Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Schematic for domain structures of Ect2 wild-type (Ect2-wt), N-terminally truncated active (ΔN-Ect2) and the catalytically inactive variant with PVQR->AAAA (564-567) substitutions (ΔN-Ect2-DHmut). B) Representative maximum intensity projections of 3D-SIM images of U2OS cells expressing EGFP control or the indicated EGFP-tagged Ect2 constructs. Upper panels show EGFP or EGFP-tagged Ect2 localization, middle panels display F-actin stained with phalloidin, and lower panels present magnified views of the regions depicted in the actin images. Scale bar: 10 µm. n = 7-15 cells. C) Quantification of mean phalloidin fluorescence intensity in wide-field images of cells expressing the indicated constructs. Cells were stained with WGA to visualize cell morphology and with phalloidin to label F-actin. Transfected cells were identified EGFP fluorescence. F-actin intensity was quantified by automated image analysis as described in the Methods. Data represent n ≥ 276 cells from 4 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. D and H) Representative TIRF images of the Rho activity sensor (mCherry-Rhotekin RBD) co-expressing either EGFP control, constitutively active Ect2 (EGFP-ΔN-Ect2) (D) or constitutively active GEF-H1 C53R (H) . Two phenotypes induced by active Ect2 are shown in (D) : (top) reduced pulsatory Rho sensor signal dynamics and (bottom) peripheral enrichment of Rho sensor signal with slow circumferential movement (white arrow). Frame rate: 3 frames/min, scale bar, 20 µm. E and F) Percent cells with peripheral Rho sensor enrichment (E) and the frequency of Rho sensor pulses in the central cell region (F) ; n ≥ 32 cells from 4 independent experiments. (G) Average pulse frequency of the Rho activity sensor signal upon co-expression of EGFP-control, active EGFP-ΔN-Ect2 or a mutant which cannot bind to active Rho (EGFP-ΔN-Ect2-RBmut); n ≥ 25 cells from 3 independent experiments. Bars indicate mean ± SEM.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Variant Assay, Expressing, Control, Construct, Staining, Fluorescence, Transfection, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: Background-corrected average EGFP intensity in U2OS cells expressing EGFP control (black dots) or EGFP-ΔN-Ect2 (green dots), plotted against the normalized mean pulse frequency of the RBD sensor. Each dot represents a single cell. Data are from n ≥52 cells across three independent experiments.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Expressing, Control, Single Cell

Journal: bioRxiv

Article Title: Constitutive plasma membrane interaction of active Rho GEF Ect2 inhibits cortex contraction pulses

doi: 10.64898/2026.06.03.729549

Figure Lengend Snippet: A) Representative spinning disk confocal images of fixed U2OS cells transfected with either EGFP control, active Ect2 (EGFP-ΔN-Ect2), or a variant lacking the C-terminal PBC region (EGFP-ΔN-Ect2-ΔPBC). A schematic of the constructs is shown in . Upper panels: single central z-plane, lower panels: side views show orthogonal (x-z) projections of the z-stack along the line indicated in the corresponding upper panels. Scale bars: 10 µm (xy), 5 µm (z), n=50-56 cells from 3 independent experiments. B) Average pulse frequency of the Rho activity sensor signal (mCherry-Rhotekin-RBD) in cells expressing the indicated Ect2 variants. Red dots mark cells that generate fast high-amplitude pulses. n ≥ 51 cells from 3 independent experiments. using one-way ANOVA followed by Tukey’s post hoc test. Bars indicate mean ± SEM. C, E, G) Representative TIRF images of cells expressing the Rho activity sensor (right) and the indicated constructs (left), respectively. Frame rate: 3 frames/min, scale bar = 20 µm. D, F, H) Mean normalized intensity values of the Rho sensor signal in the corresponding cell regions (white boxes) in (C, E, F) . I) Proposed interplay between Ect2 and pulsatory Rho contraction signal network dynamics. Left: Pulsatory Rho contraction depends on a positive feedback loop in which Lbc-GEFs are recruited to the plasma membrane through binding to active Rho (right angled blue arrow). Curved red arrow points to enzymatic activation. Middle: In contrast, Ect2 is constitutively associated with the plasma membrane, independently of active Rho . Increased local concentration of Ect2 may result in a higher effective on-rate, thereby conferring a kinetic advantage that can outcompete Lbc-GEFs and thereby suppress Rho activity pulses. Curved red arrows illustrate enzymatic activation and allosteric activation. Right: Loss of the C-terminal PBC reduces Ect2 plasma membrane association, shifting its properties towards a Lbc-GEF-like phenotype, that is based on plasma membrane recruitment to active Rho, and that stimulates Rho activity pulses.

Article Snippet: Human U2OS osteosarcoma cells (HTB-96; ATCC) were maintained at 37 °C and 5 % CO 2 humidified atmosphere using standard cell culture techniques (DMEM + GlutaMAXTM medium, 10 % FBS, Life technologies; Gibco).

Techniques: Transfection, Control, Variant Assay, Construct, Activity Assay, Expressing, Clinical Proteomics, Membrane, Binding Assay, Activation Assay, Concentration Assay