Journal: bioRxiv

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

doi: 10.64898/2026.03.10.710749

Figure Lengend Snippet: Inhibition of branched actin leads to increased endosome size. (A-J) . HeLa cells were ( A-C ) untreated, ( D-E ) treated with the formin inhibitor SMIFH2 (25 µM), ( F-G ) treated with the inactive control CK-689 (300 µM), or ( H-J ) the ARP2/3 branched actin inhibitor CK-666 (300 µM) for 50 min. Cells were fixed and stained with EEA1 and cortactin. Merged images (panels C and J ) show decreased cortactin at endosomes upon CK-666 treatment. (K) . Quantification of the effects of the inhibitors on endosome size as depicted in A-J. Imaris software was used to render EEA1-decorated endosomes as surfaces, and endosome size for each treatment (µm 2 ) was normalized to the average endosome size of the untreated group. Quantification represents 30 images from three independent experiments, including ∼50,000 endosomes per treatment. A two-tailed Mann-Whitney nonparametric test was used to determine p -values between treatment groups.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Control, Staining, Software, 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 recycling is impaired upon ARP2/3 inhibition. (A-D) . HeLa cells on coverslips were incubated with fluorophore-labeled transferrin (Tf-488) for 10 minutes of uptake. Cells were ( A ) untreated, ( B ) treated with 25 µM SMIFH2, ( C ) treated with 300 µM CK-689, or ( D ) treated with 300 µM CK-666 during the transferrin uptake. Images are representative of a treated coverslip after uptake. (E-H) . Cells were chased in ( E ) complete media, ( F ) complete media containing 25 µM SMIFH2, ( G ) complete media containing 300 µM CK-689, or ( H ) complete media containing 300 µM CK-666 for 40 minutes to allow recycling. Images are representative of cells on an treated coverslip after chase. (I) . The arithmetic mean intensity of each image was analyzed using Zeiss Zen Blue software after uptake and chase. For each treatment condition, the internalized mean for the uptake was set at 100%, and the arithmetic mean intensity for each recycling image was expressed as a percentage of the normalized uptake for that condition. Quantification represents 30 images from three independent experiments. Statistical significance was determined using a two-tailed unpaired t -test.

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Labeling, Software, Two Tailed Test

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 and EGF are delayed at early endosomes upon ARP2/3 inhibition. (A-D) . HeLa cells were incubated with Tf-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( A-B ) complete media or ( C-D ) in media containing 300 µM CK-666. Representative images are from the 30 min chase time point. (E). The percentage of Tf-488 fluorescence in EEA1 endosomes was calculated by measuring the area of Tf-EEA1 overlap as a percentage of total Tf fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 30 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 15 min time point. (F). A bar graph showing the individual data points from the 30 min time point in ( E ). (G-J) . HeLa cells were serum starved for 1 h, then incubated with EGF-488 diluted in complete media for 10 min. Following uptake, cells were chased in ( G-H ) complete media or ( I-J ) in complete media containing 300 µM CK-666. Representative images are from the 45 min time point of media chase. (K). The percentage of EGF-488 fluorescence in EEA1-marked endosomes was calculated by measuring the area of EGF-EEA1 overlap as a percentage of total EGF fluorescence area. Statistical analysis was performed between the untreated and CK-666-treated groups at each time point. Quantification represents 24 images from three independent experiments. Statistical significance for the 15 min and 45 min time points was determined using a two-tailed Mann-Whitney nonparametric test, and an unpaired two-tailed t -test was used for the 30 min time point. (L). A bar graph showing the individual data points from the 45 min time point in ( K ).

Article Snippet: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Incubation, Fluorescence, 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: 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: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

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: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

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: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

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: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

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: HeLa cells (ATCC-CCL-2) were cultured with complete DMEM (high glucose) (ThermoFisher Scientific, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich), 1× penicillin-streptomycin, 100 μg/mL Normicin, and 2 mM L-glutamine at 37°C in a humidified incubator with 5% CO2.

Techniques: Inhibition, Transfection, Incubation, Two Tailed Test