Journal: Cell Reports

Article Title: The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition

doi: 10.1016/j.celrep.2021.110103

Figure Lengend Snippet:

Article Snippet: Taqman assay: Cav1 , ThermoFisher Scientific , Mm00483057_m1.

Techniques: Blocking Assay, Virus, Recombinant, Modification, Reverse Transcription, TaqMan Assay, DNA Library Preparation, Gene Expression, Control, RNA Sequencing, Mutagenesis, Knock-Out, Software, Membrane

Journal: eLife

Article Title: A synaptic F-actin network controls otoferlin-dependent exocytosis in auditory inner hair cells

doi: 10.7554/eLife.10988

Figure Lengend Snippet: (A) Confocal images from averaged Z-stack projection (20 slices of 0.25 µm) of P13-IHCs labeled in blue with otoferlin-immuno-reactivity. Directly visualized with fluorescent-phalloidin (purple), F-actin intensively labelled the cuticular plate and the stereocilia but also in a punctated manner the synaptic basal pole of the IHCs. In this latter area, at higher magnification (averaged Z-stack projection of 8 slices of 0.25 µm), the synaptic F-actin forms a mesh of cages (see right panel where the blue channel of otoferlin is omitted; the cages are indicated by the white asterisks). At each border of the synaptic F-actin cages was generally attached one synaptic ribbon (red) and one associated Cav1.3 patch (green) as indicated in the lower left panel. The graph represents an example of fluorescent intensity profile through the white dashed line crossing the ribbon and the associated Cav1.3. (B) The graph indicates the Gaussian distribution of the larger axis (double white arrow head) of each F-actin cage. (C) A 45 min treatment with extracellular latrunculin-A disrupted the synaptic F-actin cages. The black holes at the base of the IHCs likely indicated swollen IHC active zones produced by the synaptic F-actin disorganization. At higher magnification (right panel), note also the disorganization of the Cav1.3 clusters (green) at the ribbons, as indicated by a larger distance in their respective fluorescent intensity profile distribution (bottom graph). (D) Comparative Gaussian distribution of the center mass distance between Cav1.3 and ribbon in controls (black, n = 71 active zones) and latrunculin-treated (orange, n = 102 active zones) IHCs. The inset histogram indicates the mean ± SEM distance in both conditions. *p<0.05. DOI: http://dx.doi.org/10.7554/eLife.10988.003

Article Snippet: Synaptic ribbons (CtBP2), Cav1.3 channels and otoferlin were simultaneously labeled with anti-CtBP2 (Goat polyclonal, Santa Cruz, USA; cat # SC-5966), anti-Cav1.3 (Rabbit polyclonal, Alomone labs, Jerusalem, Israel; cat # ACC-005) and anti-otoferlin (Mouse monoclonal, Abcam, Paris, France; cat # ab53233) antibodies, respectively.

Techniques: Labeling, Produced

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A localizes to large macropinocytic cup-like structures before RAB5A recruitment. (See .) (A–C) P16-GFP-CYRI-A in HEK293T (scale bar = 20 µm; n = 204 events in 18 cells for cups/vesicles and n = 24 events in 10 cells for tubules) decorates structures resembling macropinocytic cups (yellow arrowheads, diameter ranging from 0.4 to 2.9 µm; scale bar = 5 µm). Tubule length 0.7–7 µm. Average lifetime of CYRI-A on cups, 50 s ( n = 58 events in 5 cells). Red lines represent the average value. (D) Still images of COS-7 cells (scale bar = 20 µm) expressing P16-GFP-CYRI-A showing the diffuse pool of CYRI-A (yellow doubled arrow) near the leading edge. Dotted square denotes time sequence on the right (scale bar = 5 µm). (E) Time-lapse sequence of COS-7 cells expressing P16-GFP-CYRI-A (cyan) and mCherry-RAB5A (magenta). Scale bar = 20 µm (full size) or 5 µm (zoom). (F and G) Time sequence of CYRI-A and RAB5A recruitment to the macropinocytic cups (CYRI-A only, n = 75 events in 8 cells; CYRI-A with RAB5A, n = 68 events in 8 cells). (H–J) Dextran uptake assay (scale bar = 5 µm). Quantification of the percentage of CYRI-A–positive cups/vesicles containing dextran ( n = 9 cells) and the size (cups/vesicles, n = 15 events in 7 cells; tubules, n = 10 events in 7 cells). Two-tailed unpaired t test.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Expressing, Sequencing, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI proteins localize to macropinocytic structures prior to RAB5 arrival. (See .) (A and B) Representative images of live COS-7 cells expressing P17-GFP-CYRI-B (scale bar = 10 µm). Tubular and vesicular structures are highlighted in zoomed panels, and quantification of their sizes is shown in B (vesicles, n = 16 events in 3 cells; tubules, n = 22 events in 4 cells; scale bar = 5 µm). (C–E) Time sequence of live HEK293T cells (scale bar = 10 µm) coexpressing P16-GFP-CYRI-A (cyan) and mCherry-RAB5A WT (magenta). Arrowhead points to vesicular structures (C; scale bar = 5 µm). The dynamics of each protein is reported by its normalized intensity plot (D) and lifetime ( n = 84 events in 7 cells; E). (F and G) COS-7 cells (scale bar = 10 µm) coexpressing P17-GFP-CYRI-B (cyan) and mCherry-RAB5A WT (magenta). Time sequence corresponding to the white dotted square area is shown in the bottom panel (scale bar = 5 µm). Arrowhead points to tubular and vesicular structures, and intensity profile along the yellow line is plotted in G. (H–M) Time sequence images HEK293T (H) and CHL-1 cells (K) expressing P16-GFP-CYRI-A and incubated with dextran 70 kD (scale bar = 10 µm). Yellow arrowheads indicate macropinocytic events positive for both CYRI-A and dextran signals (scale bar = 5 µm). Quantification showing the majority of CYRI-A–positive vesicles are also dextran-positive in HEK293T (I; 88%, n = 6 cells) and CHL-1 (L; 100%, n = 6 cells) and their sizes (J and M; n = 53 events in 6 cells in HEK293T; n = 57 events in 6 cells in CHL-1). Red line indicates the average value. (N and O) HEK293T cells expressing either GFP control or P16-GFP-CYRI-A and incubated with dextran 70 kD. The size of dextran-positive vesicles in GFP ( n = 163 events in 4 cells) is the same as that of CYRI-A–positive vesicles ( n = 75 events in 7 cells). Events from each cell are color-coded. Unpaired t test. Mean ± SD.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Expressing, Sequencing, Incubation

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A- colocalizes with plasma membrane-associated nascent macropinocytic structures. (See .) (A–D) Time sequence images of COS-7 cells expressing P16-GFP-CYRI-A or P17-GFP-CYRI-B (cyan) and either mCherry-tagged CLC15 (clathrin light chain 15; A and B), Caveolin-1 (C), or ARF1 (D). Scale bar = 10 µm for full-size image and 5 µm for zooms. (E and F) Time sequence images of live COS-7 cells coexpressing either P16-mCherry-CYRI-A WT or P16-mCherry-CYRI-A RRDD mutant (magenta) and P16-GFP-CYRI-A WT (cyan). (G–L) COS-7 cells coexpressing P16-GFP-CYRI-A (cyan) and two independent PIP3 reporters (magenta), PH-Grp1 (G–I) or PH-Btk (J–L; n = 31 events in 3 cells for Grp1; n = 9 events in 1 cell for Btk). Red line represents the average value. Scale bar = 10 µm for full-size image and 5 µm for zooms. (M and N) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mScarlet-Lck (labeling the plasma membrane; magenta). The time Lck resides on the vesicles before CYRI-A is recruited is quantified in N ( n = 48 events in 10 cells). Scale bar = 10 µm for full-size image and 3 µm for zooms. Red line indicates the average value.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Sequencing, Expressing, Mutagenesis, Labeling

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A regulates actin dynamics at macropinocytic structures. (See , , and .) (A) Still images of COS-7 cells coexpressing either GFP (negative control) or P16-GFP-CYRI-A (cyan) and LifeAct-RFP (magenta). Scale bar = 10 µm for full-sized image and 5 µm for zooms. (B) Time sequence images showing the dynamics of P16-GFP-CYRI-A and actin at the macropinocytic structure in COS-7 cells. Graph shows normalized signal intensities over time. Black arrows denote peak actin and CYRI-A signals. Scale bar = 5 µm. (C) Normalized signal intensities over time between P16-GFP-CYRI-A and LifeAct signal in HEK293T cells. (D) Lifetime of actin before and after P16-GFP-CYRI-A is recruited in HEK293T cells (before CYRI-A, n = 25 events in 10 cells; with CYRI-A, n = 34 events in 10 cells). Red lines indicate the average value. (E–G) Lifetime of actin on macropinocytic structures ± expression of P16-GFP-CYRI-A in CYRI DBKD COS-7 cells. Scale bar = 1 µm. Number of actin-positive structures in cells ± P16-GFP-CYRI-A expression ( n = 9 cells; F). Lifetime of the actin signal on macropinocytic structures ± P16-GFP-CYRI-A signal (actin alone, n = 43 events in 9 cells; actin with CYRI-A, n = 33 events in 8 cells). (H) Macropinocytosis assay in siRNA-treated COS-7 cells. Scr, scramble. Black dots are internalized dextran. Black dashed lines indicate the boundary of the cell clusters. Scale bar = 30 µm. (I) Macropinocytic index of H. Data are from at least 10 different fields of view per experiment from a total of three independent experiments (color-coded by experiment). Two-tailed unpaired t test. Mean ± SD. (J and K) Expression of P16-mCherry-CYRI-A in control COS-7 cells, DBKD COS-7 cells, and P16-mCherry-CYRI-A RRDD mutant (non-RAC1 binding mutant) cells showing dextran 70-kD uptake capacity of the cells. Data are from ≥10 different fields of view for a total of three independent experiments. Each experiment is color-coded. Mean ± SD. Kruskal–Wallis test with Dunn’s multiple comparison test. ns, P > 0.05. (L and M) Lifetime of P16-GFP-CYRI-A on macropinosomes ± 1 µM of Latrunculin A (LatA) or Cytochalasin D (CytoD) in COS-7 and HEK293T cells. At least five cells per experiment from three independent experiments (color-coded). Mean ± SD. Mann–Whitney U test.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Negative Control, Sequencing, Expressing, Two Tailed Test, Mutagenesis, Binding Assay, MANN-WHITNEY

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A is recruited to macropinocytic structures by active RAC1. (See .) (A–C) Time sequence images of HEK293T cell coexpressing P16-mCherry-CYRI-A (cyan) and GFP-RAC1 WT (magenta). (B) Normalized signal intensity of RAC1 and CYRI-A at macropinocytic structure. (C) Lifetime of RAC1 signal on the macropinocytic structures ( n = 37 events in 4 cells). Scale bar = 10 µm for full-sized image and 5 µm for zooms. (D and E) Time sequence images of HEK293T cell coexpressing P16-GFP-CYRI-A (cyan) and CFP-PBD (magenta). (E) Normalized signal intensities of CYRI-A and PBD over time. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (F–K) Time sequence images of HEK293T cells coexpressing either WT or RRDD mutant of P16-mCherry-CYRI-A (magenta) with the WT P16-GFP-CYRI-A (cyan; F and I). Colocalization of the signals between the two WT constructs (G) and lack of colocalization between WT and mutant constructs (J). Percentage of colocalization events ( n = 5 cells; H and K). Scale bar = 10 µm for full-sized image and 5 µm for zooms. Two-tailed unpaired t test. Mean ± SEM. **, P < 0.01; ****, P < 0.0001.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Sequencing, Mutagenesis, Construct, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRI-A’s recruitment to macropinocytic structures is dependent on PI3K signaling. (See .) (A–F) HEK293T cells were cotransfected with P16-mCherry-CYRI-A (cyan) and either GFP-PH-Grp1 (magenta) or GFP-PH-Btk (magenta) as specific markers for PIP3 (A and D). Line graphs show the sequential events between PIP3 reporters and CYRI-A (B and E). Black arrows indicate the peaks of each normalized signal. Scatter plots show the average lifetime of PIP3 reporter signal before CYRI-A is recruited to macropinocytic structures (Grp1, n = 9 events in 3 cells; Btk1, n = 57 events in 6 cells). Red lines represent the average value. Scale bar = 10 µm for full-sized images and 5 µm for zooms. (G and H) Time sequence images showing COS-7 cells expressing P16-GFP-CYRI-A before and after the addition of 20 µM of LY294002. Quantification shows a significant decrease in the number of P16-GFP-CYRI-A–positive cups/vesicles formed upon PI3K inhibition ( n = 9 cells). Scale bar = 10 µm. Statistical analysis using paired t test.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Sequencing, Expressing, Inhibition

Journal: The Journal of Cell Biology

Article Title: CYRI-A limits invasive migration through macropinosome formation and integrin uptake regulation

doi: 10.1083/jcb.202012114

Figure Lengend Snippet: CYRIs affect integrin α5β1 trafficking. (See .) (A and B) Flow cytometry analysis of surface expression of active integrin α5, detected using the SNAKA51 antibody (A) and MT1MMP (B) comparing the control pLKO and CYRI-A/B DBKO A-673 cells. Data from three independent experiments. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. (C–E) Immunofluorescence images of the control pLKO and DBKO A-673 cells stained for active integrin α5 (magenta) and actin (cyan). The average area of integrin clusters or the number of clusters per cell are quantified in D and E. Data from three independent experiments with at least 10 cells per experiment. Each experiment is color-coded. Mean ± SD. Statistical analysis use one-way ANOVA with Tukey’s multiple comparison test. Scale bars = 20 µm. (F–H) Non-RAC1-binding mutant P16-mCherry-CYRI-A RRDD does not rescue the spreading phenotype of CYRI-B KO COS-7. Quantification of the cell spread area (G) and the Arp2/3 signal accumulating at the cell periphery (H) show that WT CYRI-A rescued these phenotypes in CYRI-B KO COS-7, while RRDD mutant did not. Data from at least 10 random fields of view in a total of three independent experiments. Each experiment is color-coded. Statistical analysis using one-way ANOVA with Tukey’s multiple comparison test. Mean ± SD. ns, P > 0.05. (I) Time sequence images of HEK293T cells coexpressing P16-GFP-CYRI-A (cyan) and mApple-integrin α5 (magenta) showing integrin α5 signal present on CYRI-A–positive vesicles. Scale bar = 10 µm for full-sized image and 5 µm for zooms. (J–M) Immunofluorescence images of endogenous integrins α5 and β1 in A-673 cells with the P16-GFP-CYRI-A or P17-GFP-CYRI-B constructs along with actin (yellow). Graphs show the colocalization of CYRI-A, integrins, and filamentous actin (phalloidin) on the vesicles. Scale bars = 10 µm. In C, F, and J–M: DAPI for DNA.

Article Snippet: mApple-α-5-Integrin-12 (plasmid #54864; RRID:Addgene_54864; Addgene), mCherry-Clathrin LC-15 (plasmid #55019; RRID:Addgene_55019; Addgene), and mCherry-Rab5a-7 (plasmid #55126; RRID:Addgene_55126; Addgene) were gifts from Michael Davidson ( ); pcDNA3/hArf1(WT)-mCherry (plasmid #79419; RRID:Addgene_79419; Addgene) was a gift from Kazuhisa Nakayama ( ); CAV1-mCherry (plasmid #27705; RRID:Addgene_27705; Addgene) was a gift from Ari Helenius ( ); and PH-Btk-GFP (plasmid #51463; RRID:Addgene_51463; Addgene) was a gift from Tamas Balla ( ).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Staining, Binding Assay, Mutagenesis, Sequencing, Construct