24 h later. (A) NF-B reporter gene expression. (B) IL-8 secretion. Values are the mean of triplicates ± s.e. P-values are compared against empty vector treatment. Significance levels, ns P>0.05, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Unless otherwise stated, p-values are p<0.001. (C) Immunoblotting for c-Jun, phospho-c-Jun, JNK, phospho-JNK, p38 and phospho-p38 as indicated. Actin was used as a loading control. Data are representative of two independent experiments.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 2

Figure 2. MALT1 interacts with CARD14 and enables CARD14 signaling, which are enhanced by pathogenic CARD14 mutations.

(A) MALT1 interacts with CARD14sh in a BCL10-dependent manner. HEK293T cells were transfected with FLAG-CARD14sh, Myc-MALT1 and FLAG-BCL10 as indicated. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1. CARD14sh and BCL10 co-immunoprecipitation with MALT1 was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-myc. The closed arrowhead shows the position of CARD14, the double arrowhead shows the position of BCL10. The asterisk indicates a non-specific band. Total expression levels of transfected proteins are shown by immunoblotting of a fraction of the cell lysates with the indicated antibodies (bottom panel). (B) MALT1 co-expression potentiates CARD14sh-induced JNK and p38 MAP kinase activation. HEK293T cells were transfected with the indicated concentrations of FLAG-tagged CARD14sh, with or without MALT1 (30ng). Transfection with FLAG-TRAF6 and FLAG-CARD11(L232LI) were used as a positive control. c-Jun accumulation, JNK and p38 phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control. (C) MALT1 co-expression potentiates NF-B activation by pathogenic CARD14 mutants. HEK293T were transfected with NF-B reporter plasmid and wild-type (WT) FLAG-CARD14 or the indicated psoriasis-associated CARD14 mutants (15ng), with or without MALT1 (15ng). Luciferase activity in cell lysates was measured after 24 h. Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.01 and ***P<0.001 by student t test. Expression of transfected proteins was verified by western blotting (bottom panel). (D) CARD14(E138A) mutation enhances MALT1 binding. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14sh or the indicated CARD14sh mutants and Myc-MALT1. 24 h later, MALT1 was immunoprecipitated (IP) from cell lysates with anti-MALT1 and CARD14 co-immunoprecipitation was detected by immunoblotting with anti-FLAG. Immunoprecipitated MALT1 was detected by immunoblotting with anti-MALT1. The asterisk indicates a non-specific band. Immunoprecipitation with a non-relevant antibody (rIgG) was used as a negative control (first lane). Total expression levels of transfected proteins are shown by immunoblotting of total cell lysates (bottom panel). Data are representative of two independent experiments.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 3

Figure 3. MALT1 deficiency prevents CARD14sh-induced NF-B/JNK signaling and inflammatory gene expression.

(A) MALT1 expression is essential for CARD14sh-induced NF-B activation. MALT1-deficient HEK293T cells were transfected with NF-B reporter plasmid and the indicated expression plasmids. Luciferase activity in cell lysates was measured 24 h later. CARD11(L232LI) and TRAF6 transfection were used as MALT1-dependent and MALT1-independent controls, respectively. Values are the mean of triplicates ± s.e. Expression of the transfected plasmids was verified by immunoblotting (lower panel). (B) MALT1 expression is essential for CARD14sh-induced JNK activation. MALT1-deficient HEK293T cells were transfected with FLAG-CARD14sh (30ng) either alone or together with either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A) as indicated. c-Jun accumulation and JNK phosphorylation were analyzed by immunoblotting with the antibodies indicated. Actin was used as a loading control. (C) MALT1 deficiency inhibits CARD14-induced gene expression in HaCaT keratinocytes. HaCaT cells were transfected with scrambled (scr) or MALT1-targeting siRNA prior to doxycycline-induced CARD14 expression as described in materials and methods. IL-8 and MCP-1 concentration in the cell supernatants was measured 8 h (IL-8) or 24 h (MCP-1) later by ELISA. Values are the mean of triplicates ± s.e. Doxycycline-induced CARD14 expression and MALT1 knock-down were verified by immunoblotting (right panel). Data are representative of two independent experiments.

Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 4

Figure 4. CARD14 signaling activates MALT1 proteolytic activity.

(A) CARD14 signaling induces CYLD and A20 cleavage. HEK293T cells were transfected with different concentrations of FLAG-CARD14, with or without MALT1 as indicated. CARD11(L232LI) transfection was used as a positive control. Endogenous CYLD and A20 processing was analyzed by immunoblotting. CYLD and A20 cleavage fragments are indicated by an arrow. (B) CARD14 signaling induces CYLD cleavage after R324. HEK293T cells were transfected with different concentrations of FLAG-CARD14sh and either wild-type (CYLDWT) or non-cleavable CYLD (CYLDR324A). CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow. (C) Expression of catalytically active MALT1 is necessary for CARD14-induced CYLD cleavage. MALT1-deficient HEK293T cells in which MALT1 expression was restored by transfection with different concentrations of either wild-type (MALT1WT) or catalytically inactive MALT1 (MALT1C/A), were co-transfected with FLAG-CARD14sh and CYLD as indicated. CYLD processing was analyzed by immunoblotting. The CYLD cleavage fragment is indicated by an arrow. (D) Psoriasis-associated CARD14 mutation enhances activation of MALT1 proteolytic activity. HEK293T cells were transfected with wild-type (WT) FLAG-CARD14 or different psoriasis-associated CARD14 mutants (20ng), with or without MALT1 (20 ng) as indicated. Endogenous CYLD and A20 processing, as well as IκBα phosphorylation, were analyzed by immunoblotting. The CYLD and A20 cleavage fragments are indicated by an arrow. (E) Pharmacological inhibition of MALT1 proteolytic activity prevents CARD14-induced gene expression in HaCaT keratinocytes. CARD14 expression was induced by doxycycline (dox) treatment in the absence or presence of different concentrations of mepazine as indicated. The next morning, cell culture medium was refreshed and IL-8 and MCP-1 concentration in the cell supernatant was measured 8 h later by ELISA. TNF stimulation (MALT1-independent) was used as a negative control. Values are the mean of triplicates ± s.e. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 5

Figure 5. CARD14sh-induced NF-B signaling is negatively regulated by CYLD, A20 and ABIN1.

(A-B) CYLD and A20 inhibit CARD14sh-induced NF-B activation. HEK293T cells were transfected with NF-B reporter plasmid and FLAG-tagged CARD14sh (30ng), with or without the indicated concentrations of CYLD (A) or A20 (B). Luciferase activity in cell lysates was measured 24 h later. (C) A20 and ABIN-1 inhibit CARD14-induced NF-B activation. HEK293T cells were transfected with NF-B reporter plasmid, A20 (3 ng) or ABIN1 (15ng) and the indicated concentrations of FLAG-tagged CARD14sh. Luciferase activity in cell lysates was measured 24 h later. Values are the mean of triplicates ± s.e. Protein expression of transfected plasmids was verified by western blot and is shown in the accompanying panels. Closed arrowheads indicate full length A20 and CYLD; arrows indicate cleaved A20 and CYLD. Data are representative of three independent experiments. (D) A20 inhibits CARD14-induced NF-B activation through its ZnF domains. HEK293T cells were transfected with NF-B reporter plasmid, FLAG-CARD14sh (20ng) and wild-type (WT) or mutant A20 expression plasmids (3ng) as indicated. Luciferase activity in cell lysates was measured after 24 h. CARD14 and A20 expression was measured by immunoblotting (bottom panel). Data are representative of two independent experiments.

Values are the mean of triplicates ± s.e. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test. In panel D, p-values are compared against the ‘CARD14 only’ set up.
The paracaspase MALT1 mediates CARD14-induced signaling in keratinocytes (2016)
Figure 6

Figure 6. Pathogenic mutant CARD14-induced gene expression and A20 cleavage in human primary keratinocytes is MALT1-dependent.

(A) CARD14(E138A) induces cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with empty vector (EV) or CARD14(E138A) as indicated. 24 h later, the secretion of 36 different human cytokines, chemokines, and acute phase proteins was analyzed via a multiplex antibody array. Quantification was done using ImageJ software and is expressed as relative CARD14/EV values for a selected number of proteins (bottom panel). (B) MALT1 deficiency inhibits CARD14(E138A)-induced cytokine and chemokine expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14(E138A). After 24 h, cytokine and chemokine expression was analyzed and is expressed as described in (A). (C) Pathogenic CARD14 mutation enhances MALT1-dependent CARD14-induced IL-8 expression in human primary keratinocytes. Cells were transfected with scrambled (scr) or MALT1-targeting siRNA as indicated, 48 h later followed by transfection with CARD14 wild-type (WT), CARD14(G117S) or CARD14(E138A) as indicated. TNF treatment was used as a control. After 24 h, IL-8 concentration in the cell supernatant was analyzed via ELISA. Values are the mean of triplicates ± s.e. Expression levels of MALT1 and CARD14 variants is verified by immunoblotting (bottom panel). (D) Pharmacological inhibition of MALT1 proteolytic activity inhibits CARD14-induced IL-8 production and A20 processing in human primary keratinocytes. Cells were transfected with different CARD14 variants in the presence or absence of mepazine as indicated. TNF treatment was used as a control. 24 h later, IL-8 levels in the cell supernatant were measured by ELISA. Values are the mean of triplicates ± s.e. Expression of different CARD14 variants and CARD14-induced processing of endogenous A20 were analyzed by immunoblotting. Data are representative of two independent experiments. Significance levels, *P<0.05, **P<0.001, ***P<0.001 by student t test.
Downregulation of basal myosin-II is required for cell shape changes and tissue invagination (2018)
Figure 2

Figure 2. Stabilization of basal myosin-II inhibits ventral furrow formation despite normal apical myosin-II accumulation.

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm.

(A) Top view showing apical myosin-II 10 min after the initial activation. While cells in the non-activated region have internalized and disappeared from the apical focal planes (right), cells in the activated region failed to internalize despite showing myosin-II accumulation at the apical surface (left). Red and Blue dotted lines indicate the location of the cross-section panels displayed in (C) and (D).

(B) Quantification of apical myosin-II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin-II accumulation as the average apical myosin-II level per cell did not significantly differ between the non-activated (NR) and activated region (AR). The intensity values were normalized using the median value of the combined cell population in the NR and AR. (N = 3 embryos; nNR: 94, nAR: 78). Significances (by two-sample t-Test): n.s. indicates no statistically significant differences. In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point and the '+' symbol indicates an outlier.

(C, D) z-projections (transverse cross section) of myosin-II time-lapse recordings of the non-activated (C) and activated region (D). While cells in the non-activated region internalized, resulting in a complete closure of the furrow (C), stabilization of basal myosin-II levels prevented invagination (D). Scale bars, 20 µm.
Downregulation of basal myosin-II is required for cell shape changes and tissue invagination (2018)
Figure 5

Figure 5. Engineering a basal-specific optogenetic anchor to enable basal-specific photo-activation in a curved tissue.

(A) Schematic illustration of two alternative approaches to control myosin-II activation in a curved tissue. In order to restrict the region of activation in a curved tissue to the basal surface using a uniformly distributed optogenetic anchor such as the CIBN-pm anchor (left), the photo-activation pattern needs to account for the tissue curvature and the region of activation needs to be corrected in 3-D using adaptive optics and holography. An alternative approach is to engineer an optogenetic anchor that specifically localizes to the cell base. In this scenario, a bigger focal volume can be photo-activated while still resulting in the restricted recruitment of RhoGEF2-CRY2 to the cell base.

(B) Three different proteins, which have been reported to localize to the cell base during early Drosophila development, were expressed as CIBN fusion proteins in different configurations. Bottleneck (CIBN::Bnk::GFP, CIBN::GFP::Bnk), Slam (GFP-CIBN-Slam) and PatJ (PatJPDZ-CIBN::GFP, PatJ::CIBN, PatJ-CIBN::GFP-CAAX).

(C-E) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2-CRY2::mCherry were imaged during late cellularization. (C) z-projections of confocal image stacks show the localization of the respective GFP-tagged optogenetic anchor. Please note that the construct PatJ-CIBN (indicated by asterisk) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2-CRY2::mCh. All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation. Scale bars, 20 μm. (D) A region of interest (right side of the dashed line) in a 10 µm-sized stack was photo-activated using two-photon illumination for 30 s before acquiring the RhoGEF2-CRY2::mCherry signal using confocal microscopy. All anchor proteins were able to recruit RhoGEF2-CRY2::mCherry specifically to the activated region. Scale bars, 10 μm. (E) Although all basal anchors were effective at recruiting RhoGEF2-CRY2::mCherry to the cell base, only PatJ-CIBN::GFP-pm resulted in effective myosin-II recruitment and actomyosin ring constriction.