mcherry cav 1 Search Results


cav1 mcherry  (Addgene inc)
93
Addgene inc cav1 mcherry
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 <t>mCherry-RAB5A</t> (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.
Cav1 Mcherry, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cav1 mcherry/product/Addgene inc
Average 93 stars, based on 1 article reviews
cav1 mcherry - by Bioz Stars, 2026-05
93/100 stars
  Buy from Supplier
cav-1-megfp  (3i - Intelligent Imaging)
90
3i - Intelligent Imaging cav-1-megfp
The association of actin filaments with cytoplasmic <t>CAV-1.</t> ( A ) Time-lapse imaging of U2OS cells expressing CAV-1-mEGFP and mCherry-actin revealing the motility of cytoplasmic CAV-1 vesicles along actin filaments. Two magnified regions (yellow boxes-defined Roi1 and Roi2) are chosen. White and yellow circles in the magnified regions indicate the starting and ending positions of discrete CAV-1-tagged vesicles. Scale bar, 10 µm (in cell image), 2 µm (magnified yellow box Roi 1 image), and 2 µm (magnified yellow box Roi 2 image), respectively. ( B ) The percentage of CAV-1 colocalized with actin. n = 30 cells are used for quantification. ( C ) Pearson’s coefficient of CAV-1 and actin. n = 30 cells are used for quantification. ( D ) Moving trajectories of CAV-1-positive vesicles 1, 2, and 3 in A. Red dots and green arrows indicate the starting and ending points. Scale bar, 1 μm. ( E ) The kymograph analysis of CAV-1-positive vesicles 1, 2, and 3 in A . White arrow in vesicle 3 illustrates the fast-moving trail of CAV-1 signals. The vertical scale bar represents 1 min and the horizontal scale bar represents 1 µm. ( F ) The percentage of distinct mobility types of cytoplasmic CAV-1. n = 16 cells are used for quantification. The data are presented as mean ± SEM. ( G ) The mean length of ‘dwelling’ ( n = 34 vesicles) and ‘go and dwelling’ ( n = 37 vesicles) CAV-1-positive vesicles. Data are represented as mean ± SEM. * P ≤ 0.05 ( t -test).
Cav 1 Megfp, supplied by 3i - Intelligent Imaging, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/cav-1-megfp/product/3i - Intelligent Imaging
Average 90 stars, based on 1 article reviews
cav-1-megfp - by Bioz Stars, 2026-05
90/100 stars
  Buy from Supplier
cav1-megfp-mcherry (plasmid #27763)  (Addgene inc)
N/A
Standard format: Plasmid sent in bacteria as agar stab
   Buy from Supplier

Image Search Results


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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

The association of actin filaments with cytoplasmic CAV-1. ( A ) Time-lapse imaging of U2OS cells expressing CAV-1-mEGFP and mCherry-actin revealing the motility of cytoplasmic CAV-1 vesicles along actin filaments. Two magnified regions (yellow boxes-defined Roi1 and Roi2) are chosen. White and yellow circles in the magnified regions indicate the starting and ending positions of discrete CAV-1-tagged vesicles. Scale bar, 10 µm (in cell image), 2 µm (magnified yellow box Roi 1 image), and 2 µm (magnified yellow box Roi 2 image), respectively. ( B ) The percentage of CAV-1 colocalized with actin. n = 30 cells are used for quantification. ( C ) Pearson’s coefficient of CAV-1 and actin. n = 30 cells are used for quantification. ( D ) Moving trajectories of CAV-1-positive vesicles 1, 2, and 3 in A. Red dots and green arrows indicate the starting and ending points. Scale bar, 1 μm. ( E ) The kymograph analysis of CAV-1-positive vesicles 1, 2, and 3 in A . White arrow in vesicle 3 illustrates the fast-moving trail of CAV-1 signals. The vertical scale bar represents 1 min and the horizontal scale bar represents 1 µm. ( F ) The percentage of distinct mobility types of cytoplasmic CAV-1. n = 16 cells are used for quantification. The data are presented as mean ± SEM. ( G ) The mean length of ‘dwelling’ ( n = 34 vesicles) and ‘go and dwelling’ ( n = 37 vesicles) CAV-1-positive vesicles. Data are represented as mean ± SEM. * P ≤ 0.05 ( t -test).

Journal: Journal of Molecular Cell Biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: The association of actin filaments with cytoplasmic CAV-1. ( A ) Time-lapse imaging of U2OS cells expressing CAV-1-mEGFP and mCherry-actin revealing the motility of cytoplasmic CAV-1 vesicles along actin filaments. Two magnified regions (yellow boxes-defined Roi1 and Roi2) are chosen. White and yellow circles in the magnified regions indicate the starting and ending positions of discrete CAV-1-tagged vesicles. Scale bar, 10 µm (in cell image), 2 µm (magnified yellow box Roi 1 image), and 2 µm (magnified yellow box Roi 2 image), respectively. ( B ) The percentage of CAV-1 colocalized with actin. n = 30 cells are used for quantification. ( C ) Pearson’s coefficient of CAV-1 and actin. n = 30 cells are used for quantification. ( D ) Moving trajectories of CAV-1-positive vesicles 1, 2, and 3 in A. Red dots and green arrows indicate the starting and ending points. Scale bar, 1 μm. ( E ) The kymograph analysis of CAV-1-positive vesicles 1, 2, and 3 in A . White arrow in vesicle 3 illustrates the fast-moving trail of CAV-1 signals. The vertical scale bar represents 1 min and the horizontal scale bar represents 1 µm. ( F ) The percentage of distinct mobility types of cytoplasmic CAV-1. n = 16 cells are used for quantification. The data are presented as mean ± SEM. ( G ) The mean length of ‘dwelling’ ( n = 34 vesicles) and ‘go and dwelling’ ( n = 37 vesicles) CAV-1-positive vesicles. Data are represented as mean ± SEM. * P ≤ 0.05 ( t -test).

Article Snippet: The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner.

Techniques: Imaging, Expressing

Inhibition of formin results in reduced density and motility of cytoplasmic CAV-1. ( A and B ) The transcriptional level of CAV-1 ( A ) and the translational level of CAV-1 and cavin-1 ( B ) are examined by real-time quantitative PCR and western blotting analysis in the wild-type U2OS (Ctrl) cells and formin-inhibited cells. GAPDH is probed for equal sample loading. ( C ) Immunofluorescence staining of endogenous F-actin and CAV-1-positive vesicles in wild-type and formin-inhibited cells. Scale bar, 10 µm. ( D ) Magnified images indicated by yellow box in C illustrate the changes of CAV-1 vesicles. Scale bar, 4 µm. The representative analysis of CAV-1-positive dots was detected by Imaris. Identified dots are marked as balls, which are randomly colored in the lower panel. The size of the color balls indicates the calculated CAV-1 vesicle size. ( E ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the Ctrl ( n = 20) and formin inhibition ( n = 24) groups. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( F ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each group of size is divided by the total CAV-1 number of the same cell. n = 25602 vesicles from 32 cells (Ctrl) and 12035 vesicles from 30 cells (formin inhibition). Data are represented as mean ± SEM. ( G ) The representative dot tracking analysis of CAV-1-positive vesicles in formin-inhibited cells by Imaris. White dashed line indicates the outline of the cell. Color-coded bar from blue to red indicates the tracked mean speeds ranging from 0 to 0.3 μm/sec. Scale bar, 10 μm. ( H ) Quantification of the movement rate of CAV-1-marked vesicles in Ctrl ( n = 23) and formin-inhibited ( n = 22) cells. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test).

Journal: Journal of Molecular Cell Biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: Inhibition of formin results in reduced density and motility of cytoplasmic CAV-1. ( A and B ) The transcriptional level of CAV-1 ( A ) and the translational level of CAV-1 and cavin-1 ( B ) are examined by real-time quantitative PCR and western blotting analysis in the wild-type U2OS (Ctrl) cells and formin-inhibited cells. GAPDH is probed for equal sample loading. ( C ) Immunofluorescence staining of endogenous F-actin and CAV-1-positive vesicles in wild-type and formin-inhibited cells. Scale bar, 10 µm. ( D ) Magnified images indicated by yellow box in C illustrate the changes of CAV-1 vesicles. Scale bar, 4 µm. The representative analysis of CAV-1-positive dots was detected by Imaris. Identified dots are marked as balls, which are randomly colored in the lower panel. The size of the color balls indicates the calculated CAV-1 vesicle size. ( E ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the Ctrl ( n = 20) and formin inhibition ( n = 24) groups. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( F ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each group of size is divided by the total CAV-1 number of the same cell. n = 25602 vesicles from 32 cells (Ctrl) and 12035 vesicles from 30 cells (formin inhibition). Data are represented as mean ± SEM. ( G ) The representative dot tracking analysis of CAV-1-positive vesicles in formin-inhibited cells by Imaris. White dashed line indicates the outline of the cell. Color-coded bar from blue to red indicates the tracked mean speeds ranging from 0 to 0.3 μm/sec. Scale bar, 10 μm. ( H ) Quantification of the movement rate of CAV-1-marked vesicles in Ctrl ( n = 23) and formin-inhibited ( n = 22) cells. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test).

Article Snippet: The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining

The effects of formin components FHOD1 and Dia1 on the density, size, and motility of cytoplasmic CAV-1 vesicles. ( A ) Western blotting analysis of endogenous FHOD1 and Dia1 levels in total cell lysates of wild-type (Ctrl), FHOD1 knockdown (FD1 siRNA), Dia1 knockdown (Dia1 siRNA), and FHOD1/Dia1 double knockdown (FD1 + Dia1 siRNA) U2OS cells, respectively. GAPDH is probed for equal sample loading. ( B ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the Ctrl ( n = 24), FD1 siRNA ( n = 12), Dia1 siRNA ( n = 18), and FD1 + Dia1 siRNA ( n = 23) U2OS cells. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( C ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 17602 vesicles from 24 cells (Ctrl), 6432 vesicles from 12 cells (FD1 siRNA), 9543 vesicles from 18 cells (Dia1 siRNA), and 10231 vesicles from 23 cells (FD1 + Dia1 siRNA). Data are represented as mean ± SEM. ( D ) Quantification of the movement rate of CAV-1-marked vesicles in the Ctrl ( n = 25), FD1 siRNA ( n = 13), Dia1 siRNA ( n = 14), and FD1 + Dia1 siRNA ( n = 24) cells. ( E ) Quantification of the ratio of ‘go and dwelling’ to ‘dwelling’ in the Ctrl ( n = 9), formin-inhibited ( n = 9), FD1 siRNA ( n = 9), and Dia1 siRNA ( n = 9) cells. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( F ) Immunofluorescence staining of endogenous F-actin and CAV-1-positive vesicles in U2OS cells expressing GFP, active GFP-FHOD1, and active GFP-Dia1, respectively. Scale bar, 10 and 5 µm in images and magnified images, respectively. ( G ) Quantification of the number of CAV-1-positive vesicles per µm 2 in each group. n = 30 cells. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test). ( H ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 16597 vesicles from 20 cells (Ctrl), 15737 vesicles from 20 cells (GFP), 20623 vesicles from 20 cells (active GFP-FHOD1), and 19405 vesicles from 20 cells (active GFP-mDia1). Data are represented as mean ± SEM.

Journal: Journal of Molecular Cell Biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: The effects of formin components FHOD1 and Dia1 on the density, size, and motility of cytoplasmic CAV-1 vesicles. ( A ) Western blotting analysis of endogenous FHOD1 and Dia1 levels in total cell lysates of wild-type (Ctrl), FHOD1 knockdown (FD1 siRNA), Dia1 knockdown (Dia1 siRNA), and FHOD1/Dia1 double knockdown (FD1 + Dia1 siRNA) U2OS cells, respectively. GAPDH is probed for equal sample loading. ( B ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the Ctrl ( n = 24), FD1 siRNA ( n = 12), Dia1 siRNA ( n = 18), and FD1 + Dia1 siRNA ( n = 23) U2OS cells. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( C ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 17602 vesicles from 24 cells (Ctrl), 6432 vesicles from 12 cells (FD1 siRNA), 9543 vesicles from 18 cells (Dia1 siRNA), and 10231 vesicles from 23 cells (FD1 + Dia1 siRNA). Data are represented as mean ± SEM. ( D ) Quantification of the movement rate of CAV-1-marked vesicles in the Ctrl ( n = 25), FD1 siRNA ( n = 13), Dia1 siRNA ( n = 14), and FD1 + Dia1 siRNA ( n = 24) cells. ( E ) Quantification of the ratio of ‘go and dwelling’ to ‘dwelling’ in the Ctrl ( n = 9), formin-inhibited ( n = 9), FD1 siRNA ( n = 9), and Dia1 siRNA ( n = 9) cells. The data are presented as mean ± SEM. ** P ≤ 0.01 ( t -test). ( F ) Immunofluorescence staining of endogenous F-actin and CAV-1-positive vesicles in U2OS cells expressing GFP, active GFP-FHOD1, and active GFP-Dia1, respectively. Scale bar, 10 and 5 µm in images and magnified images, respectively. ( G ) Quantification of the number of CAV-1-positive vesicles per µm 2 in each group. n = 30 cells. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test). ( H ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 16597 vesicles from 20 cells (Ctrl), 15737 vesicles from 20 cells (GFP), 20623 vesicles from 20 cells (active GFP-FHOD1), and 19405 vesicles from 20 cells (active GFP-mDia1). Data are represented as mean ± SEM.

Article Snippet: The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing

The linear elongated actin network by formins is critical for CAV-1 disappearance upon hypo-osmotic shock. ( A ) Time-lapse imaging of U2OS cells expressing CAV-1-mCherry cultured under routine culture condition (iso-osmosis) with formin inhibition or FHOD1 + Dia1 double knockdown, respectively. ( B ) Time-lapse imaging of U2OS cells expressing CAV-1-mCherry upon hypo-osmotic shock under Ctrl, formin inhibition, or FHOD1 + Dia1 double knockdown condition, respectively. In A and B , white dash lines indicate the outline of the cells. CAV-1-positive vesicles were detected by Imaris at different time points. Vanished CAV-1 vesicles upon 2- and 5-min iso-osmotic ( A ) or hypo-osmotic ( B ) shock are labeled by green and orange dots, respectively. Red dots illustrate the remaining CAV-1-positive vesicles after 5-min hypo-osmotic treatment. Scale bar, 10 µm. ( C ) Quantification of the percentage of CAV-1-positive vesicles left upon 5-min hypo- or iso-osmotic shock under Ctrl, formin inhibition, or FHOD1 + Dia1 double knockdown conditions, respectively. The data are presented as mean ± SEM. ** P ≤ 0.01, *** P ≤ 0.001 ( t -test). ( D ) Western blotting analysis of CAV-1, cavin-1, and actin levels in U2OS cells with formin inhibition upon hypo-osmotic shock. The blot is also probed with GAPDH antibody to verify equal sample loading. The obtained intensity value from wild-type cells was set to 1. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA).

Journal: Journal of Molecular Cell Biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: The linear elongated actin network by formins is critical for CAV-1 disappearance upon hypo-osmotic shock. ( A ) Time-lapse imaging of U2OS cells expressing CAV-1-mCherry cultured under routine culture condition (iso-osmosis) with formin inhibition or FHOD1 + Dia1 double knockdown, respectively. ( B ) Time-lapse imaging of U2OS cells expressing CAV-1-mCherry upon hypo-osmotic shock under Ctrl, formin inhibition, or FHOD1 + Dia1 double knockdown condition, respectively. In A and B , white dash lines indicate the outline of the cells. CAV-1-positive vesicles were detected by Imaris at different time points. Vanished CAV-1 vesicles upon 2- and 5-min iso-osmotic ( A ) or hypo-osmotic ( B ) shock are labeled by green and orange dots, respectively. Red dots illustrate the remaining CAV-1-positive vesicles after 5-min hypo-osmotic treatment. Scale bar, 10 µm. ( C ) Quantification of the percentage of CAV-1-positive vesicles left upon 5-min hypo- or iso-osmotic shock under Ctrl, formin inhibition, or FHOD1 + Dia1 double knockdown conditions, respectively. The data are presented as mean ± SEM. ** P ≤ 0.01, *** P ≤ 0.001 ( t -test). ( D ) Western blotting analysis of CAV-1, cavin-1, and actin levels in U2OS cells with formin inhibition upon hypo-osmotic shock. The blot is also probed with GAPDH antibody to verify equal sample loading. The obtained intensity value from wild-type cells was set to 1. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way ANOVA).

Article Snippet: The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner.

Techniques: Imaging, Expressing, Cell Culture, Inhibition, Labeling, Western Blot

Inhibition of formin promotes the reduced density, increased size, and decreased motility of cytoplasmic CAV-1 vesicles when grown on soft matrix. ( A and B ) Representative live cell imaging of actin and CAV-1-positive vesicles in U2OS cells grown on glass or soft substrate 25 or 0.5 kPa, with or without formin inhibitor treatment, respectively. Scale bar, 10 µm. ( C ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the cells grown on glass ( n = 29 for Ctrl, n = 22 for formin inhibition) or 25 kPa ( n = 25 for Ctrl, n = 19 for formin inhibition) and 0.5 kPa ( n = 30 for Ctrl, n = 17 for formin inhibition) substrates. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test). ( D ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 25602 vesicles from 32 Ctrl cells and 16786 vesicles from 29 formin-inhibited cells (glass), 13467 vesicles from 31 Ctrl cells and 8546 vesicles from 33 formin-inhibited cells (25 kPa), and 14567 vesicles from 32 Ctrl cells and 7658 vesicles from 34 formin-inhibited cells (0.5 kPa). Data are represented as mean ±SEM. ** P < 0.01, *** P ≤ 0.001 ( t -test). ( E ) Quantification of the movement rate of CAV-1-positive vesicles in the cells grown on glass ( n = 26 for Ctrl, n = 22 for formin inhibition) and 25 kPa ( n = 12 for Ctrl, n = 13 for formin inhibition) and 0.5 kPa ( n = 14 for Ctrl, n = 10 for formin inhibition) substrates. The data are presented as mean ± SEM. ( F ) The schematic working model. CAV-1 vesicles are associated with and can move on actin filaments in normal cells. Formin inhibition results in decreased CAV-1 vesicle numbers, enlarged vesicle areas, and slowed movement speeds. When cells are challenged with hypo-osmotic shock or softer matrix, CAV-1 vesicles become less in number, larger in area, and slower in speed in order to resist the external environment pressure. Formin inhibition expedites the changes of CAV-1 vesicles in all aspects.

Journal: Journal of Molecular Cell Biology

Article Title: Actin nucleator formins regulate the tension-buffering function of caveolin-1

doi: 10.1093/jmcb/mjab070

Figure Lengend Snippet: Inhibition of formin promotes the reduced density, increased size, and decreased motility of cytoplasmic CAV-1 vesicles when grown on soft matrix. ( A and B ) Representative live cell imaging of actin and CAV-1-positive vesicles in U2OS cells grown on glass or soft substrate 25 or 0.5 kPa, with or without formin inhibitor treatment, respectively. Scale bar, 10 µm. ( C ) Quantification of the number of CAV-1-positive vesicles per µm 2 in the cells grown on glass ( n = 29 for Ctrl, n = 22 for formin inhibition) or 25 kPa ( n = 25 for Ctrl, n = 19 for formin inhibition) and 0.5 kPa ( n = 30 for Ctrl, n = 17 for formin inhibition) substrates. The data are presented as mean ± SEM. *** P ≤ 0.001 ( t -test). ( D ) The length distribution of CAV-1-positive vesicles. The number of vesicles in each size group is divided by the total CAV-1 number of the same cell. n = 25602 vesicles from 32 Ctrl cells and 16786 vesicles from 29 formin-inhibited cells (glass), 13467 vesicles from 31 Ctrl cells and 8546 vesicles from 33 formin-inhibited cells (25 kPa), and 14567 vesicles from 32 Ctrl cells and 7658 vesicles from 34 formin-inhibited cells (0.5 kPa). Data are represented as mean ±SEM. ** P < 0.01, *** P ≤ 0.001 ( t -test). ( E ) Quantification of the movement rate of CAV-1-positive vesicles in the cells grown on glass ( n = 26 for Ctrl, n = 22 for formin inhibition) and 25 kPa ( n = 12 for Ctrl, n = 13 for formin inhibition) and 0.5 kPa ( n = 14 for Ctrl, n = 10 for formin inhibition) substrates. The data are presented as mean ± SEM. ( F ) The schematic working model. CAV-1 vesicles are associated with and can move on actin filaments in normal cells. Formin inhibition results in decreased CAV-1 vesicle numbers, enlarged vesicle areas, and slowed movement speeds. When cells are challenged with hypo-osmotic shock or softer matrix, CAV-1 vesicles become less in number, larger in area, and slower in speed in order to resist the external environment pressure. Formin inhibition expedites the changes of CAV-1 vesicles in all aspects.

Article Snippet: The time-lapse images of cells with transient transfection of CAV-1-mEGFP, mCherry-actin, and CAV-1-mCherry were acquired with 3I Marianas imaging system (3I intelligent Imaging Innovations), consisting of an inverted spinning disk confocal microscope Zeiss Axio Observer Z1 (Zeiss) and a Yokogawa CSU-X1 M1 confocal scanner.

Techniques: Inhibition, Live Cell Imaging