Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Increased integrin α5 expression correlates with enhanced bacterial adherence and uptake in vinculin -/- cells. (A) Vinculin WT and vinculin -/- cells were seeded on fibronectin coated glass bottom imaging dishes, next day fixed and stained for vinculin expression using α-human vinculin antibody (clone hVIN1). Total amounts of integrins were stained using integrin β1 antibody (clone HMβ1-1). TIRF microscopy was used to assess the localization of vinculin and β1 integrins. Bars represent 10 μm. (B) Equal amounts of protein in the whole cell lysates (WCL) of vinculin WT and vinculin -/- cells were separated by SDS-PAGE and processed for Western blotting using antibodies against talin, FAK, vinculin, paxillin, Src, or β-tubulin as indicated. Representative blots are shown. (C and D) Cells were seeded on gelatine coated 24 well plates and infected with S. aureus or S. carnosus with MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D) , respectively. Values are means ± SEM from 5 independent experiments done in quadruplicate (n=20). Statistical significance of data was assessed by Mann-Whitney test. (E) Cells were stained for surface expression of β1, α5, αv, or β3 integrins and examined by flow cytometry. Data are shown as mean fluorescence intensity (MFI) and are corrected for the background MFI of integrin β1 -/- fibroblasts (β1 integrin staining) or corrected for the MFI of cells stained in the absence of primary antibody (α5, αv, and β3 integrin stainings). Results are shown as mean ± SEM from 3 independent experiments. (F) mRNA levels of β1, α5, αv, or β3 integrin transcripts in vinculin WT or vinculin -/- cells were analyzed by qRT-PCR. Integrin expression was normalized to GAPDH expression and was set to 1 for vinculin WT cells. Bars represent the mean of two independent experiments each performed in triplicate.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Expressing, Imaging, Staining, Microscopy, SDS Page, Western Blot, Infection, Bacteria, MANN-WHITNEY, Flow Cytometry, Fluorescence, Quantitative RT-PCR

Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Transient vinculin overexpression in 293 cells does not interfere with bacterial uptake. (A) 293 cells were transfected with constructs encoding GFP or GFP-vinculin. 48 h post-transfection whole cell lysates (WCLs) were prepared and analyzed for vinculin and β-tubulin expression by immunoblotting. Representative blots are shown. (B) Cells transfected as in (A) were compared for integrin α5 surface expression by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibody and a rhodamine-coupled secondary antibody (blue – GFP; red – GFP-vinculin) or cells stained with the secondary antibody only (grey lines). (C and D) Cells as in (A) were infected with S. aureus and S. carnosus for 2 h (MOI 20). Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (C) or gentamicin protection assays (D) , respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical significance testing.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Over Expression, Transfection, Construct, Expressing, Western Blot, Flow Cytometry, Staining, Infection, Bacteria, MANN-WHITNEY

Journal: BMC Cell Biology

Article Title: Integrin-mediated internalization of Staphylococcus aureus does not require vinculin

doi: 10.1186/1471-2121-14-2

Figure Lengend Snippet: Knock-down of vinculin in wildtype fibroblasts does not affect S. aureus uptake. (A) Murine fibroblasts were transduced with lentiviral particles harboring either pLKO.1 or pLKO.1 encoding shRNA against mouse vinculin (pLKO-1 sh-mvinc). WCL of the stably transduced cell lines were analyzed for FAK, vinculin, paxillin, Src and β-tubulin protein expression by Western blotting. Representative blots are shown. (B) Integrin α5 surface expression in cells from (A) was analyzed by flow cytometry. Shown is a representative histogram of cells stained with α-integrin α5 antibodies and a rhodamine-coupled secondary antibody (blue – pLKO.1; red – pLKO.1 sh-mvinc) or cells stained with the secondary antibody only (contr.; grey lines). (C) pLKO.1 or pLKO.1 sh-mvinc cells were seeded on gelatine coated 24 well plates. Next day, cells were infected with S. aureus at MOI 20 for 2 h. Total cell associated or recovered viable intracellular bacteria were determined by bacterial cell adhesion (top) or gentamicin protection assays (bottom), respectively. Values are means ± SEM from 2 independent experiments done in quadruplicate, n=8. Mann-Whitney test was applied for statistical testing.

Article Snippet: Integrin β1 (clone CBL1333F) was obtained from Cymbus Biotechnology (London, UK) and integrin β3 antibody (clone 2C9.G3) from eBioscience (San Diego, CA).

Techniques: Knockdown, Transduction, shRNA, Stable Transfection, Expressing, Western Blot, Flow Cytometry, Staining, Infection, Bacteria, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Domain architecture of an active (open headpiece/extended) integrin α5β1 heterodimer. α5 Subunit is grey; β1 subunit headpiece and leg are, respectively, in shades of blue and green. The localization of three different mAb epitopes, exposed only in the conformationally active β1 subunit, is represented. Epitopes of mAb 12G10 and mAb HUTS4, respectively, lie in the βI domain and hybrid domain of the headpiece, whereas mAb 9EG7 epitope is in the I-EGF2 domain. (B) Confocal immunofluorescence microscopy analysis of the subcellular localization of the three different anti-active β1 integrin mAbs employed to stain fixed ECs. All three mAbs bind to active β1 integrins mainly located within typical elongated fibrillar adhesions. Scale bar 20 µ m; magnification scale bar 10 µ m. (C) Confocal immunofluorescence microscopy analysis of anti-active β1 integrin mAbs localization after 10 min of incubation on living ECs. Anti-I-EGF2 domain mAb 9EG7 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, whereas anti-βI domain mAb 12G10 recognizes active β1 integrins located both outside and inside highly fragmented and tiny adhesions. Similar to mAb 9EG7, the anti-hybrid domain mAb HUTS4 preferentially binds to active β1 integrins located within elongated fibrillar adhesions, hinting that anti-active headpiece mAb-elicited fragmentation specifically depends on βI domain binding. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Selected frames from (top row) and (bottom rows), respectively, illustrating dynamic mAb 9EG7-Alexa Fluor 488 binding to active β1 integrins over time upon live incubation on ECs either in the absence (top row) or in the presence (bottom rows) of mAb 12G10–Alexa Fluor 647. When incubated alone (top row), mAb 9EG7–Alexa Fluor 488 preferentially binds active β1 integrins located within fibrillar adhesions and remains stable over time. When mAb 12G10–Alexa Fluor 647 is pre-incubated on ECs, mAb 9EG7–Alexa Fluor 488 does no longer localize in fibrillar adhesions. Scale bar 20 µ m. (E) Representative g -STED confocal microscopy pictures of anti-active β1 integrin 9EG7 mAb localization after 10-min incubation on living ECs either in the absence (top left panel) or the presence (middle left panel) of 12G10 or TS2/16 (bottom left panel). To thoroughly analyze the morphology of ECM adhesion sites, g -STED confocal images were acquired close to the basal EC surface. 9EG7-labeled adhesions were then analyzed with ImageJ software (right panels) and classified, according to their shape factor (SF), into elongated (red) and round (yellow) structures. 9EG7-labeled adhesions were classified as elongated, if their SF was < 0.5, and round, if the SF was ≥ 0.5. Scale bar 20 µ m. The maximum Feret’s diameter was measured to quantify the morphological features of 9EG7 + elongated structures. Compared with control ECs incubated live with 9EG7 alone, 9EG7 + elongated structures were significantly shortened in the presence of 12G10 or TS2/16. Data are mean ± SD, n ≥ 20 cells per condition pooled from two independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Staining, Incubation, Binding Assay, Confocal Microscopy, Labeling, Software, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Low magnification confocal immunofluorescence microscopy analysis of ECs that were treated live for 10 min with either mAb 9EG7 mAb ( green ) alone or in combination with mAb 12G10 ( red ). Scale bar 100 µ m.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Representative confocal microscopy analysis of SNAKA51 + active α5 integrin (A), β3 integrin (B), and soluble rhodamine-FN (C) localization in ECs that were incubated (bottom panels) or not (top panels) for 10 min with the anti-βI domain mAb 12G10. Scale bar 20 µ m; magnification scale bar 10 µ m. (A, C) When compared with untreated control (CTL) ECs, the mFD of SNAKA51 + fibrillar adhesions or rhodamine-FN + fibrils was significantly reduced in ECs treated with mAb 12G10. Data are mean ± S.D, n ≥ 34 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (B) The incubation of cultured ECs with mAb 12G10 did not influence number, mean area, or mFD of β3 integrin + focal adhesions. Data are mean ± S.D, n ≥ 32 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Representative g -STED confocal microscopy analysis of tensin 1 localization in ECs that were incubated or not for 10 min with the anti-active β1 integrin mAb 9EG7 or mAb 12G10 or the Fab fragment of mAb 12G10 (12G10-Fab) or mAb TS2/16. Scale bar 20 µ m; magnification scale bar 10 µ m. When compared with untreated control (CTL) ECs or those treated with mAb 9EG7, the mFD of tensin 1 + fibrillar adhesions was significantly reduced in ECs treated with either mAb 12G10, 12G10-Fab or mAb TS2/16. Data are mean ± SD, n ≥ 22 cells per condition pooled from three independent experiments. The number of structures was normalized on cell area and on those in control cells. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (B) MAb 9EG7 affects the lifetime of FN–α5β1 bonds. AFM measurement of mAb 9EG7 effect on force-dependent lifetime of single bonds between a FNIII 7–10 fragment and an integrin α5β1-Fc fusion protein. Lifetime versus force plots of α5β1-Fc–functionalized Petri dish dissociating from FNIII 7–10 -coated cantilever tips in Mn 2+ either in the absence (grey) or the presence (green) of 10 µ g/ml mAb 9EG7 mAb. Data are mean ± SEM of several tens to several hundreds of measurements per point. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Control

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A, B, C) Selected frames from (A), (B), and (C), monitoring mAb 12G10 ( red ) binding to active β1 integrins on living: control ECs (A); ECs previously oligofected with tensin-EGFP (B); ECs pre-incubated for 10 min with mAb 9EG7 (C). (A, B, C) As expected, mAb 12G10 mAb binds active β1 integrins located within and outside fragmented adhesion sites (A), but, either when Tensin-EGFP is overexpressed (B) or upon pre-incubation with mAb 9EG7 (C), mAb 12G10 mAb localizes instead within fibrillar adhesions that remain stable over time. Scale bar 20 µ m; magnification scale bar 10 µ m. (D) Representative confocal images showing pre-bleaching, post-bleaching, and recovery on the region of interest (indicated by arrows) of tensin-EGFP–positive fibrillar adhesions in ECs treated with DMSO (as control) or with the FAK inhibitor PF-562271. Scale bar 5 µ m. Recovery rate was measured, and data were normalized by employing as reference the fluorescence intensity acquired on the same ROI before bleaching. Data were then normalized on control DMSO-treated samples. Data are mean ± SD, n ≥ 31 adhesions per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.001 ***. The effectiveness of inhibition of FAK autophosphorylation by PF-562271 was verified by Western blot analysis of EC lysates. (E) Representative confocal microscopy images of anti-active β1 mAb 12G10 ( green ) localization in ECs treated or not with the FAK inhibitor PF-562271; ECs were also stained for auto-phosphorylated FAK on tyrosine 397 (pFAK-Y397, red ). Scale bar 20 µ m; magnification scale bar 10 µ m. Measurement of mFD of 12G10 + adhesions revealed that, compared with control EC incubated with DMSO, 12G10 + adhesive structures are significantly longer in presence of FAK inhibitor PF-562271. Data are mean ± SD, n ≥ 19 cells per condition pooled from three independent experiments. Statistical analysis: unpaired t test, P ≤ 0.0001 ****. (F) Western blot analysis of FAK autophosphorylation (Y397) levels in ECs treated with or without mAb 9EG7 or mAb 12G10 for 2 or 15 min. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Binding Assay, Control, Incubation, Fluorescence, Inhibition, Western Blot, Confocal Microscopy, Staining, Adhesive

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Representative confocal microscopy analysis of the impact of mAb 12G10 on living ECs previously incubated for 10 min either with intact mAb 9EG7–Alexa Fluor 488 (9EG7) or its Fab (9EG7-Fab). Scale bar 10 µ m; magnification scale bar 10 µ m. Upon pre-incubation with intact dimeric 9EG7, but not monomeric 9EG7-Fab, mAb 12G10 localizes within fibrillar adhesions. Quantitative analysis showed that the incubation of cultured ECs with 12G10 shortens the mFD of elongated active β1 integrin + clusters only when cells are pre-incubated with monomeric 9EG7-Fab, but not intact dimeric 9EG7. Data are mean ± S.D, n ≥ 22 cells per condition pooled from three independent experiments. Statistical analysis: one-way ANOVA, P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Confocal Microscopy, Incubation, Cell Culture

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: (A) Confocal immunofluorescence microscopy analysis of ECs live treated with mAb 9EG7 ( green ) alone or in combination with mAb 12G10 ( blue ). The internalization of mAb 9EG7–bound active β1 integrins in EEA1 + early endosomes ( red ) was quantified by Pearson correlation coefficient (PCC). Treatment with the anti-βI domain mAb 12G10 promotes mAb 9EG7–bound active β1 integrin endocytosis. Data are mean ± SD, n ≥ 23 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. (B) Time-course analysis of the relative amounts of endocytosed 9EG7 + active β1 integrins in control (CTL; green ) versus mAb 12G10–treated ( light blue ) ECs, evaluated by internalization and capture ELISA assays. Treating living ECs with the anti-βI domain mAb 12G10 elicits a strong increase in 9EG7 + active β1 integrin endocytosis. Data are mean ± SD, of eight technical replicates per condition pooled from three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.0001 ****. (C, D) Real-time analysis of EC haptotactic migration towards FN (xCELLigence RTCA DP system) either in the absence (CTL) or the presence of anti-active β1 integrin mAb 9EG7 alone (C, D) or mAb12G10 alone (C) or combined mAb 9EG7 and mAb 12G10 (D). Data are mean ± SD, n ≥ 14 technical replicates per condition pooled from four independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis; P ≤ 0.05 * , # ; P ≤ 0.01 ** , ## ; P ≤ 0.001 *** , ### ; P ≤ 0.0001 **** , #### . (C, D) *CTL versus mAb 9EG7; # CTL versus mAb 12G10 (C); or CTL versus mAb 9EG7 + mAb 12G10 (D). Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy, Control, Enzyme-linked Immunosorbent Assay, Migration

Journal: Life Science Alliance

Article Title: The βI domain promotes active β1 integrin clustering into mature adhesion sites

doi: 10.26508/lsa.202201388

Figure Lengend Snippet: Confocal immunofluorescence microscopy analysis of ECs live treated with mAb SNAKA51 ( red ) alone or in combination with mAb 12G10 ( green ). The internalization of mAb SNAKA51–bound active α5 integrins in EEA1 + early endosomes ( blue ) was quantified by Pearson correlation coefficient. Treatment with the anti-βI domain mAb 12G10 promotes mAb SNAKA51–bound active α5 integrin endocytosis. Data are mean ± SD, n ≥ 26 cells per condition pooled from three independent experiments. Scale bar 20 µ m; magnification scale bar 10 µ m. Statistical analysis: unpaired t test; P ≤ 0.0001 ****. Source data are available for this figure.

Article Snippet: Mouse mAbs anti-active β1 integrin clone 12G10 and clone HUTS4 and mouse mAb anti-active α5 integrin clone SNAKA51 were from Merck Millipore.

Techniques: Immunofluorescence, Microscopy

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is necessary for epiblast cell survival upon implantation and pluripotency transition (A–C) Analysis of apoptosis by cleaved caspase-3 staining in wild-type (fl/fl, top) and mutant (Δ/Δ, bottom) mESC spheroids at 24 (A), 48 (B), and 72 h (C) of culture. Steady increase in the number of c-caspase-3 + structures in mutants over time. Fisher’s exact test: (A) p = ns (number of spheroids n = 42 [fl/fl], n = 44 [Δ/Δ]); (B) ∗∗∗∗ p < 0.0001 (n = 38 [fl/fl], n = 48 [Δ/Δ]); and (C) ∗∗∗∗ p < 0.0001 (n = 26 [fl/fl], n = 28 [Δ/Δ]). (D–G) In vivo -recovered post-implantation embryos at E5.5, as shown in Figure S1 D: epiblast is either fl/fl or Δ/Δ; visceral endoderm (VE) and extraembryonic ectoderm (ExE) are +/+. In contrast to wild-type embryos (Epi:fl/fl) (D), embryos deficient for integrin β1 (Epi:Δ/Δ) either have a small epiblast (E), show few residual Oct4 + cells (F), or lack epiblast completely. Quantification of the number of embryos with epiblast versus degenerated/lethal—Fisher’s exact test: ∗∗∗ p = 0.0003 (number of embryos n = 15 [fl/fl], n = 25 [Δ/Δ], 4 replicates). (H–J) Assessment of apoptotic cells in chimeric blastocysts at E4.5–4.75, Epi:fl/fl (H, top) or Epi:Δ/Δ (H, bottom): embryos are sequentially re-stained to assess NANOG cells in the c-caspase-3 channel. Mutant embryos (Epi:Δ/Δ) contain a significantly higher number of apoptotic cells within the epiblast compartment: quantification of the percentage of c-caspase-3 + cells over total epiblast cell number (I), and percentage of apoptotic cells in wild type (median = 1.7%) versus mutants (median = 14%), Mann-Whitney test; ∗∗∗∗ p < 0.0001 (number of embryos n = 37 [fl/fl], n = 29 [Δ/Δ]). Correlation between the percentage of c-caspase-3 + cells with the percentage of either OTX2 (top) or NANOG + cells (bottom) within the epiblast (J): exit from naive pluripotency (increase in OTX2 and decrease in NANOG cells) correlates with the increase in the percentage of c-caspase-3 + cells (number of embryos n = 23 [fl/fl], n = 15 [Δ/Δ]). (K and L) Wild-type and mutant mESCs display comparable minimal levels of apoptotic cells when cultured in 2iLIF (naive pluripotency status, absence of OTX2 expression). (K) Fisher’s exact test: p = ns (number of spheroids n = 33 [fl/fl], n = 37 [Δ/Δ]). In the absence of 2iLIF (L), cells transit to formative pluripotency as shown by the expression of OTX2 and activate the apoptotic pathway in the absence of integrin β1 (Δ/Δ). Scale bars: 5 μm (A), 10 μm (B, C, K, and L), 15 μm (H, inset), 25 μm (H, inset, and D–F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Staining, Mutagenesis, In Vivo, MANN-WHITNEY, Cell Culture, Expressing

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Fine-tune spatial segregation between integrin β1 and actomyosin (A) Configuration of the mouse epiblast before implantation (E4.5): cells are apolar and display heterogenous localization of integrin β1 (Itg-β1) and actomyosin, as shown by the distribution of phalloidin (F-actin) and phosphorylated non-muscle myosin (pMLC-II). (B) Configuration of the mouse epiblast upon implantation (E5.0): segregation in a mutually exclusive manner between integrin β1 and actomyosin while cells acquire a wedge-shape morphology. (C) Configuration of the mouse epiblast at post-implantation (E5.5): cells of the mature epiblast epithelium are columnar in shape surrounding a central pro-amniotic cavity. Integrin β1 localizes on the basolateral domain and actomyosin on the apical side. (D) Integrin β1 and actomyosin are heterogeneously distributed in mouse embryonic stem cells (mESCs) maintained in naive pluripotency (+2iLIF, left panel), similarly to E4.5. Integrin β1 and actomyosin become spatially segregated during formative pluripotency following 72 h of removal of 2iLIF (−2iLIF, right panel), similarly to E5.5. Scale bars: 25 μm (A–D). Magnified/inset areas indicated by arrows (A and B).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques:

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Loss of integrin β1 causes basal actomyosin accumulation and morphogenesis failure (A and B) Distribution of actomyosin in mESC after 24 (A) and 48 h of culture (B). Wild-type cells (fl/fl) display enrichment of F-actin and p-myosin at the central point of the apical membrane initiation site (AMIS) at 24 h (A, top). Apical actomyosin is maintained after 48 h at the center of the rosette configuration (B, top). Mutant cells (Δ/Δ) display ectopic accumulation of actomyosin basally (A, bottom), giving rise to a large basal actomyosin cable after 48 h of culture (B, bottom). (C) Blebs and ectopic E-cadherin junctions appear on the basal domain of mutant mESCs (arrows). (D) Distribution of actomyosin in the mouse blastocyst at E4.5, chimeric for either fl/fl (top) or Δ/Δ (bottom) alleles of Itgβ1 . Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the central point of apical constriction, forming a rosette configuration (top). Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the rosette (bottom). (E) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E4.5 shows significant increase of basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 21 [fl/fl], n = 23 [Δ/Δ]). (F) Assessment of actomyosin localization in post-implantation embryos at E5.5. Wild-type epiblasts (Epi:fl/fl) accumulate actomyosin at the apical side of the epiblast epithelium. Mutant epiblasts (Epi:Δ/Δ) accumulate actomyosin basally and fail to form the central pro-amniotic cavity. (G) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) at E5.5: significant increase in basal myosin in mutant epiblasts. Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). (H) Quantification of percentage of embryos forming cavity in the epiblast at E5.5: all mutant epiblasts fail to form the cavity: Fisher’s exact test: ∗∗∗∗ p = 0.0001 (number of embryos n = 14 [fl/fl], n = 8 [Δ/Δ]). Scale bars: 5 μm (A), 10 μm (B and C), 25 μm (D), and 50 μm (F).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Membrane, Mutagenesis, Fluorescence, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: Integrin β1 is dispensable for the establishment but necessary for the maintenance of apicobasal polarity (A) Distribution of the polarity marker PAR3 in mESCs at 1- and 2-cell stage (24 h of culture in −2iLIF). Wild-type cells (fl/fl, top) recruit PAR3 toward the AMIS similarly to mutant cells (Δ/Δ, bottom). (B) Quantification of the orientation of apicobasal polarity at 24 h: Fisher’s exact test: p = ns (number of mESC spheroids n = 42 [fl/fl], n = 39 [Δ/Δ]). (C and D) Distribution of centrosomes, as shown by γ-tubulin staining, at 2-cell stage and quantification of the angles along the nuclear-centrosome axis. Centrosome-nuclear axis angle: means ± SEMs (red). Test: Mann-Whitney test: p = ns (number of mESC spheroids n = 26 [fl/fl], n = 31 [Δ/Δ]). (E and F) Assessment of polarization in agarose at 24 h by Golgi and PAR3 localization. Fisher’s exact test: p = ns (number of spheroids n = 38 [fl/fl], 38 [Δ/Δ]). Both wild-type and mutant cells display correct apicobasal polarity at 24 h of culture, even in the absence of ECM components. (G) Assessment of polarization at 48 h. PAR6 is recruited at the apical site where actomyosin accumulated in wild type. PAR6 is recruited basally at the site of actomyosin accumulation in mutant cells. (H) In wild type, podocalyxin vesicles are recruited apically at PAR6 site. In mutant, PAR6 localizes basally, and podocalyxin vesicles are secreted basally at this latter site. (I) Quantification of the orientation of apicobasal polarity at 48 h, assessed by Golgi and PAR6 localization. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (n = 30 [fl/fl], n = 29 [Δ/Δ]). Scale bars: 5 μm (A, C, and E) and 10 μm (G and H).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Marker, Mutagenesis, Staining, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vitro rescue of integrin-mediated survival and morphogenesis via FGF/IGF stimulation and ROCK inhibition (A) ROCK inhibition by Y27632 rescues lumen initiation in mESCs deficient for integrin β1 (Δ/Δ, bottom): Golgi is correctly oriented apically, podocalyxin vesicles are secreted apically, polarity marker aPKC is recruited at the apical domain, and actomyosin is re-established apically. Quantification of the percentage of mESC spheroids undergoing lumenogenesis shows no significant differences between wild-type and mutant cells: Fisher’s exact test: p = ns (number of spheroids n = 65 [fl/fl], n = 68 [Δ/Δ]). (B) Assessment of structures showing apoptotic cell death following ROCK inhibition treatment: mutant cells activate apoptosis despite rescue of polarity and lumen formation. Fisher’s exact test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 35 [fl/fl], n = 39 [Δ/Δ]). (C) Stimulation of the survival pathways via supplementation of FGF2, IGF1, and GSK3i is not sufficient to prevent initiation of apoptosis in integrin β1 mutant cells (Δ/Δ): Fisher’s exact test: ∗∗∗ p = 0.0002 (n = number of spheroids = 35 [fl/fl], n = 35 [Δ/Δ]). (D) Inhibition of ROCK coupled to supplementation of FGF2, IGF1, and GSK3i restores both morphogenesis and survival in spheroids deficient for integrin β1 (Δ/Δ, bottom): Fisher’s exact test: p = ns (number of spheroids n = 47 [fl/fl], n = 40 [Δ/Δ]). (E and F) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids cultured for 48 h in medium only shows myosin ectopic localization on the basal side of mutant cells (ratio <1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗∗∗ p < 0.0001 (number of spheroids n = 31 [fl/fl], n = 30 [Δ/Δ]). (G and H) Fluorescence intensity quantification of apicobasal p-myosin (pMLC-II) in mESCs spheroids supplemented with FGF2, IGF1, GSK3i, and ROCKi for 48 h shows re-establishment of myosin in the apical domain in mutant cells (ratio >1). Fluorescence intensity = mean ± SEM, Mann-Whitney test: ∗∗ p = 0.002 (number of spheroids n = 35 [fl/fl], n = 31 [Δ/Δ]). Despite the increase in the ratio of apicobasal p-myosin, mutants still differ significantly from wild types. Scale bars: 10 μm (A–D, F, and H),

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vitro, Inhibition, Marker, Mutagenesis, Fluorescence, Cell Culture, MANN-WHITNEY

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet: In vivo rescue of survival and morphogenesis in mouse embryos and spatial segregation of integrin β1 and actomyosin in the human embryo (A) Culture of mouse embryos from pre- to post-implantation as shown in Figure S3 F: wild-type controls (Epi:fl/fl) develop into post-implantation egg-cylinders (left), mutant epiblasts deficient for integrin β1 (Epi:Δ/Δ) fail to undergo lumenogenesis and to survive during post-implantation development in normal culture conditions (center). Supplementation with ROCKi, FGF2, IGF1, and GSK3i restores lumenogenesis and survival of the epiblast compartment in integrin β1-deficient embryos (right). (B) Quantification of the percentage of embryos undergoing lumenogenesis in wild-type and mutant embryos cultured in normal conditions compared to mutant embryos cultured in the presence of ROCKi, FGF2, IGF1, and GSK3i. Fisher’s exact test: fl/fl versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i p = ns; Δ/Δ medium versus Δ/Δ ROCKi/FGF2/IGF1/GSK3i ∗∗∗∗ p < 0.0001 (number of embryos n = 26 [fl/fl], n = 16 [Δ/Δ], n = 51 [Δ/Δ, ROCKi/FGF2/IGF1/GSK3i]). (C) Schematic summary of results. Wild-type (left): integrin β1-mediated adhesion to the basement membrane leads to suppression of actomyosin basally, allowing its apical localization. Activation of actomyosin at the apical side leads to the apical localization of PAR6 ( Figure 4 G) and secretion of podocalyxin vesicles and initiation of lumenogenesis ( Figure 4 H). Integrin β1 promotes epiblast survival via stimulation of FGF/ERK and IGF1/AKT pathways. Mutant (right): loss of integrin β1 leads to ectopic actomyosin accumulation basally. The basal actomyosin recruits PAR6 basally, which directs podocalyxin vesicles toward the basal side, preventing lumenogenesis. Loss of integrin β1 leads to apoptosis. (D) Assessment of the localization of integrin β1 and actomyosin in human embryos at day 8 d.p.f. shows initiation of spatial segregation between the 2 complexes in a mutually exclusive manner (arrows). At 10 d.p.f., the 2 are fully segregated, with integrin β1 localizing on the basolateral domain, while actomyosin is confined on the apical side of the epiblast epithelium facing the central amniotic cavity. Scale bars: 25 μm (A) and 50 μm (D).

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: In Vivo, Mutagenesis, Cell Culture, Membrane, Activation Assay

Journal: Cell Reports

Article Title: Integrin β1 coordinates survival and morphogenesis of the embryonic lineage upon implantation and pluripotency transition

doi: 10.1016/j.celrep.2021.108834

Figure Lengend Snippet:

Article Snippet: Integrin β1 (for human embryos) , Merck Millipore , MABT821.

Techniques: Recombinant, Software