EMBO/soda-nel huggingface dataset:
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 2

Figure 2. CA10 prevents HS addition to neurexin.

(A-E) HEK293 cells: (A) Schematic outline of the experiment shown in (B). HA-Nrxn1α or HA-Nrxn1α (S>A), a mutant of the modified serine were expressed alone or in combination with V5-tagged CA10 in HEK293 cells. Neurexins were immunoprecipitated for HA and treated with heparinases ('Heps'). (B) Representative immunoblots (of three independent experiments) with antibodies against HA, the 3G10 epitope (for the HS stub) and V5. (C) Schematic outline of the experiment shown in (D). HA-Nrxn1α was expressed alone or in combination with CA10-V5 in HEK293 cells, lysates were immunoprecipitated for HA (for Nrxn1α) or V5 (for CA10). (D) Representative immunoblots (of three independent experiments) analyzed as described for (B). (E) Schematic illustration of how endogenous neurexin-HS was analyzed. HS-modified α-neurexin migrates at higher apparent molecular weight (HMW) than non-modified low molecular weight (LMW; white arrowheads) species (left). Following treatment with heparinases (right), both HS-modified and non-modified α-neurexin migrates as LMW species, but HS-modified α-neurexin can be detected by the 3G10 monoclonal.

(F-H) Primary cortical neurons: Cortical neurons were infected with control lentivirus (empty vector) or lentivirus expressing FLAG-tagged CA10, and cell lysates subjected to immunoprecipitation for neurexin and heparinase treatment. (F) Samples were analyzed by immunoblotting using antibodies against neurexins, 3G10 (for the HS stub) and FLAG (for CA10). Stars indicate non-neurexin bands (see results). (G) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (H) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests (n=3).

(I-K) Mouse brains: Neurexins and CA10 were immunoprecipitated from total mouse brain lysates and subject to heparinase treatment. (I) Samples were analyzed by immunoblotting using antibodies against neurexins, the 3G10 epitope (for the HS stub) and CA10. Stars indicate non-neurexin bands (see results). (J) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total neurexin amounts [upper band (line) + LMW]. Data shown as mean ±SEM (n=3 independent experiments); *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests. (K) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Data shown as mean ±SEM of 3 biological replicates. *, p< 0.05 by 2-way ANOVA and Holm-Sidak tests.
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 3

Figure 3. A direct CA10-neurexin interaction is required to prevent neurexin HS addition, and a non-covalent interaction is sufficient.

(A) Sequences of the Nrxn1β mutants used, color-coded as in Fig 1A, with residues previously mapped to bind CA10 indicated. Right, table summarizing properties of the recombinant variants: 'HS?' indicates if HS-modified when expressed alone (Fig 1B), 'Binds CA10?', indicates if found to bind CA10, according to (et al,Sterky 2017), and whether the HS modification is blocked by CA10 (this figure). Asterisk denotes non-covalent interaction (see discussion).

(B) Representative immunoblot of the indicated proteins harvested from HEK293 cell media and subjected to heparinase treatment ('Heps'). Samples were analyzed by reducing SDS/PAGE and immunoblotting with antibodies against Fc (for Nrxn1β), the 3G10 epitope (for the HS stub) and V5 (for CA10). Input samples were analyzed separately for CA10 to ensure equal expression (lower panel).

(C) Quantification of the 3G10 epitope signal, normalized to Fc (Nrxn1β). Data shown are mean ±SEM of 3 independent replicates. *, p< 0.05; by Mann-Whitney tests comparing each Nrxn1 mutant with and without CA10 (n=3). No significance difference was observed for S&T>G (p=0.1143), GIGG (p=0.9714) or DIRR (p=0.4857) variants.

(D-H) Fc-tagged (secreted) Nrxn1β and HA-tagged (transmembrane) Nrxn1α were co-expressed with CA10 and a CA10 mutant for the reactive cysteine CA10(C310A). (D) Summary of CA10 variants used, and their properties (this figure). Asterisk denotes non-covalent interaction (see discussion). Panels (E) and (G) show representative immunoblots analyzed using antibodies against the Fc-tag (for Nrxn1β-Fc) or the HA tag (Nrxn1α-HA), respectively, the 3G10 epitope (for the HS stub) and V5 (CA10-V5), as indicated. CA10 in input samples is shown for demonstration of equal expression. (F, H) Quantification of the 3G10 epitope signal normalized to Fc (for Nrxn1β-Fc) or HA (for Nrxn1α-HA) signals, respectively. Data shown are mean ±SEM of 3 biological replicates. *, p< 0.05; ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests.
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 4

Figure 4. Analysis of the Nrxn1 stalk and the neurexin-CA10 interaction by NMR. (A) Sequence of the Nrxn1 peptide used for the analysis. Residues previously mapped to bind CA10 (et al,Sterky 2017) and the site where alternative splice site 5 (SS5; et al,Ushkaryov 1992) inserts three residues (GGL) are indicated.

(B) Solution structure of the Nrxn1 stalk, with cysteines labeled yellow. The 10 models of lowest energy with respect to the target function are shown (ensemble average RMSD 2.95 Å).

(C) Chemical shift changes induced by CA10, qualitative mapped onto the Nrxn1 solution structure. Residues with the strongest changes are labeled.

(D) Comparison of 1H-13C HSQC spectra of Nrxn1 stalk (red) and Nrxn1 stalk/CA10 (black) at a 2:1 molar ratio (50 mM phosphate buffer, pH 7.4, 0.5 mM d10-DTT, 10% D2O). Nrxn1 residues with stronger chemical shift changes are indicated blue boxes. Asterisk (*) indicates an impurity from glycerol.
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 5

Figure 5. CA10 prevents neurexin xylosylation.

(A) Schematic representation of the intermediate steps of HS biosynthesis. Heparinase cleavage and the neo-epitope detected by the 3G10 antibody following cleavage et al,(David 1992) are indicated.

(B-C) Extracted ion chromatograms (EIC) of DDILVASAECPSDDE peptides and its glycosylated forms, from Nrxn1β expressed in HEK293 cells. Fragment ion spectra for all observed glycosylated peptides are shown in Fig EV5.

(D) Quantification, expressed as % of total occupancy of each glycosylation form, upon control and CA10 expression conditions. Bar graph shows means ±SEM of 4 biological replicates; *, p<0.05 by Mann-Whitney tests comparing the occupancy of each glycosylation with and without CA10 (n=4 biological replicates).
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 6

Figure 6. Neurexin HS modification is differentially regulated between brain regions.

(A) Immunoblot α-showing neurexin isoform distribution in cortex, hippocampus , midbrain and cerebellum dissected from 8-week-old mice.

(B) Quantification of lower molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ± SEM of 3 independent replicates***, p<0.001 by 2-way ANOVA and Holm-Sidak tests, comparing to levels in cerebellum.

(C) α-Neurexin immunoprecipitated from forebrain (Fb) and cerebellum (Cb) using an anti-pan-neurexin antibody was heparinase-treated ('Heps') and analyzed by immunoblotting with antibodies against neurexin and the 3G10 monoclonal (to detect the HS stub). Stars indicate non-neurexin bands (see results).

(D) Quantification of low molecular weight neurexin (LMW; white arrowhead) in relation to total levels of α-neurexins [upper band (line) + LMW]. Bar graphs show mean ±SEMof 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.

(E) Quantification of the 3G10 signal normalized to the signal of the corresponding neurexin band. Bar graphs show mean ±SEM of 3 biological replicates. ***, p<0.001 by 2-way ANOVA and Holm-Sidak tests comparing forebrain and cerebellum.
CA10 Regulates Neurexin Heparan Sulfate Addition via a Direct Binding in the Secretory Pathway (2021)
Figure 7

Figure 7. CA10 reduces affinity to postsynaptic partners LRRTM2 and neuroligin-1 and attenuates neurexin-induced synapse formation.

(A-B) Cell surface binding to endogenous neurexins. (A) Recombinant Fc-tagged LRRTM2 was added to day 10 hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction. Surface-bound protein was quantified as the Fc signal of and normalized to a staining for endogenous neurexin (n=4 cultures; three fields imaged and averaged per well). Scatchard analysis for LRRTM2 revealed an apparent Kd of 29.5 nM and a Bmax of 0.49 nM for control and Kd 35.3 nM, Bmax 0.28 nM upon expression of CA10; significant (p=0.0006) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4). (B) Increasing amounts of recombinant, Fc-tagged Nlgn1 was added to immature hippocampal neurons expressing CA10 or control (empty vector) by lentiviral transduction, and analyzed as in (A). The apparent Kd for Nlgn1 was 186 nM and Bmax 1.65 nM for control and Kd 223 nM, Bmax 1.49 nM when CA10 was overexpressed. The difference was not significant (p=0.20) by non-linear regression. Data shown as mean ± SEM of biological replicates (n=4).

(C-D) Artificial synapse formation assays. (C) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured with HEK293 cells expressing GFP, Nlgn1-YFP, LRRTM2-YFP or TrkC-YFP. Cells were stained with antibodies against the synaptic marker SV2 (red) and somatodendritic MAP2 (blue); GFP/YFP is shown in green. Scale bar 20 μm. (D) Quantifications of SV2 signal overlapping GFP/YFP-positive cells. Data shown as means ±SEM with the n, reflecting number of independent cover slips analyzed, indicated in each bar with the total number of cells imaged in parenthesis. Comparisons were made by one-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups (GFP condition was omitted from analysis due to unequal variance; ***, p<0.001.

(E-F) Analysis of cerebellin(Cbln)-induced artificial synapse formation. (E) Confocal images of hippocampal neurons transduced to express CA10-FLAG or control (empty vector) and co-cultured exposed to anti-V5-coupled beads pre-incubated with recombinant V5-tagged Cbln1 or empty anti-V5 beads. Cells were stained with antibodies against the synaptic marker synapsin (green), somatodendritic MAP2 (blue); beads are colored red and indicated by arrowheads. Scale bar 10 μm. (F) Quantifications of the synapsin signal overlapping red beads. Data shown as means ±SEM (n=3 independent experiments, each data point corresponding to 58-171 analyzed beads), with comparisons made by two-way ANOVA with Holm-Sidak's posthoc test for multiple comparisons between the indicated groups.
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules (2016)
Figure 1

Figure 1. CITK controls mitotic spindle orientation in developing mouse and fly brains.

(A) WT and CITK -/- embryonic (E14.5) mouse cerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.

(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.

(C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2.

(D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype).

(E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm.

(F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype).

(G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.

(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).

Data shown in histograms are means ± SEM, statistical significance was assessed using a two tails Student T-test. ** P < 0.01; * P < 0.05.
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules (2016)
Figure 2

Figure 2. CITK controls mitotic spindle orientation in HeLa cells.

(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.

(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.

(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments).

(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.

(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).

(F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).

Scale bars, 5μm. Data shown in histograms are means ± SEM. Statistical significance was assessed using a two tails Student T-test. *** P < 0.001, ** P < 0.01.
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules (2016)
Figure 3

Figure 3. CITK is associated with mitotic spindle poles through ASPM.

(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm

(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm

(C) Western blot of centrosome-containing fractions from HeLa cells, showing that CITK co-purifies with γ‑tubulin.

(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).

(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).

(F) Close physical proximity between CITK and ASPM, revealed by PLA on ASPM-GFP-expressing HeLa cells, incubated with GFP and CITK antibodies and developed with PLA-specific secondary reagents.

(G) Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red) and DNA (blue).

(H) Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red) and DNA (blue).

(I) Quantification of CITK positive centrosomes and of the ratio of CITK spindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells. (n ≥ 75 cells, 4 independent experiments).

Data presented are means ± SEM. *** P < 0.001; ** P < 0.01. Statistical significance was assessed using a two tails Student T-test. All cell images represent maximum intensity projections of confocal z-stacks. Scale bars, 5μm.
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules (2016)
Figure 4

Figure 4. CITK regulates astral MT and spindle orientation downstream of ASPM and through a kinase-dependent mechanism.

(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.

(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments).

(C) Quantification of the rescue of astral MT number after CITK overexpression in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).

(D) Quantification of spindle orientation rescue after overexpression of CITK, CITN (lacking the kinase domain) or CKD (Citron kinase dead) mutants in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).

(E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).

Data presented in (B), (C), (D) and (E) are means ± SEM. Statistical significance was assessed using a two tails Student T-test. *** P < 0.001. All cell images represent maximum intensity projections of confocal z-stacks. Scale bars, 5μm.
ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules (2016)
Figure 5

Figure 5. Loss of CITK decreases astral MT length, stability and nucleation.

(A) Exemplar image of EB3–tdTomato HeLa cell used for kymograph analyses. To assess astral MT number and dynamics a line (blue) was added to images from the pole to the cortex.

(B) Kymographs of pole (bottom)-to-cortex (top) line scans of control and CITK-depleted EB3–tdTomato HeLa cells. The maximum projection of a 20-pixel-wide line (exemplified above) was plotted at 500 ms time intervals from left to right. MT growth results in diagonal lines.

(C) Quantification of astral MT dynamics. MT stability, length, growth speed (n > 250 MT, 30 cells) and nucleation frequency were calculated (n > 30 cells, 3 independent experiments).

(D) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0, 1, and 2 min after nocodazole washout. Control and CITK-depleted cells were immunostained for α-tubulin (red) and DNA (blue).

(E) Percentage of cells showing detectable MT nucleation (aster size > 1 µm) 0, 1, and 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).

(F) Quantification of aster size 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).

Data are presented as mean ± SEM. Statistical significance was assessed using a two tails Student T-test. ** P < 0.01; *** P < 0.001. Scale bars, 5 μm.
Cell softness regulates tumorigenicity and stemness of cancer cells (2020)
Figure 3

Figure 3. Tumorigenic cells are characterized by mechanical softness.

(A) The cellular stiffness of ALDH- and ALDH+ 4T1 or MCF-7 cells sorted by flow cytometry were determined by AFM. n = 100.

(B) The stiff or soft cells were isolated from ALDH- or ALDH+ 4T1 cells by microfluidic chip. Then, these cells were injected into the mammary fat pads of NSG or BALB/c mice mice (100 cells/mouse). Twelve weeks later, the tumor formation was recorded. n = 10.

(C, D) The 100 stiff ALDH+ or soft ALDH- 4T1 cells were injected into the mammary fat pads of NSG mice. Eight weeks later, the lung sections were H&E stained. The lung metastatic mice were counted (C) and the metastasis index was calculated (D). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor.

(E) The side-population (SP) or non-SP (NSP) from 4T1 or MCF-7 cells were sorted by flow cytometry, and then the cellular stiffness of those cells were measured by AFM. n = 100.

(F) The 100 soft NSP or stiff SP- 4T1, MCF-7, B16 or MP-1 cells were injected into the mammary fat pads (4T1and MCF-7) or subcutaneous tissue (B16 and MP-1) of NSG mice. The tumor formation in the lungs was recorded.

(G, H) The 100 soft NSP or stiff SP- 4T1 or MP-1 cells were injected into the mammary fat pads (for 4T1) of NSG mice or by tail vein into NSG mice (for MP-1). The tumor formation in the lungs was recorded (G). Six to eight weeks later, mice were sacrificed and the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (H). Scale bar, 0.5 mm. n = 5 mice with metastatic tumor.

Data information: Mann-Whitney test (A and E), or two-tailed Paired Student's t test (D and H). The data represent mean ± SD.
Cell softness regulates tumorigenicity and stemness of cancer cells (2020)
Figure 5

Figure 5. BCL9L is the key mediator to regulate the stemness and tumorigenicity of soft tumor cells.

(A) The expression of BCL9L or β-catenin in stiff or soft 4T1, MCF-7, B16 or MP-1 cells was determined by western blot.

(B) The expression of β-catenin and BCL9L from SGGFP, BCL9L-SGs-4T1, MCF-7, B16 or MP-1 cells were determined by western blot.

(C) 500 soft SGGFP or BCL9L-SGs- 4T1, MCF-7, B16-F1 or MP-1 cells were seeded in a 90 Pa soft 3D fibrin gel for 6 days. The number of formed colonies was counted. n = 10.

(D) The soft SGGFP or BCL9L-SGs- 4T1, MCF-7, B16 or MP-1 cells (100 cells) were injected into the mammary fat pads (4T1 and MCF-7) or subcutaneous tissue (B16 and MP-1) of NSG mice. Tumor formation was recorded. n = 10 or 12 mice as indicated.

(E, F) The 100 soft SGGFP or BCL9L-SGs- 4T1, B16 or MP-1 cells were injected into the mammary fat pads (4T1) of BALB/c mice or tail vein (B16 and MP-1) of NSG mice. The tumor formation was recorded (E). Six to eight weeks later, the lung sections were H&E stained. The metastatic micronodules in the lung were counted and the metastasis index was calculated (F). Scale bar, 0.5 mm. n = 3 (for B16-F1) or 5 (for MP-1) mice with metastatic tumor.

Data information: Bonferroni test (C and F). The data represent mean ± SD. n = 3 independent experiments in (A and B).