Journal: Nature Communications

Article Title: A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

doi: 10.1038/s41467-024-49543-z

Figure Lengend Snippet: a CD147 internalization was monitored in HeLa cells subjected to N-WASP-KD, RTN3-KD or treatment with CK666 and stimulated with high dose EGF. Left, representative IF images, CD147 (green), EGF (red), DAPI (blue), Bar, 10 µm. Right upper, quantification of relative CD147 fluorescence intensity expressed as % of control. N = number of cells: Control/N = 338 ( n = 4); RTN3 = KD/N = 158, NWASP-KD/N = 237, CK666/N = 250 ( n = 3). Right lower, Alexa647-Tf internalization was monitored for 8 min at 37 °C in HeLa cells treated as above. The data reported for RTN3 KD are the same as displayed in Fig. . N=number of cells: Control/N = 246 ( n = 6); RTN3-KD/N = 185, NWASP-KD/N = 276, CK666/N = 295 ( n = 3). Box plots are defined as in Fig. . b , c EM morphometric analysis of the length of EGFR gold-positive TIs ( b ) and CCPs ( c ) in HeLa cells subjected to dynamin KD (Dyn-KD) and/or treated with CK666. Data are expressed as Fig. , mean ± SD; N = cell profiles. Control/N = 10, CK666/N = 10, Dyn-KD/N = 9, Dyn-KD/CK666/N = 10 ( b ); Control/N = 10, CK666/N = 10, Dyn-KD/N = 9, Dyn-KD/CK666/N = 10 (c); a representative experiment of n = 2 is shown. d Ratiometric analysis of membrane proximal actin (MPA) density. HeLa cells were subjected or not to RTN3 KD, followed by MPAct-mCherry and YFP-CaaX co-transfection, and stimulated with high or low EGF or left unstimulated (-EGF). Left, two representative images of the ratiometric analysis of MPA density (MPAct signal normalized over the CaaX PM marker). Bar, 10 μm. e Right, mean raw integrated fluorescence intensity ±SD is reported as a percentage relative to control cells. N = number of cells: -EGF/N = 424, LowEGF/N = 495, HighEGF/N = 568 ( n = 7). f , g Ratiometric analysis of MPA density as in d , in HeLa cells subjected to the indicated KDs ( f ) or inhibitor treatments ( g ) and stimulated with high dose EGF. Data are expressed as mean ± SD. Inhibitors were present during the stimulation. N = number of cells: ( f ) -EGF/N = 316/n = 5, Control/N = 356/n = 5, RTN3-KD/N = 409/n = 5, RTN4-KD/N = 311/n = 4, NWASP-KD/N = 370/n = 4; ( g ) Control/N = 306, CK666/N = 228, XeC/N = 231, MCUi11/N = 232, OMY/N = 189 ( n = 3). Exact p -values (Each pair Student’s t test, two-tailed) are shown in all panels; ns, not significant; n=biological replicates. Source data are provided as a Source Data file.

Article Snippet: Oligomycin (OMY), histamine, and succinate were from Sigma, xestospongin C (XeC) was from abcam, CK666 and bongkrekic acid were from Merck Life Technology S.R.L., MCUi11 was from CliniSciences and Gefitinib was from Merck Life Technology S.R.L., Ciliobrevin D was from TargetMol (T14965).

Techniques: Fluorescence, Control, Membrane, Cotransfection, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism

doi: 10.1038/s41467-024-49543-z

Figure Lengend Snippet: a CD147 internalization in HaCaT cells subjected to the indicated KDs or mock transfection (Ctrl). Internalized CD147 was measured by IF after stimulation with high dose Alexa647-EGF for 12 min at 37 °C, as described in Fig. . Mean integrated fluorescence intensity is reported as a percentage of the control cells. N = number of cells: Control/N = 301/n = 6, RTN3-KD/N = 97/n = 3, RTN4-KD/N = 179/n = 5, MCU-KD/N = 221/n = 4, IP3R-KD/N = 210/n = 4. b HaCaT cells expressing the Ca 2+ sensor PM-GCaMP6f were stimulated with high or low EGF. Left, Representative single-cell Ca 2+ response curves presented as the ratio of fluorescence emission at 490/406 nm. Right, AUC of the Ca 2+ response. N=number of cells: High EGF/N = 17, Low EGF/N = 20 ( n = 2). c Ca 2+ response in HaCaT cells subjected to RTN3 or RTN4 KD or to mock transfection (Ctrl) and stimulated with high EGF dose as in b . AUC is shown. N = number of cells: Control/N = 18, RTN3-KD/N = 25, RTN4-KD/N = 14. A representative experiment of three independent replicates is shown. d ΔΨm in HaCaT cells stimulated or not with low or high EGF, measured using TMRM. Total peak area is reported. N=number of cells: -EGF/N = 206, Low EGF/N = 253, High EGF/N = 327. A representative experiment of n = 2 is shown. e Wound healing in sub-confluent HaCaT cells stimulated or not with low or high EGF was monitored by time-lapse video microscopy. Left, representative images at the indicated time points. Middle, distance covered by cells in the different conditions corrected to the baseline at t = 0. Right, wound closure velocity (μm/h) at t = 24 h is shown. N=number of cells: -EGF/N = 15, Low EGF/N = 23, HighEGF/N = 34 ( n = 3). f Wound closure velocity (μm/h) in sub-confluent HaCaT cells stimulated with high EGF dose and subjected to RTN3 KD or the indicated treatments for 24 h. N = number of cells: Control/N = 98/n = 6, DMSO/N = 24/n = ), RTN3-KD/N = 40/n = 2, OMY/N = 44/n = 3, MCUi11/N = 22/n = 2, CK666/N = 28/n = 2, XeC/N = 30/n = 2). g Schematic showing the crosstalk between EGFR-NCE, ER, and mitochondria in controlling EGFR endocytosis, receptor fate and cell migration. Box plots in all panels are defined as in Fig. . Exact p -values (each pair Student’s t -test, two-tailed) are shown; ns not significant, n biological replicates. Source data are provided as a Source Data file.

Article Snippet: Oligomycin (OMY), histamine, and succinate were from Sigma, xestospongin C (XeC) was from abcam, CK666 and bongkrekic acid were from Merck Life Technology S.R.L., MCUi11 was from CliniSciences and Gefitinib was from Merck Life Technology S.R.L., Ciliobrevin D was from TargetMol (T14965).

Techniques: Transfection, Fluorescence, Control, Expressing, Single Cell, Microscopy, Migration, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Type V myosin focuses the polarisome and shapes the tip of yeast cells

doi: 10.1083/jcb.202006193

Figure Lengend Snippet: Actin is required for polarisome focusing. (A) myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2 RD from the genomic URA3 locus were spotted in 10-fold serial dilutions on SD-Ura − plates containing 70 µM methionine and incubated for 3 d at either 25°C (upper panel) or 30°C (lower panel). (B) myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2 RD from the genomic URA3 locus and coexpressing Spa2-mCherry were analyzed by fluorescence microscopy at either 27°C (left panel) or 31°C (right panel). Insets show the DIC image of the same cell in reduced magnification. (C) Intensity profiles of the Spa2-mCherry signals of the bud circumference of the cells in B at 27°C. (D) The polarisome distribution in the bud of the cells from B was classified as focused (gray), cortically spread (spread, blue), or diffuse (black; 27°C: n empty = 226, n MYO2 = 232, n myo2RD = 225; 31°C: n MYO2 = 223, n myo2RD = 224). (E) Left: Fluorescence microscopy of WT, bni1Δ , or bni1 Δ 1240–end cells expressing Spa2-GFP. Right: Intensity profiles of Spa2-GFP of the bud circumference of the cells from the left panel. (F) The polarisome distribution of the cells from E were classified as either focused (gray) or cortically spread (spread, blue; n WT = 234, n bni1Δ = 234, n bni1Δ1240–end = 227). (G) The actin cytoskeleton of WT (upper panel) or myo2 RD cells (lower panel) was stained with Alexa Fluor 488–phalloidin in the absence (left) or presence (right) of CK-666. All scale bars: 5 µm.

Article Snippet: Actin patches were removed by addition of 100 µM CK-666 (Merck) to the cell medium at 30°C, 10 min before cells were fixed.

Techniques: Plasmid Preparation, Expressing, Incubation, Fluorescence, Microscopy, Staining

Journal: Science Advances

Article Title: Actin kinetics shapes cortical network structure and mechanics

doi: 10.1126/sciadv.1501337

Figure Lengend Snippet: ( A ) Individual actin-GFP molecules appear as dot-like structures in the cortex of HeLa and M2 cells. Arrows indicate individual molecules. ( B to E ) Distribution of actin-GFP molecule lifetimes in wild-type (wt) HeLa cells (B), after application of the formin inhibitor SMIFH2 (C), and after application of the Arp2/3 inhibitor CK666 (D). Circles, experimental values; triangles, values from single-filament simulation of the processes illustrated in (E). Red lines give an analytical approximation of the latter. Parameter values are ω off,A = 0.23 s −1 , k off = 29 sub s -1 , and k on,0 = 28 sub s -1 . Insets give a log-linear presentation of the data. (C and D) Insets: Data from untreated cells are given in gray for comparison. (E) Illustration of the molecular processes accounted for in the quantitative analysis of actin molecule dynamics. This included free and formin-mediated, barbed-end elongation at rates k on,0 and k on,F , release of an elongation factor from the barbed end at rate ω off,F , release of a capping factor from the pointed end at rate ω off,A , and filament pointed-end disassembly at rate k off . These processes capture the dynamics of filaments with free barbed ends, as well as the effects of formin-mediated elongation, Arp2/3-complex capping, and of factors involved in filament disassembly.

Article Snippet: SMIFH2, CK666, and Y27632 were purchased from Merck Biosciences.

Techniques: Comparison

Journal: Science Advances

Article Title: Actin kinetics shapes cortical network structure and mechanics

doi: 10.1126/sciadv.1501337

Figure Lengend Snippet: ( A ) Inferred filament length distributions for HeLa (red) and M2 cells (blue). Symbols, finite cortex simulations; lines, analytical approximations (Supplementary Materials). In HeLa cells, formin-nucleated filaments had an average length of 1200 nm (M2: 600 nm), whereas the average length of Arp2/3-nucleated filaments was 120 nm (M2: 60 nm). Parameter values are r on = 9 s −1 μM −1 , r on,F = 45 s −1 μM −1 , k off = 28 s −1 , ω off,F = 0.12 s −1 , ω off,A = 0.3 s −1 , c tot = 80 μM, c form = 0.2 nM, and c arp = 24 nM. ( B ) Scanning electron micrographs of the actin cortex of control HeLa cells (top), cells treated with the formin inhibitor SMIFH2 (middle), and cells treated with the Arp2/3 inhibitor CK666 (bottom), with (right) and without (left) dashed lines indicating complete actin filaments. In control cells, areas of long and highly oriented filaments were clearly visible. These could not be seen in SMIFH2-treated cells. In contrast, in CK666-treated cells, long and highly oriented filaments were visible but short ones were not. Scale bar, 500 nm. ( C ) Snapshots of stochastic simulations of actin cortex turnover. Scale bar, 10 μm. ( D ) Experimental FRAP curve for HeLa cells (main panel) and M2 cells (inset) averaged over 28 experiments. Red curves, simulation results; see the Supplementary Materials for parameter values. Experimental data points are indicated in gray. Bars, SDs.

Article Snippet: SMIFH2, CK666, and Y27632 were purchased from Merck Biosciences.

Techniques: Control