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Addgene inc mcherry rab5 q79l

Branched actin inhibition, but not formin inhibition, decreases actin at <t>RAB5</t> QL endosomes. (A-P) . HeLa cells were transfected <t>with</t> <t>mCherry-RAB5</t> <t>Q79L</t> and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Mcherry Rab5 Q79l, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/mcherry+rab5+q79l/bio_rxiv__64898__2026__03__10__710749-175-1-4?v=Addgene+inc
Average 94 stars, based on 31 article reviews

mcherry rab5 q79l - by Bioz Stars, 2026-07

94/100 stars

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1) Product Images from "Branched actin constrains endosomal cargo to control sorting and fission"

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

Journal: bioRxiv

doi: 10.64898/2026.03.10.710749

Branched actin inhibition, but not formin inhibition, decreases actin at RAB5 QL endosomes. (A-P) . HeLa cells were transfected with mCherry-RAB5 Q79L and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Figure Legend Snippet: Branched actin inhibition, but not formin inhibition, decreases actin at RAB5 QL endosomes. (A-P) . HeLa cells were transfected with mCherry-RAB5 Q79L and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Techniques Used: Inhibition, Transfection, Staining, Two Tailed Test, MANN-WHITNEY

Figure Legend Snippet: Transferrin is bounded by cortactin at endosomes. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. (M-P) . Representative fluorescence intensity profiles of endosomes from ( M ) untreated, ( N ) SMIFH2-treated cells, ( O ) CK-689-treated cells, or ( P ) CK-666-treated cells. The intensity profile of Tf is in green, and the intensity profile of cortactin is in magenta. Tf vertices above 130% that occur within 20 degrees of a cortactin value above 130% are represented with a red circle; these peaks are “bounded”. Tf vertices above 130% that are not within 20 degrees of a cortactin value above 130% are represented with a grey circle and are “not bounded”. (Q). The percentage of “bounded” Tf peaks was quantified from fluorescence intensity profiles (as represented in ( M-P )). Quantification is from three independent experiments, and from 37 endosomes for the untreated group, 43 endosomes for SMIFH2-treated, 38 endosomes for CK-689-treated, and 37 endosomes from the CK-666-treated group. Statistical significance was determined using a two-tailed unpaired t -test (ns: p > 0.05). (R, S) . Representative models for the quantification and results of the experiment. A circle was drawn around the endosome membrane (red) that intersects with the regions of Tf (green) and cortactin (magenta), and fluorescence intensity at each degree around the circle was measured. ( R ) Untreated cells have Tf in confined regions on the endosome and are adjacent to regions of cortactin ∼60% of the time. ( S ) CK-666-treated cells have regions of Tf that are broader and “not bounded” by cortactin.

Techniques Used: Transfection, Incubation, Fluorescence, Two Tailed Test, Membrane

Figure Legend Snippet: Tf occupies less discrete regions on the endosome when branched actin is inhibited. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show Tf localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of Tf on the endosome (blue arrows). (M). Model for quantification. A circle (blue) was drawn around the endosome membrane (red) that intersects with the regions of Tf, and the fluorescence intensity at each degree around the circle was measured. (N-Q) . Representative fluorescence intensity profiles of Tf on the endosome from ( N ) untreated, ( O ) SMIFH2-treated, ( P ) CK-689-treated, and ( Q ) CK-666-treated cells. The profiles depicted are from the endosomes indicated with a magenta-colored star ( A-L ). (R). Tf-containing regions from the fluorescence intensity profiles ( N-Q ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of Tf-containing regions are Tf “peaks”. The graph shows the average profile of 72 Tf peaks from 37 endosomes for the control group, 70 peaks from 43 endosomes from SMIFH2-treated cells, 73 peaks from 38 endosomes from CK-689-treated cells, and 70 peaks from 37 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of Tf peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV3 for statistical information.

Techniques Used: Transfection, Incubation, Membrane, Fluorescence, Control

Figure Legend Snippet: EGF segregation on the endosome is affected by branched actin inhibition. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) SMIFH2, ( G-I ) CK-689, or ( J-L ) CK-666 for 4 min. Following the pre-treatment, cells were incubated with EGF-488 (and the respective inhibitor) for 17 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show EGF localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of EGF on the endosome (blue arrows). (M-P) . Representative fluorescence intensity profiles of EGF on the endosome from the ( M ) untreated, ( N ) SMIFH2-treated, ( O ) CK-689-treated, and ( P ) CK-666 treated cells. The profiles depicted are from the endosomes indicated with a magenta star in ( A-L ). (Q) . EGF-containing regions from the fluorescence intensity profiles ( M-P ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of EGF-containing regions are EGF “peaks”. The graph shows the average profile of 84 EGF peaks from 47 endosomes that were analyzed for the control group, 86 peaks from 51 endosomes from SMIFH2-treated cells, 72 peaks from 39 endosomes from CK-689-treated cells, and 86 peaks from 49 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of EGF peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV4 for statistical information.

Techniques Used: Inhibition, Transfection, Incubation, Fluorescence, Control

The degradative and retrieval subdomains on endosomes coalesce upon branched actin inhibition. (A-F) . HeLa cells were transfected with mCherry-RAB5 Q79L and were co-incubated with EGF-488 and anti-CD59 antibody for 20 min. During the last 5 min of uptake, either ( A-C ) no inhibitor or ( D-F ) 300 µM CK-666 was added to the media. Pearson’s and Manders’ correlation coefficients for each representative image are listed. (G). ImageJ was used to calculate Pearson’s correlation coefficient for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test. (H, I) . ImageJ was used to calculate Manders’ correlation coefficients (M1 and M2) for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test.

Figure Legend Snippet: The degradative and retrieval subdomains on endosomes coalesce upon branched actin inhibition. (A-F) . HeLa cells were transfected with mCherry-RAB5 Q79L and were co-incubated with EGF-488 and anti-CD59 antibody for 20 min. During the last 5 min of uptake, either ( A-C ) no inhibitor or ( D-F ) 300 µM CK-666 was added to the media. Pearson’s and Manders’ correlation coefficients for each representative image are listed. (G). ImageJ was used to calculate Pearson’s correlation coefficient for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test. (H, I) . ImageJ was used to calculate Manders’ correlation coefficients (M1 and M2) for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test.

Techniques Used: Inhibition, Transfection, Incubation, Two Tailed Test

Image Search Results

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Branched actin inhibition, but not formin inhibition, decreases actin at RAB5 QL endosomes. (A-P) . HeLa cells were transfected with mCherry-RAB5 Q79L and were either ( A-D ) untreated or treated with ( E-H ) 25 µM SMIFH2, ( I-L ) 300 µM CK-689, or ( M-P ) 300 µM CK-666 for 20 min. Cells were fixed and co-stained with cortactin and phalloidin to visualize the actin network. (Q) . Quantification of ( A-P ). RAB5 Q79L endosomes that colocalized with phalloidin or cortactin were counted as a percentage of total RAB5 Q79L endosomes. Quantification represents 15 images from three independent experiments. A two-tailed Mann-Whitney nonparametric test was used to determine significance between treatment groups. Data comparisons without error bars are not significant ( p > 0.05).

Article Snippet: The mCherry-RAB5 Q79L (35138, Addgene) plasmid was used to generate enlarged endosomes.

Techniques: Inhibition, Transfection, Staining, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Transferrin is bounded by cortactin at endosomes. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. (M-P) . Representative fluorescence intensity profiles of endosomes from ( M ) untreated, ( N ) SMIFH2-treated cells, ( O ) CK-689-treated cells, or ( P ) CK-666-treated cells. The intensity profile of Tf is in green, and the intensity profile of cortactin is in magenta. Tf vertices above 130% that occur within 20 degrees of a cortactin value above 130% are represented with a red circle; these peaks are “bounded”. Tf vertices above 130% that are not within 20 degrees of a cortactin value above 130% are represented with a grey circle and are “not bounded”. (Q). The percentage of “bounded” Tf peaks was quantified from fluorescence intensity profiles (as represented in ( M-P )). Quantification is from three independent experiments, and from 37 endosomes for the untreated group, 43 endosomes for SMIFH2-treated, 38 endosomes for CK-689-treated, and 37 endosomes from the CK-666-treated group. Statistical significance was determined using a two-tailed unpaired t -test (ns: p > 0.05). (R, S) . Representative models for the quantification and results of the experiment. A circle was drawn around the endosome membrane (red) that intersects with the regions of Tf (green) and cortactin (magenta), and fluorescence intensity at each degree around the circle was measured. ( R ) Untreated cells have Tf in confined regions on the endosome and are adjacent to regions of cortactin ∼60% of the time. ( S ) CK-666-treated cells have regions of Tf that are broader and “not bounded” by cortactin.

Article Snippet: The mCherry-RAB5 Q79L (35138, Addgene) plasmid was used to generate enlarged endosomes.

Techniques: Transfection, Incubation, Fluorescence, Two Tailed Test, Membrane

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Tf occupies less discrete regions on the endosome when branched actin is inhibited. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) 25 µM SMIFH2, ( G-I ) 300 µM CK-689, or ( J-L ) 300 µM CK-666 for 4 min. Following the pre-treatment, cells were incubated with Tf-488 (and their respective inhibitor) for 6 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show Tf localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of Tf on the endosome (blue arrows). (M). Model for quantification. A circle (blue) was drawn around the endosome membrane (red) that intersects with the regions of Tf, and the fluorescence intensity at each degree around the circle was measured. (N-Q) . Representative fluorescence intensity profiles of Tf on the endosome from ( N ) untreated, ( O ) SMIFH2-treated, ( P ) CK-689-treated, and ( Q ) CK-666-treated cells. The profiles depicted are from the endosomes indicated with a magenta-colored star ( A-L ). (R). Tf-containing regions from the fluorescence intensity profiles ( N-Q ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of Tf-containing regions are Tf “peaks”. The graph shows the average profile of 72 Tf peaks from 37 endosomes for the control group, 70 peaks from 43 endosomes from SMIFH2-treated cells, 73 peaks from 38 endosomes from CK-689-treated cells, and 70 peaks from 37 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of Tf peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV3 for statistical information.

Article Snippet: The mCherry-RAB5 Q79L (35138, Addgene) plasmid was used to generate enlarged endosomes.

Techniques: Transfection, Incubation, Membrane, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: EGF segregation on the endosome is affected by branched actin inhibition. (A-L) . HeLa cells were transfected with mCherry-RAB5 Q79L and were incubated with ( A-C ) no inhibitor, ( D-F ) SMIFH2, ( G-I ) CK-689, or ( J-L ) CK-666 for 4 min. Following the pre-treatment, cells were incubated with EGF-488 (and the respective inhibitor) for 17 min of uptake. Untreated, SMIFH2-treated, and CK-689-treated cells show EGF localized to discrete regions on the endosome (yellow arrows). CK-666-treated cells have broader regions of EGF on the endosome (blue arrows). (M-P) . Representative fluorescence intensity profiles of EGF on the endosome from the ( M ) untreated, ( N ) SMIFH2-treated, ( O ) CK-689-treated, and ( P ) CK-666 treated cells. The profiles depicted are from the endosomes indicated with a magenta star in ( A-L ). (Q) . EGF-containing regions from the fluorescence intensity profiles ( M-P ) were identified, and the fluorescence values ± 20 degrees from the maximum were normalized. These normalized profiles of EGF-containing regions are EGF “peaks”. The graph shows the average profile of 84 EGF peaks from 47 endosomes that were analyzed for the control group, 86 peaks from 51 endosomes from SMIFH2-treated cells, 72 peaks from 39 endosomes from CK-689-treated cells, and 86 peaks from 49 endosomes from CK-666-treated cells. Data are from three independent experiments. The average profile of EGF peaks in the CK-666-treated cells is broader (less discrete) than the other treatment groups. See table in Figure EV4 for statistical information.

Article Snippet: The mCherry-RAB5 Q79L (35138, Addgene) plasmid was used to generate enlarged endosomes.

Techniques: Inhibition, Transfection, Incubation, Fluorescence, Control

Journal: bioRxiv

Article Title: Branched actin constrains endosomal cargo to control sorting and fission

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: The degradative and retrieval subdomains on endosomes coalesce upon branched actin inhibition. (A-F) . HeLa cells were transfected with mCherry-RAB5 Q79L and were co-incubated with EGF-488 and anti-CD59 antibody for 20 min. During the last 5 min of uptake, either ( A-C ) no inhibitor or ( D-F ) 300 µM CK-666 was added to the media. Pearson’s and Manders’ correlation coefficients for each representative image are listed. (G). ImageJ was used to calculate Pearson’s correlation coefficient for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test. (H, I) . ImageJ was used to calculate Manders’ correlation coefficients (M1 and M2) for 47 ROIs in the untreated group and 42 ROIs in the CK-666-treated group from three independent experiments. Statistical significance was determined using an unpaired two-tailed t -test.

Article Snippet: The mCherry-RAB5 Q79L (35138, Addgene) plasmid was used to generate enlarged endosomes.

Techniques: Inhibition, Transfection, Incubation, Two Tailed Test

( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

( A and B ) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells ( N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. ( D ) Role of GDI2 in α V β 6 -dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. ( E ) Haptotactic migration analysis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of α V β 6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM ( N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells ( N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. ( D ) Role of GDI2 in α V β 6 -dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. ( E ) Haptotactic migration analysis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of α V β 6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM ( N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Techniques: Transfection, Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Migration, Negative Control, Blocking Assay

( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Fig. 4. Integrin αVβ6 engagement triggers internalization and vesicular accumulation of surface-labeled HER2 and modulates RAB5 activity in trastuzumab-sensitive cells. (A and B) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate αVβ6 integrin and trigger αVβ6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells (N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization dif- ferences. (C) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min (N = 3; 16 to 28 cells per condition); scale bar, 10 μm. (Ca) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. (D) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM (N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. (E and F) Affibody-chase experiments in (E) siControl Trastuzumab- Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quan- titation represents cytoplasmic HER2 fluorescence intensity (N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Repre- sentative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: Science advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance.

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: Fig. 4. Integrin αVβ6 engagement triggers internalization and vesicular accumulation of surface-labeled HER2 and modulates RAB5 activity in trastuzumab-sensitive cells. (A and B) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate αVβ6 integrin and trigger αVβ6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells (N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization dif- ferences. (C) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min (N = 3; 16 to 28 cells per condition); scale bar, 10 μm. (Ca) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. (D) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM (N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. (E and F) Affibody-chase experiments in (E) siControl Trastuzumab- Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quan- titation represents cytoplasmic HER2 fluorescence intensity (N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Repre- sentative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 (125) [mcherry- RAB5CA(Q79L), Addgene plasmid #35138], dominant- negative RAB5 [mCherry- RAB5DN(S34N), Addgene plasmid #35139] (125), dominant- negative RAB7 [DsRedRAB7 DN(T22N), Addgene plasmid #12662] (126), or empty pmCherry- C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Activity Assay, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Expressing, Dominant Negative Mutation, Plasmid Preparation

Fig. 5. GDI2 regulates RAB5 activity and controls αVβ6-dependent HER2 endocytosis and cell migration. (A and B) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate αVβ6 integrin and trigger αVβ6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells (N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. (C) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab- sensitive and trastuzumab-resistant BT474 cells (N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. (D) Role of GDI2 in αVβ6-dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM (N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. (E) Haptotactic migration analy- sis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of αVβ6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM (N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: Science advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance.

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: Fig. 5. GDI2 regulates RAB5 activity and controls αVβ6-dependent HER2 endocytosis and cell migration. (A and B) Affibody-chase experiments: siControl-transfected or siGDI2-transfected BT474 cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate αVβ6 integrin and trigger αVβ6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells (N = 3; 74 to 160 cells per condition); scale bars, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. (C) GDI2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab- sensitive and trastuzumab-resistant BT474 cells (N = 3; >120 cells per condition); scale bars, 5 μm. GDI2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM), two-sided t test. (D) Role of GDI2 in αVβ6-dependent RAB5 activity modulation. Trastuzumab-sensitive and trastuzumab-resistant BT474 cells transfected with siRNA against GDI2 (siGDI2 #1 and #2) or control siRNA. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM (N = 3), normalized to 0-min trastuzumab-sensitive cells. N = 4 independent replicate experiments. Two-way ANOVA with Šídák’s multiple comparison tests. (E) Haptotactic migration analy- sis of BT474 cells (Trastuzumab-Sensitive and Trastuzumab-Resistant) in Transwell coated with FN or BSA as a negative control. Cells were transfected with siRNA against GDI2 (siGDI2 #1 and #2) or siRNA control. Migration was assessed over 24 hours in the presence or absence of αVβ6 integrin blocking antibody or trastuzumab. Data shown are means ± SEM (N = 3). One-way ANOVA with Šídák’s multiple comparison tests. [(A), (B), (D), and (E)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (E)] Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Activity Assay, Migration, Transfection, Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Negative Control, Blocking Assay

Fig. 6. RAB5, RAB7A, and GDI2 differentially regulate invasion and TGFβ activity in trastuzumab-sensitive and trastuzumab-resistant cells. (A) Invasion of trastuzumab-sensitive versus trastuzumab-resistant through the cross-linked collagen-rich and FN-rich ECM (N = 3). Two-sided t test. (B and C) Invasion of siControl, siRAB5A, siRAB7A, and siGDI2 trastuzumab-sensitive (B) or trastuzumab-resistant (C) cells, in the presence or absence of integrin αVβ6 blocking antibody (10 μg/ml) or trastuzumab (10 μg/ml). Note different y axis scales: (B) 0 to 4; (C) 0 to 20. N = 4. (D and E) Invasion/dissemination of CFSE-labeled siRAB5A, siRAB7A, siGDI2, or siControl BT474 trastuzumab-sensitive (D) or resistant cells (E) in a zebrafish xenograft model. Xenografts imaged 48 hours after injection. Images: maximum intensity projections; scale bars, 30 μm. n = 20 to 35 animals per condition. [(B) to (E)] Welch’s ANOVA with Dunnett’s multiple comparisons test. (F) TGFβ activity coculture assay comparing BT474 trastuzumab-sensitive and trastuzumab-resistant cells (N = 3). Two-sided t test. (G and H) Invasion of trastuzumab-sensitive (G) or trastuzumab-resistant (H) BT474 cells in the presence or absence of αVβ6 integrin blocking antibody (10 μg/ml), trastuzumab (10 μg/ml), or TGFβ receptor 1/2 inhibitor (LY2109761; 10 μM) (N = 3). (I and J) Invasion of trastuzumab-sensitive AU565 cells (I) or trastuzumab-resistant JIMT1 cells (J) in the presence or absence of αVβ6 integrin blocking antibody (10 μg/ml), trastuzumab (10 μg/ml), or TGFβ receptor 1/2 inhibitor (10 μM) (N = 6). (K) TGFβ activity analysis of siGDI2 and siControl trastuzumab-sensitive and trastuzumab-resistant BT474 cells treated with αVβ6 integrin blocking antibody or trastuzumab (N = 4; 4 wells per biological replicate). [(G) to (K)] One-way ANOVA with Tukey’s multiple comparison tests. (L and M) TGFβ activation assays with trastuzumab-sensitive AU565 (L) and trastuzumab-resistant JIMT1 (M) cells expressing siGDI2 or siControl treated in the presence or absence of αVβ6 integrin antibody (10 μg/ml) or trastuzumab (10 μg/ml) (N = 3; 5 wells per biological replicate). Two-way ANOVA with Šídák’s multiple comparison test. [(A) to (M)] Data are arbitrary units (AU) normalized to control means ± SEM. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Journal: Science advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance.

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: Fig. 6. RAB5, RAB7A, and GDI2 differentially regulate invasion and TGFβ activity in trastuzumab-sensitive and trastuzumab-resistant cells. (A) Invasion of trastuzumab-sensitive versus trastuzumab-resistant through the cross-linked collagen-rich and FN-rich ECM (N = 3). Two-sided t test. (B and C) Invasion of siControl, siRAB5A, siRAB7A, and siGDI2 trastuzumab-sensitive (B) or trastuzumab-resistant (C) cells, in the presence or absence of integrin αVβ6 blocking antibody (10 μg/ml) or trastuzumab (10 μg/ml). Note different y axis scales: (B) 0 to 4; (C) 0 to 20. N = 4. (D and E) Invasion/dissemination of CFSE-labeled siRAB5A, siRAB7A, siGDI2, or siControl BT474 trastuzumab-sensitive (D) or resistant cells (E) in a zebrafish xenograft model. Xenografts imaged 48 hours after injection. Images: maximum intensity projections; scale bars, 30 μm. n = 20 to 35 animals per condition. [(B) to (E)] Welch’s ANOVA with Dunnett’s multiple comparisons test. (F) TGFβ activity coculture assay comparing BT474 trastuzumab-sensitive and trastuzumab-resistant cells (N = 3). Two-sided t test. (G and H) Invasion of trastuzumab-sensitive (G) or trastuzumab-resistant (H) BT474 cells in the presence or absence of αVβ6 integrin blocking antibody (10 μg/ml), trastuzumab (10 μg/ml), or TGFβ receptor 1/2 inhibitor (LY2109761; 10 μM) (N = 3). (I and J) Invasion of trastuzumab-sensitive AU565 cells (I) or trastuzumab-resistant JIMT1 cells (J) in the presence or absence of αVβ6 integrin blocking antibody (10 μg/ml), trastuzumab (10 μg/ml), or TGFβ receptor 1/2 inhibitor (10 μM) (N = 6). (K) TGFβ activity analysis of siGDI2 and siControl trastuzumab-sensitive and trastuzumab-resistant BT474 cells treated with αVβ6 integrin blocking antibody or trastuzumab (N = 4; 4 wells per biological replicate). [(G) to (K)] One-way ANOVA with Tukey’s multiple comparison tests. (L and M) TGFβ activation assays with trastuzumab-sensitive AU565 (L) and trastuzumab-resistant JIMT1 (M) cells expressing siGDI2 or siControl treated in the presence or absence of αVβ6 integrin antibody (10 μg/ml) or trastuzumab (10 μg/ml) (N = 3; 5 wells per biological replicate). Two-way ANOVA with Šídák’s multiple comparison test. [(A) to (M)] Data are arbitrary units (AU) normalized to control means ± SEM. Statistical significance: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Techniques: Activity Assay, Blocking Assay, Labeling, Injection, Co-culture Assay, Comparison, Activation Assay, Expressing, Control

Fig. 7. Integrin αVβ6/HER2 cross-talk and trafficking drive breast cancer invasion and are dysregulated by trastuzumab resistance. (A) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to αVβ6 IACs and coordinates HER2 and αVβ6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between αVβ6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. (B) Trastuzumab-Resistant Cells: GDI2 is excluded from αVβ6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/αVβ6 cross-talk is im- paired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and αVβ6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Journal: Science advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance.

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: Fig. 7. Integrin αVβ6/HER2 cross-talk and trafficking drive breast cancer invasion and are dysregulated by trastuzumab resistance. (A) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to αVβ6 IACs and coordinates HER2 and αVβ6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between αVβ6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. (B) Trastuzumab-Resistant Cells: GDI2 is excluded from αVβ6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/αVβ6 cross-talk is im- paired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and αVβ6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Molecular Biology of the Cell

Article Title: Endosomal actin branching, fission and receptor recycling require FCHSD2 recruitment by MICAL-L1

doi: 10.1091/mbc.e24-07-0324

Figure Lengend Snippet: FIGURE 2: FCHSD2 is recruited to RAB5 QL endosomes by MICAL-L1. (A–F) HeLa cells were cotransfected with GFP-FCHSD2 and the mCherry-RAB5 Q79L mutant, which remains GTP-locked and active. Cells were fixed, imaged, and analyzed in ImageJ. B, D, and F represent insets of A, C, and E, respectively. A profile 2.81 μm in length was drawn from the cytoplasm into the enlarged mCherry-RAB5 Q79L endosomes, and the fluorescence intensities of both channels were collected along the line. Background subtraction was performed and the fluorescence intensities along each profile were normalized. (G) The data (quantified from from A–F) were plotted as relative intensity over the distance from peak mCherry-Rab5 Q79L intensity (red) and demonstrates that FCHSD2 staining overlaps with RAB5 Q79L on the endosomal membrane. (H) Immunoblot validation of MICAL-L1 knockout in the CRISPR/Cas9 gene-edited knockout cell line. (I–N) HeLa WT parental or MICAL-L1 knockout cells were transfected with the GFP-RAB5 Q79L mutant. Cells on coverslips were fixed after transfection and immunostained with anti-FCHSD2 to visualize endogenous FCHSD2 (J, M). Confocal images were captured and analyzed with Imaris software. As demonstrated, endogenous FCHSD2 coats RAB5 Q79L endosomes in parental cells (I–K; yellow arrows) but is largely absent from the endosomes in MICAL-L1 knockout cells (L–N). (O) Segmentation strategy for quantification of the percentage of FCHSD2 in contact with RAB5 Q79L endosomes. Square ROIs were made to include the maximal endosomal area within the ROI. (P) Quantification of I-N. In a demarked ROI around the RAB5 Q79L endosomes, the volume of FCHSD2 puncta that made contact with GFP-RAB5 Q79L endosomes was represented as a percentage of the total FCHSD2 volume in that region.

Article Snippet: The following plasmid constructs were used: GFP-RAB5 Q79L (Roberts et al., 1999), mCherry-RAB5 Q79L (35138, Addgene), FLAG-FCHSD2 (GenScript), FCHSD2GFP (Almeida-Souza et al., 2018), FCHSD2 Y576S+F607S – GFP (Almeida-Souza et al., 2018), and FCHSD2 YY478/480AAGFP (Almeida-Souza et al., 2018).

Techniques: Mutagenesis, Fluorescence, Staining, Membrane, Western Blot, Biomarker Discovery, Knock-Out, CRISPR, Transfection, Software

FIGURE 7: Visualization of decreased branched actin at endosomes in FCHSD2 knockdown cells. (A–O) WT parental (A–C), FCHSD2 knockout (D–F), FCHSD2 knockout + WT FCHSD2 rescue (G–I), FCHSD2 knockout + FCHSD2 SH3 A mutant rescue (J–L), and FCHSD2 knockout + FCHSD2 SH3 B mutant rescue (M–O) were transfected with mCherry-RAB5 Q79L. Cells were fixed and immunostained with an antibody against cortactin to mark branched actin. WT parental cells showed a robust cortactin localization at RAB5 Q79L endosomes (B, yellow arrows), whereas the FCHSD2 knockout cells show a significant decrease in cortactin localized to endosomes (E). Transfection of WT FCHSD2 rescued cortactin localization to the RAB5 endosomes (H, yellow arrows). Transfection with the FCHSD2 SH3 A mutant displayed impaired rescue of cortactin at endosomes (K), whereas transfection of FCHSD2 SH3 B mutant rescued cortactin endosomal localization (N, yellow arrows). These data suggest that the FCHSD2 SH3 A domain is required to promote branched actin polymerization at endosomes. (P) Quantification of A–O. Confocal Z-stack images were captured and analyzed by Imaris software using the surfaces function. mCherry-RAB5 Q79L structures were 3D rendered and a ROI around the enlarged endosomes was demarked. Cortactin puncta in this region were also 3D rendered. Cortactin surfaces that contacted RAB5 structures were filtered by setting the maximal “shortest distance to surface” at 1 × 10−7 nm. The volume of cortactin structures that contacted RAB5 structures was summed and represented as a percentage of the total cortactin volume in the designated ROI. (Q) Model for the role of FCHSD2 in fission at endosomes. FCHSD2 is recruited to and/or stabilized at endosomes through an interaction with MICAL-L1. FCHSD2 induces branched actin polymerization to promote endosome fission and receptor recycling. Insets in I, L and O indicate transfected cells.

Journal: Molecular Biology of the Cell

Article Title: Endosomal actin branching, fission and receptor recycling require FCHSD2 recruitment by MICAL-L1

doi: 10.1091/mbc.e24-07-0324

Figure Lengend Snippet: FIGURE 7: Visualization of decreased branched actin at endosomes in FCHSD2 knockdown cells. (A–O) WT parental (A–C), FCHSD2 knockout (D–F), FCHSD2 knockout + WT FCHSD2 rescue (G–I), FCHSD2 knockout + FCHSD2 SH3 A mutant rescue (J–L), and FCHSD2 knockout + FCHSD2 SH3 B mutant rescue (M–O) were transfected with mCherry-RAB5 Q79L. Cells were fixed and immunostained with an antibody against cortactin to mark branched actin. WT parental cells showed a robust cortactin localization at RAB5 Q79L endosomes (B, yellow arrows), whereas the FCHSD2 knockout cells show a significant decrease in cortactin localized to endosomes (E). Transfection of WT FCHSD2 rescued cortactin localization to the RAB5 endosomes (H, yellow arrows). Transfection with the FCHSD2 SH3 A mutant displayed impaired rescue of cortactin at endosomes (K), whereas transfection of FCHSD2 SH3 B mutant rescued cortactin endosomal localization (N, yellow arrows). These data suggest that the FCHSD2 SH3 A domain is required to promote branched actin polymerization at endosomes. (P) Quantification of A–O. Confocal Z-stack images were captured and analyzed by Imaris software using the surfaces function. mCherry-RAB5 Q79L structures were 3D rendered and a ROI around the enlarged endosomes was demarked. Cortactin puncta in this region were also 3D rendered. Cortactin surfaces that contacted RAB5 structures were filtered by setting the maximal “shortest distance to surface” at 1 × 10−7 nm. The volume of cortactin structures that contacted RAB5 structures was summed and represented as a percentage of the total cortactin volume in the designated ROI. (Q) Model for the role of FCHSD2 in fission at endosomes. FCHSD2 is recruited to and/or stabilized at endosomes through an interaction with MICAL-L1. FCHSD2 induces branched actin polymerization to promote endosome fission and receptor recycling. Insets in I, L and O indicate transfected cells.

Techniques: Knockdown, Knock-Out, Mutagenesis, Transfection, Software

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