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

Journal: Microorganisms

Article Title: Herpes Simplex Virus Type 1 Infection Induces the Formation of Tunneling Nanotubes

doi: 10.3390/microorganisms11081916

Figure Lengend Snippet: Fluorescence microscopy images showed the effect of CK666 on the formation of TNTs. ( A , B ) HCECs and Vero cells were stained with phalloidin green to visualize the nanotubes. Three images (per experiment) were taken per condition, with −100 cells examined, and the number of TNTs was counted. ( C ) Graph shows a significant reduction in the number of TNTs in the presence of CK666 (50 μM). Data are presented as mean ± SD. Student’s t -test was performed to compare between the groups, and the following significance values were considered: * p < 0.05, *** p < 0.001. ( D ) Cell viability of human corneal epithelial cells and Vero cells in the presence of CK666. Cells were treated with different concentrations of inhibitors, 0.1–100 μM, for 24 h, and the cell viability was measured by MTT assays. Only 100 μM was found to be toxic in two types of cells. Data shown are of three replicates.

Article Snippet: The 10 4 HCECs and Vero cells in 96-wells plate were treated with various concentrations of CK666 (AdooQ Bioscience LLC., Irvine, CA, USA) (0, 0.1, 1, 5, 10, 20, 50, 100 μM) and 0.1% dimethyl sulfoxide (DMSO, as a drugs vehicle) (Sigma, Cream Ridge, NJ, USA) for 24 h. After incubation with CK666 and DMSO, cells were incubated with 5 mg/mL concentration of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Beyotime Biotechnology Co., Ltd., Haimen, China) reagent in media for 4 h. Then, cells were incubated with Formazan solvent for 4 h and absorbance recorded using Multiskan Spectrum at 570 nm (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Techniques: Fluorescence, Microscopy, Staining

Journal: Microorganisms

Article Title: Herpes Simplex Virus Type 1 Infection Induces the Formation of Tunneling Nanotubes

doi: 10.3390/microorganisms11081916

Figure Lengend Snippet: CK666 affects the transmission of HSV-1. Confluent monolayers of cell plates in 24-well culture were infected with HSV-1 for 1 h, then treated with/without 50 μM CK666 for 24 h in HCECs ( A ) and Vero cells ( B ) and processed for the plaque assay. ( C ) Graph shows a reduction in the number of plaques in HCECs and Vero cells treated with CK666 compared to control cells. ( D ) HCECs and Veros were infected with wild-type HSV-1 (MOI of 1), and the effect of the addition of 50 μM CK666 on the HSV-1 DNA level in HCECs and Vero cells was quantified with qPCR. Data represent mean ± SD of triplicate assays. Student’s t -test was used to compare between the groups. ** p < 0.01, *** p < 0.001.

Article Snippet: The 10 4 HCECs and Vero cells in 96-wells plate were treated with various concentrations of CK666 (AdooQ Bioscience LLC., Irvine, CA, USA) (0, 0.1, 1, 5, 10, 20, 50, 100 μM) and 0.1% dimethyl sulfoxide (DMSO, as a drugs vehicle) (Sigma, Cream Ridge, NJ, USA) for 24 h. After incubation with CK666 and DMSO, cells were incubated with 5 mg/mL concentration of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Beyotime Biotechnology Co., Ltd., Haimen, China) reagent in media for 4 h. Then, cells were incubated with Formazan solvent for 4 h and absorbance recorded using Multiskan Spectrum at 570 nm (Multiskan GO, Thermo Scientific, Waltham, MA, USA).

Techniques: Transmission Assay, Infection, Plaque Assay, Control

Journal: bioRxiv

Article Title: Keratins and Plakin family cytolinker proteins control the length of epithelial microridge protrusions

doi: 10.1101/2020.02.18.954933

Figure Lengend Snippet: (A) SIM optical sections of a Krt17-GFP[BAC]-expressing zebrafish periderm cell at 48 hpf. Cartoon shows the relative location of Apical (A) and Medial (M) optical sections. (B) Oblique optical section through a cell with endogenously-tagged Krt17. This section shows both the microridge-like pattern at the apical surface of the cell (arrow), and the filamentous pattern deeper in the cell (arrowhead). (C) Projections and orthogonal views of SIM images of Krt17-GFP[BAC]- and Lifeact-Ruby-expressing cells at the indicated developmental stages. White boxes, regions of magnification in zoom panels. Orthogonal views (XZ) at 19hpf show Krt17 is not in pegs, but at 48 hpf localizes alongside Actin in microridges. (D) Dot and box plot of average microridge length per cell in cells over-expressing Krt17-GFP[BAC] at 48hpf. *p<0.05, Wilcoxon rank-sum test. n=24 cells from 4 fish. Blue line shows the median average microridge length per cell in WT animals (see ). (E) Dot and box plot of average microridge length per cell in cells at 48hpf after 30min treatment with DMSO or CK666. ***p<0.005, Wilcoxon rank-sum test. n=11-12 cells from 3 fish. (F) Dot and box plot of microridge and Keratin overlap coefficients, comparing the colocalization of each protein at 0 min with its localization after 30 min DMSO or CK666 treatment. **p<0.01, ***p<0.005, Wilcoxon rank-sum test. n=11-12 cells from 3. (G) Krt17-GFP[BAC]- and Lifeact-Ruby-expressing cells at 48hpf treated with CK666 for 30 minutes. Arrows show that the F-actin microridge pattern was disrupted after 30min of treatment. Arrowheads show that the Krt17 microridge pattern was retained. See for images of control DMSO treatments. (H) GFP-PH-PLC and Krt17-mRuby[BAC] in periderm cells treated with CK666 for 30 minutes at 48hpf. GFP-PH-PLC was expressed by crossing BAC(ΔNp63:Gal4FF) la213 to UAS:GFP-PH-PLC, and Krt17-mRuby[BAC] was expressed by transient transgenesis. Arrowheads show that membrane protrusions and the Krt17 microridge pattern were maintained. See for images of control DMSO treatments, and orthogonal views. Scale bars:10µm (A-C, G-H, blue line) and 1µm (C, white line).

Article Snippet: CK666 (Fisher Scientific) was dissolved in DMSO.

Techniques: Expressing

Journal: bioRxiv

Article Title: Keratins and Plakin family cytolinker proteins control the length of epithelial microridge protrusions

doi: 10.1101/2020.02.18.954933

Figure Lengend Snippet: (A) Krt17-GFP[BAC]- and Lifeact-Ruby-expressing cells in 48hpf fish at time 0 and 30 minutes after DMSO or CK666 treatment. Arrows show that the F-actin microridge pattern was disrupted after 30 min of CK666 treatment. Arrowheads show that the Keratin microridge pattern was retained after 30min of CK666 or DMSO treatment. Image of CK666 treatment is replicated from , for comparison. (B) GFP-PH-PLC (membrane) and Lifeact-Ruby in 48 hpf periderm cells at time 0 and 30min after treatment with DMSO or CK666. BAC(ΔNp63:Gal4FF) la213 to UAS:GFP-PH-PLC, and Krt5:Lifeact-Ruby was expressed by transient transgenesis. Arrows show that the membrane and Actin microridge pattern was disrupted after 30min of CK666 treatment. Arrowheads show that the membrane and Actin microridge pattern was retained in DMSO controls. (C) Projection and orthogonal views of GFP-PH-PLC and Krt17-mRuby[BAC] in 48hpf periderm cells at time 0 and 30min after treatment with DMSO or CK666. BAC(ΔNp63:Gal4FF) la213 to UAS:GFP-PH-PLC, and Krt17-mRuby[BAC] was expressed by transient transgenesis. Arrows show that the membrane and Krt17 microridge patterns were retained after 30min DMSO or CK666 treatment. Orthogonal views (XZ) show that Krt17 preserved the protrusive membrane structure after 30min CK666 treatment. Image of CK666 treatment is replicated from , for comparison. Scale bars: 10µm (A-C) and 1µm (Orthogonal images in C).

Article Snippet: CK666 (Fisher Scientific) was dissolved in DMSO.

Techniques: Expressing