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    Nikon phase contrast microscopy
    FIGURE 6 Light‐induced ChR1 degradation and starting levels in mutants affected in components of the intraflagellar transport (IFT) machinery and the motor proteins dynein and kinesin‐2. (a–c) ΔIFT88 cells were incubated along with the rescued strain CC‐5687 at the end of the night phase for 1 h in the dark in the presence of cycloheximide (CHX) before being illuminated with WL + UV (81 µmol m−2 s−1) for the indicated times. Protein extraction, further processing and ChR1 quantification were as described. (a) shows a representative immunoblot probed with anti‐ChR1 and Coomassie Brilliant Blue (Coomassie) staining of the lower part of the blot to demonstrate equal protein load. Black and white bars above the immunoblot denote whether the cells were kept in the dark or illuminated for the indicated times. (b) Quantification of the immunoblot analyses shown in (a). Data points are the mean ± SEM (n = three independent biological replicates). (c) Normalized ChR1 starting levels before the onset of illumination. (d, e) Light‐induced ChR1 degradation (d) and ChR1 starting levels (e) in cells just before illumination of the insertional mutant strain bbs4‐1 and the rescued strain CC‐4373. Treatments and ChR1 quantification were as described above. Data points are the mean ± SEM (n = four independent biological replicates). (f, g) Cells of the strain ΔLC8 along with the rescued strain CC‐3939 were treated as described in (a–c). (f) shows the light‐induced degradation kinetics and (g) the ChR1 starting level normalized to CC‐3939 just before illumination. Data plotted are the mean ± SEM of four independent biological replicates. (h, i) Cells of the kinesin‐2‐null mutant strain ΔKin2 and strain CC‐2929 (wild‐type) cells were treated and analysed as above. ΔKin2 lacks cilia completely (Matsuura et al., 2002). (h) shows the ChR1 degradation kinetics and (i) the normalized starting levels of the photoreceptor. Data points are the mean ± SEM (n = four independent biological replicates). (j, k) Cells of the temperature‐sensitive Fla10‐1 mutant of kinesin‐2 (Walther et al., 1994) were grown at 15°C and shifted for 20 h either to 33°C or kept at 15°C. Resorption of cilia at 33°C was verified by phase contrast <t>microscopy.</t> At the end of the night phase both cultures were supplemented with CHX, pre‐incubated for 1 h in the dark and the illuminated with WL + UV as above. The desired temperature was maintained during the illumination period. Data for the degradation kinetics (e) and the normalized starting levels at the end of the night phase (f) are the mean ± SEM of three independent biological replicates. [Color figure can be viewed at wileyonlinelibrary.com]
    Phase Contrast Microscopy, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 59420 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phase contrast microscopy - by Bioz Stars, 2026-09
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    1) Product Images from "Insights into degradation and targeting of the photoreceptor channelrhodopsin-1."

    Article Title: Insights into degradation and targeting of the photoreceptor channelrhodopsin-1.

    Journal: Plant, cell & environment

    doi: 10.1111/pce.15017

    FIGURE 6 Light‐induced ChR1 degradation and starting levels in mutants affected in components of the intraflagellar transport (IFT) machinery and the motor proteins dynein and kinesin‐2. (a–c) ΔIFT88 cells were incubated along with the rescued strain CC‐5687 at the end of the night phase for 1 h in the dark in the presence of cycloheximide (CHX) before being illuminated with WL + UV (81 µmol m−2 s−1) for the indicated times. Protein extraction, further processing and ChR1 quantification were as described. (a) shows a representative immunoblot probed with anti‐ChR1 and Coomassie Brilliant Blue (Coomassie) staining of the lower part of the blot to demonstrate equal protein load. Black and white bars above the immunoblot denote whether the cells were kept in the dark or illuminated for the indicated times. (b) Quantification of the immunoblot analyses shown in (a). Data points are the mean ± SEM (n = three independent biological replicates). (c) Normalized ChR1 starting levels before the onset of illumination. (d, e) Light‐induced ChR1 degradation (d) and ChR1 starting levels (e) in cells just before illumination of the insertional mutant strain bbs4‐1 and the rescued strain CC‐4373. Treatments and ChR1 quantification were as described above. Data points are the mean ± SEM (n = four independent biological replicates). (f, g) Cells of the strain ΔLC8 along with the rescued strain CC‐3939 were treated as described in (a–c). (f) shows the light‐induced degradation kinetics and (g) the ChR1 starting level normalized to CC‐3939 just before illumination. Data plotted are the mean ± SEM of four independent biological replicates. (h, i) Cells of the kinesin‐2‐null mutant strain ΔKin2 and strain CC‐2929 (wild‐type) cells were treated and analysed as above. ΔKin2 lacks cilia completely (Matsuura et al., 2002). (h) shows the ChR1 degradation kinetics and (i) the normalized starting levels of the photoreceptor. Data points are the mean ± SEM (n = four independent biological replicates). (j, k) Cells of the temperature‐sensitive Fla10‐1 mutant of kinesin‐2 (Walther et al., 1994) were grown at 15°C and shifted for 20 h either to 33°C or kept at 15°C. Resorption of cilia at 33°C was verified by phase contrast microscopy. At the end of the night phase both cultures were supplemented with CHX, pre‐incubated for 1 h in the dark and the illuminated with WL + UV as above. The desired temperature was maintained during the illumination period. Data for the degradation kinetics (e) and the normalized starting levels at the end of the night phase (f) are the mean ± SEM of three independent biological replicates. [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: FIGURE 6 Light‐induced ChR1 degradation and starting levels in mutants affected in components of the intraflagellar transport (IFT) machinery and the motor proteins dynein and kinesin‐2. (a–c) ΔIFT88 cells were incubated along with the rescued strain CC‐5687 at the end of the night phase for 1 h in the dark in the presence of cycloheximide (CHX) before being illuminated with WL + UV (81 µmol m−2 s−1) for the indicated times. Protein extraction, further processing and ChR1 quantification were as described. (a) shows a representative immunoblot probed with anti‐ChR1 and Coomassie Brilliant Blue (Coomassie) staining of the lower part of the blot to demonstrate equal protein load. Black and white bars above the immunoblot denote whether the cells were kept in the dark or illuminated for the indicated times. (b) Quantification of the immunoblot analyses shown in (a). Data points are the mean ± SEM (n = three independent biological replicates). (c) Normalized ChR1 starting levels before the onset of illumination. (d, e) Light‐induced ChR1 degradation (d) and ChR1 starting levels (e) in cells just before illumination of the insertional mutant strain bbs4‐1 and the rescued strain CC‐4373. Treatments and ChR1 quantification were as described above. Data points are the mean ± SEM (n = four independent biological replicates). (f, g) Cells of the strain ΔLC8 along with the rescued strain CC‐3939 were treated as described in (a–c). (f) shows the light‐induced degradation kinetics and (g) the ChR1 starting level normalized to CC‐3939 just before illumination. Data plotted are the mean ± SEM of four independent biological replicates. (h, i) Cells of the kinesin‐2‐null mutant strain ΔKin2 and strain CC‐2929 (wild‐type) cells were treated and analysed as above. ΔKin2 lacks cilia completely (Matsuura et al., 2002). (h) shows the ChR1 degradation kinetics and (i) the normalized starting levels of the photoreceptor. Data points are the mean ± SEM (n = four independent biological replicates). (j, k) Cells of the temperature‐sensitive Fla10‐1 mutant of kinesin‐2 (Walther et al., 1994) were grown at 15°C and shifted for 20 h either to 33°C or kept at 15°C. Resorption of cilia at 33°C was verified by phase contrast microscopy. At the end of the night phase both cultures were supplemented with CHX, pre‐incubated for 1 h in the dark and the illuminated with WL + UV as above. The desired temperature was maintained during the illumination period. Data for the degradation kinetics (e) and the normalized starting levels at the end of the night phase (f) are the mean ± SEM of three independent biological replicates. [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Incubation, Protein Extraction, Western Blot, Staining, Mutagenesis, Microscopy

    FIGURE 7 Light‐induced ChR1 degradation and dark levels in strains affected in cilia formation. Cells of the indicated strains were pre‐incubated for 1 h in the dark with cycloheximide (CHX) and illuminated for the indicated times with WL + UV (81 µmol m−2 s−1). Further processing and quantification of ChR1 was done as described. (a, b) Light‐induced ChR1 degradation of cells possessing only the trans cilium (uni1) and two cilia lacking (bld) strains (a) and their ChR1 starting levels (b) at the end of the night phase relative to strain CC‐125 (wt). Data are the mean ± SEM of three independent biological replicates. (c–e) Light‐induced ChR1 degradation (c, d) and relative ChR1 starting levels (e) of two cilia lacking mutant strains (CC‐2906, CC‐2907) and their corresponding suppressor mutants (CC‐2908, CC‐2909). Data are the mean ± SEM of three independent biological replicates. (f) Wild‐type cells (CC‐125) were pre‐incubated at the end of the night phase in the dark for 1 h with CHX and, as indicated, with or without colchicine (CLC). Cells were then deciliated under red safety light by pH shock with acetate buffer. Deciliation was verified by phase contrast microscopy (Supporting Information S1: Figure S10). The corresponding control cultures were treated as these cells, except that Tris‐acetate phosphate (TAP) was used instead of the acetate buffer. Cell bodies were pelleted, suspended in fresh TAP supplemented with CHX and CLC as indicated and
    Figure Legend Snippet: FIGURE 7 Light‐induced ChR1 degradation and dark levels in strains affected in cilia formation. Cells of the indicated strains were pre‐incubated for 1 h in the dark with cycloheximide (CHX) and illuminated for the indicated times with WL + UV (81 µmol m−2 s−1). Further processing and quantification of ChR1 was done as described. (a, b) Light‐induced ChR1 degradation of cells possessing only the trans cilium (uni1) and two cilia lacking (bld) strains (a) and their ChR1 starting levels (b) at the end of the night phase relative to strain CC‐125 (wt). Data are the mean ± SEM of three independent biological replicates. (c–e) Light‐induced ChR1 degradation (c, d) and relative ChR1 starting levels (e) of two cilia lacking mutant strains (CC‐2906, CC‐2907) and their corresponding suppressor mutants (CC‐2908, CC‐2909). Data are the mean ± SEM of three independent biological replicates. (f) Wild‐type cells (CC‐125) were pre‐incubated at the end of the night phase in the dark for 1 h with CHX and, as indicated, with or without colchicine (CLC). Cells were then deciliated under red safety light by pH shock with acetate buffer. Deciliation was verified by phase contrast microscopy (Supporting Information S1: Figure S10). The corresponding control cultures were treated as these cells, except that Tris‐acetate phosphate (TAP) was used instead of the acetate buffer. Cell bodies were pelleted, suspended in fresh TAP supplemented with CHX and CLC as indicated and

    Techniques Used: Incubation, Mutagenesis, Microscopy, Control

    FIGURE 11 Extracellular vesicles from strains with and without cilia differ in size and protein composition. Whole‐mount electron microscopy of fixed, negatively stained extracellular vesicles from the ciliated strain CC‐2908 (a–e) and strain CC‐2906 with no cilia (f–j). The extracellular vesicles exhibit the typical cup‐shaped morphology of exosomes. No vesicles were visible in identically treated control grids with buffer. Extracellular vesicles were isolated from the supernatant of 5 billion cells transferred at the end of the night phase to an identical volume of fresh Tris‐acetate phosphate (TAP) medium after a 2 h incubation period in the dark. Scale bars: 200 nm (a–e, g–j) and 500 nm (f). (k, l) Size distribution profiles of the extracellular vesicles. Images were analysed with the TEM ExosomeAnalyzer (Kotrbová et al., 2019; settings: minimal vesicle size 10 nm and 5 nm bin size). (m) Protein patterns of extracellular vesicles (EV) isolated from the indicated strains. bld2 and CC‐2906 cells lack cilia. Protein load based on equal cell number used for the isolation of extracellular vesicles. For comparison also 6 µg of crude extracts (CEs) resolved on the same gel are shown. [Color figure can be viewed at wileyonlinelibrary.com]
    Figure Legend Snippet: FIGURE 11 Extracellular vesicles from strains with and without cilia differ in size and protein composition. Whole‐mount electron microscopy of fixed, negatively stained extracellular vesicles from the ciliated strain CC‐2908 (a–e) and strain CC‐2906 with no cilia (f–j). The extracellular vesicles exhibit the typical cup‐shaped morphology of exosomes. No vesicles were visible in identically treated control grids with buffer. Extracellular vesicles were isolated from the supernatant of 5 billion cells transferred at the end of the night phase to an identical volume of fresh Tris‐acetate phosphate (TAP) medium after a 2 h incubation period in the dark. Scale bars: 200 nm (a–e, g–j) and 500 nm (f). (k, l) Size distribution profiles of the extracellular vesicles. Images were analysed with the TEM ExosomeAnalyzer (Kotrbová et al., 2019; settings: minimal vesicle size 10 nm and 5 nm bin size). (m) Protein patterns of extracellular vesicles (EV) isolated from the indicated strains. bld2 and CC‐2906 cells lack cilia. Protein load based on equal cell number used for the isolation of extracellular vesicles. For comparison also 6 µg of crude extracts (CEs) resolved on the same gel are shown. [Color figure can be viewed at wileyonlinelibrary.com]

    Techniques Used: Electron Microscopy, Staining, Control, Isolation, Incubation, Comparison

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