Journal: bioRxiv

Article Title: Rab27-dependent egress of SARS-CoV-2 via secretory amphisomes

doi: 10.64898/2025.12.15.694351

Figure Lengend Snippet: (A) Model of Rab27-dependent SARS-CoV-2 secretion and inhibitory mechanism by Nexinhib20. Intraluminal vesicles (ILV). (B) Maximum (max) projection of total internal reflection fluorescence (TIRF) microscopy images of CD63-pHluorin secretion events in Vero E6 cells treated with DMSO (left) or 2000 nM Nexinhib20 (right). Zoom shows example of one secretion event at different timepoints. (C) Quantification of the number CD63-pHluorin secretion events per cell per minute (min) in Vero E6 cells treated with different concentrations of Nexinhib20. n=number of analyzed cells: n=14-19 per condition, 3 independent replicates (refer to supplemental table 1 for details). (D) High-content imaging-based quantification of Vero E6 cell density in the presence of different concentrations of Nexinhib20 after 24 or 48 hours compared to control, DMSO treated Vero E6 cells (e.a. a fold change of 1 indicates the cell density of the Nexinhib20 sample is similar to the DMSO treated sample). n=number of analyzed field of views: n= 4-5 FOV in independent replicates (refer to supplemental table 1 for details). (E) Vero E6 cells infected with SARS-CoV-2 and treated with DMSO (left) or 800 nM Nexinhib20 (right) and stained for CD63 (magenta) and spike (blue). Zooms show Spike-negative CD63-positive vesicles (DMSO) or Spike-positive CD63-positive vesicles (Nexinhib20). (F) Quantification of the Spike intensity in CD63-positive vesicles in SARS-CoV-2 infected Vero E6 cells normalized to the DMSO control (y axis is shown with a 4-fold increments scale). 3 independent replicates (refer to supplemental table 1 for details). (G) Vero E6 cells infected with SARS-CoV-2 and treated for with 800 nM Nexinhib20 and stained for LC3 (yellow), Spike (blue) and F-actin (phalloidin, purple). Zooms show Spike-positive LC3-positive vesicles. (H) qRT-PCR of Vero E6 cells infected with SARS-CoV-2 and treated for XX hours with indicated concentration Nexinhib20 and fixed at different timepoints (hour (h) and days (d) post infection (pi)). ΔCT is the normalized expression of the SARS-CoV-2 E gene to a reference gene. n=number of analyzed wells: n= 16 from 3 independent replicates (refer to supplemental table 1 for details). (I) High-content-based imaging of SARS-CoV-2-infected Vero E6 cells treated with DMSO or 800 nM Nexinhib20 and stained for spike (yellow) and CD63 (blue). (J) Quantification of the fraction of SARS-CoV-2 infected cells to the total number of cells on the coverslip in DMSO treated or Nexinhib20 treated cells at 1 or 2 dpi. Bar graphs represent mean±SD. In graph (H), dots represent individual wells. See also supplemental table 1. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, ns = non-significant (Student’s t -test, unpaired). Scale bars are 10 µm (B,E), 100 µm (G).

Article Snippet: After 1 h pre-incubation, CD63-pHluorin was imaged at 3 frames/s using an objective-based azimuthal ILAS2 TIRF microscope (Nikon Eclipse Ti, modified by Roper Scientific), equipped with Nikon Apo TIRF 100x N.A.

Techniques: Fluorescence, Microscopy, Imaging, Control, Infection, Staining, Quantitative RT-PCR, Concentration Assay, Expressing

Journal: Nature Communications

Article Title: Xanthomonas effector XopR hijacks host actin cytoskeleton via complex coacervation

doi: 10.1038/s41467-021-24375-3

Figure Lengend Snippet: a Representative images of the AtFH6-GFP clusters and moving trajectories in Arabidopsis . Seven-day-old seedlings were dip-inoculated with Xcc/XccΔhrcC/XccΔxopR for 24 h before imaging using VA-TIRFM. b – e Distributions of signal intensity (from left to right, n = 454, n = 406, n = 650, and n = 636 punctates), mean square displacement (MSD) and diffusion coefficient ( n = 0 movies from six seedlings for each infection assay), and percentage distribution of the bleaching step ( n = 165 punctates for mock, n = 167 for Xcc , n = 165 for XccΔhrcC , and n = 164 for XccΔxopR ) were analyzed for AtFH6-GFP foci. The relative ratios of total bleaching steps are indicated in brackets, which were normalized by mock without Xcc . Error bands and error bars in Fig. 3c, d are ± SD. Whiskers represent min to max. f Representative dual-color TIRF images of 2.5 nM AtFH1-FH1C (10% Alexa647-AtFH1-FH1C) with 2.5 nM XopR (10% XopR-mRuby2) on an immobilized supported lipid bilayer (SLB). g Signal intensity quantification of AtFH1-FH1C (50% Alexa647 labeled) on SLB for Supplementary Fig. ( n = 250 particles, Error bar, SD). h Representative dual-color confocal images of AtFH1-FH1C (5 μM, 10% Alexa647-AtFH1-FH1C) with XopR (5 μM, 10% Alexa488-XopR) that were incubated on dynamic SLB with 50 mM NaCl for 15 min before imaging. i Actin polymerization rate in the pyrene–actin assay, which was normalized by spontaneous actin polymerization, in the presence of 100 nM AtFH1-FH1C and XopR (left to right, 0, 25, 50, 100, 200, 400, and 1600 nM) at the indicated stoichiometries in the presence of 5 μM profilin AtPRF1 ( n = 4 for 0, 25, 50, 100, 200 nM, n = 6 for 400 nM, and n = 7 for 1600 nM; Error bar, SD). Scale bar: 2 μm for a , 10 μm for b – f , 2 μm for magnified images in f , 10 μm for h . Two-tailed Student’s t -test was performed assuming equal variance. Ns no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The microscope was equipped with a perfect focus system that prevents focus drift, an iLAS2 motorized TIRF illuminator (Roper Scientific, Evry Cedex, France), and Prime95b sCMOS camera (Photometrics, Tucson, AZ).

Techniques: Imaging, Diffusion-based Assay, Infection, Labeling, Incubation, Pyrene Actin Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Xanthomonas effector XopR hijacks host actin cytoskeleton via complex coacervation

doi: 10.1038/s41467-021-24375-3

Figure Lengend Snippet: a Schematic domain illustration of four XopR truncation variants, EHWH2, HH, WH2, and WH2α. b MST binding curves of LatB-G-actin titrated with different XopR peptides with three biological replicates each. n = 3; Error bar, SD. c High-speed F-actin cosedimentation assay using MBP-EHWH2-msfGFP, MBP-HH-msfGFP, MBP-WH2-msfGFP, and MBP-WH2α-msfGFP. The data were fit using a Hill equation. d Negative stain electron microscopy (EM) of F-actin bundles formed by mixing 1 μM XopR with 0.2 μM F-actin in 150 mM NaCl. Scale bar from left to right: 400, 50, and 50 nm. e Micrographs of 0.2 μM F-actin in the presence of 10 μM XopR and XopRΔHH in the indicated NaCl buffer. F-actin was labeled with Acti-stain™ phalloidin. Scale bar = 5 μm. f Low-speed F-actin cosedimentation assay with XopR and XopRΔHH in the buffer with both 150 and 200 mM NaCl. g Low-speed cosedimentation assay of XopR full-length, XopR-IDR and XopR-Cter in 150 mM NaCl solution ( n = 3 biological replicates). Data were presented as mean values ± SD. The data were fit using a Hill equation. h Representative time-lapse images of TIRF-actin polymerization over 480 s with 0.5 μM G-actin (10% Oregon-actin), 100 nM AtFH1-FH1C, and 400 nM XopR under 50 mM NaCl conditions. Scale bar = 2 μm for f .

Article Snippet: The microscope was equipped with a perfect focus system that prevents focus drift, an iLAS2 motorized TIRF illuminator (Roper Scientific, Evry Cedex, France), and Prime95b sCMOS camera (Photometrics, Tucson, AZ).

Techniques: Binding Assay, Staining, Electron Microscopy, Labeling

Journal: Nature Communications

Article Title: Xanthomonas effector XopR hijacks host actin cytoskeleton via complex coacervation

doi: 10.1038/s41467-021-24375-3

Figure Lengend Snippet: a Representative time-lapse TIRF images of F-actin depolymerization. To obtain these images, 100 nM XopR, 100 nM XopRΔCC, 0.2 μM AtADF3, and 20 μM CC peptide were used. b Mean fluorescence intensity of F-actin of a ( n = 6, data were presented as mean values with error bands which represent SD). c Representative images of Lifeact-Venus in epidermal cells of WT Arabidopsis cotyledons. Seven-day-old seedlings were flood-inoculated with Xcc or XccΔxopR , and then treated with 1 μM actin LatB for 1 h after 24 hpi. Scale bar = 10 μm. d F-actin density quantification in c ( n = 50 images from five individual seedlings (Data were presented as mean values ± SD). e Representative images of Lifeact-Venus in epidermal cells of Arabidopsis cotyledons expressing XVE-XopR. The expression of XopR was first induced for 24 h using 10 μM β−estradiol before being subjected to 1 μM LatB treatment for 1 h before imaging. Scale bar = 5 μm for a , Scale bar = 10 μm for c , e . Two-tailed Student’s t -test was performed assuming equal variance. Ns no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The microscope was equipped with a perfect focus system that prevents focus drift, an iLAS2 motorized TIRF illuminator (Roper Scientific, Evry Cedex, France), and Prime95b sCMOS camera (Photometrics, Tucson, AZ).

Techniques: Fluorescence, Expressing, Imaging, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

doi: 10.1083/jcb.202009063

Figure Lengend Snippet: High specificity and selectivity of fibrinogen micropatterning on PLL-PEG surfaces. (A) Fibrinogen-Alexa546 (50 µg/ml) and NeutrAvidin-Dylight-550 (50 µg/ml) were micropatterned on PLL-PEG–coated glass using LIMAP with identical UV exposure and micropattern shape. After washing, red fluorescence of the micropatterns was imaged by TIRF microscopy (TIRFM) using identical settings (left and middle). Alternatively, a higher dynamic range lookup table was applied to the image on the right. (B) Quantification of the effects seen in A. Mean ± SEM of the selectivity and homogeneity (see Materials and methods). Statistics were performed using a Mann–Whitney rank-sum test. n, number of micropatterns measured. Fibrinogen quantitatively micropatterns better than NeutrAvidin. (C) Scheme illustrating the different steps for sequential multiplexed micropatterning of three fibrinogens labeled with different fluorophores (ATTO488, Alexa546, and Alexa647). (D) Multiplexed micropatterning of fibrinogen-ATTO488, Alexa546, and Alexa647 (50 µg/ml) using the scheme depicted in C onto PLL-PEG–coated glass. Note that there is high specificity of the fibrinogen for their specific micropattern and minimum overlap between the three fluorescent fibrinogens. (E) Quantification of the effects seen in D. The fluorescence of each fibrinogen was measured on the three successive micropatterns and normalized to the intensity of their respective micropattern. Mean ± SEM. Statistics were performed using a one-way ANOVA test followed by a Tukey post hoc test (P < 0.0001). n, number of micropatterns measured. Note that the amount of fibrinogen deposited onto the nonintended micropatterns is minimal. Scale bars, 10 µm. n.s., not significant.

Article Snippet: Conversely, the TIRF imaging arm is composed of an azimuthal TIRF illuminator (iLas2; GATACA Systems) modified to have an extended field of view (Cairn) to match the full field of view of the camera.

Techniques: Fluorescence, Microscopy, MANN-WHITNEY, Labeling

Journal: The Journal of Cell Biology

Article Title: High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

doi: 10.1083/jcb.202009063

Figure Lengend Snippet: Fibrinogen micropatterning using LIMAP. (A) Optical design of the DMD-UV illuminator used in this study. Schematic optical path. A 385-nm high-power UV LED light source is collimated using an AR-coated aspheric lens, and the collimated UV beam is then directed toward a DMD chip at a 24° angle of incidence (corresponding to twice the tilting angle of the DMD mirrors). The image of the DMD chip is then relayed onto the conjugate of the sample plane at the backport of the microscope through a 4f imaging system (f1 = f2 = 125 mm UV fused silica bi-convex lenses, AR-coated). This intermediate image is then relayed onto the sample plane by a tube lens and the objective. To combine DMD-UV illumination with TIRF illumination, a 470-nm dichroic is placed after f2 within a custom backport assembly (Cairn). To offer a second illumination wavelength for 450-nm optogenetic stimulation, our design also contains a second 450-nm collimated LED at the symmetric −24° angle. This LED can be exchanged for any other LED to provide epifluorescence imaging. (B and C) Enhanced fibrinogen micropatterning efficiency depends on the buffer used. (B) PLL-PEG–coated glass was processed for LIMAP patterning with identical UV exposure, micropattern shape, and photoinitiator concentration (50 mM BBTB in 0.1 M sodium bicarbonate, pH 8.3). Fibrinogen-Alexa546 (50 µg/ml) was then adsorbed onto the UV-activated surface in either PBS or carbonate buffer. After washing, red fluorescence of the patterns was imaged by TIRFM using identical settings. (C) Quantification of the effects seen in B (average selectivity ± SEM; see Materials and methods). Fibrinogen quantitatively patterns better in carbonate buffer. Statistics were performed using a Mann–Whitney rank-sum test. n, number of patterns measured. (D–F) Advantages of fibrinogen for multiplexed micropatterning. (D) Scheme illustrating the different steps for sequential multiplexed micropatterning of two fibrinogens labeled with different fluorophores (ATTO488 and Alexa647). (E) Multiplexed micropatterning of fibrinogen-ATTO488 and Alexa647 (50 µg/ml) using the scheme depicted in D onto PLL-PEG–coated glass. Note that there is high specificity of the fibrinogen for their specific pattern and minimum overlap among the two fluorescent fibrinogens. In addition, binding of fibrinogen to unexposed PLL-PEG is minimal, down to a punctate, single molecule–like level (orange arrowheads). (F) Quantification of the effects seen in E: the fluorescence of each Fibrinogen was measured on the two patterns and normalized to the fluorescence of their intended pattern (i.e., first pattern for Alexa647 and second for ATTO488; mean ± SEM). Statistics were performed using a Kruskal–Wallis test followed by a Dunn post hoc test (P < 0.0001). n, number of patterns measured. Note that the vertical scale of the graph is split to better appreciate the minute amounts of fibrinogen deposited onto the nonintended patterns or the unpatterned PLL-PEG area. Scale bars, 10 µm. n.s., not significant.

Article Snippet: Conversely, the TIRF imaging arm is composed of an azimuthal TIRF illuminator (iLas2; GATACA Systems) modified to have an extended field of view (Cairn) to match the full field of view of the camera.

Techniques: Microscopy, Imaging, Concentration Assay, Fluorescence, MANN-WHITNEY, Labeling, Binding Assay

Journal: The Journal of Cell Biology

Article Title: High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

doi: 10.1083/jcb.202009063

Figure Lengend Snippet: Fibrinogen anchors enables the subcellular micropatterning of a synthetic receptor fused to a cortical protein of interest. (A) Experimental scheme: NIH/3T3 cells stably expressing GBP-TM-mScarlet were allowed to spread on dual micropatterns of fibronectin/fibrinogen-Alexa647 and fibrinogen-biotin-ATTO490LS::streptavidin-GFP-GFP, and were then imaged live by TIRF microscopy. (B) Efficient relocalization of the GBP-TM-mScarlet construct onto an area defined by the extracellular GFP micropattern in live cells. (C) Quantification of the effects seen in B (mean ± SEM). Statistics were performed using a Student’s t test. n, number of cells analyzed. (D) Cells as in B were imaged by TIRF microscopy during spreading to evaluate the kinetics of GBP-TM-mScarlet recruitment onto the GFP micropattern. Fibrinogen and GFP micropatterns are outlined in blue and green dashed lines, respectively. (E) quantification of the effects seen in D (mean ± SEM; number of cells analyzed: 11 for control and 32 for Streptavidin-GFP-GFP). Scale bar, 10 µm. Ctrl, control; Strept., streptavidin.

Article Snippet: Conversely, the TIRF imaging arm is composed of an azimuthal TIRF illuminator (iLas2; GATACA Systems) modified to have an extended field of view (Cairn) to match the full field of view of the camera.

Techniques: Stable Transfection, Expressing, Microscopy, Construct, Control

Journal: The Journal of Cell Biology

Article Title: High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

doi: 10.1083/jcb.202009063

Figure Lengend Snippet: Dynamics and controls of subcellular micropatterning of receptors. (A–C) Regular micropatterning does not allow subcellular micropatterning of receptors. (A) Experimental scheme. Stable NIH/3T3 cells constitutively expressing GBP-TM-mScarlet were allowed to spread on dual patterns of fibronectin/fibrinogen-Alexa647 and either GFP, fibrinogen-GFP (low degree of labeling of 0.5 mol GFP per mol fibrinogen), or fibrinogen-biotin::streptavidin-GFP-GFP, then imaged live by TIRF microscopy. (B) Only high-density GFP micropatterning via fibrinogen-biotin::streptavidin-GFP-GFP allows efficient relocalization of the GBP-TM-mScarlet construct onto an area defined by the extracellular pattern. Note that bottom panel corresponds to , reproduced here for convenience. Note also that the dynamic range of the GFP channel panels is not identical here. There is much more GFP when using streptavidin-GFP-GFP compared with using fibrinogen-GFP. (C) In contrast to when biotin-EGF was attached to the fibrinogen-biotin-ATTO490LS::NeutrAvidin sandwich , direct micropatterning of biotin-EGF::streptavidin-Alexa555 (1 µg/ml) showed very weak, inhomogeneous, and nonspecific micropatterning, preventing micropatterning of the second, surrounding fibronectin pattern. Dashed line, region exposed to UV. (D and E) Dynamics of GFP-Notch relocalization by Delta micropatterns. (D) U2OS cells stably expressing GFP-Notch1 were allowed to spread on dual patterns of fibronectin/fibrinogen-Alexa647 and fibrinogen-biotin-ATTO490LS::NeutrAvidin::biotin-DLL4 and GFP-Notch fluorescence was imaged live during spreading by TIRF microscopy. Elapsed time in minutes:seconds. (E) Quantification of the effects seen in D (mean ± SEM; number of cells analyzed: 27 for control and 28 for Biotin-DLL4); see also Materials and methods. Scale bars, 10 µm. Fib, fibrinogen; biot, biotin.

Article Snippet: Conversely, the TIRF imaging arm is composed of an azimuthal TIRF illuminator (iLas2; GATACA Systems) modified to have an extended field of view (Cairn) to match the full field of view of the camera.

Techniques: Expressing, Labeling, Microscopy, Construct, Stable Transfection, Fluorescence, Control

Journal: Nature communications

Article Title: Regulation of minimal spindle midzone organization by mitotic kinases.

doi: 10.1038/s41467-024-53500-1

Figure Lengend Snippet: Fig. 3 | Phosphorylation of PRC1 by CDK1 prevents stable minimal midzone organization in the presence of KIF4A. A Schematic of the minimal midzone self- organization assay. Purified PRC1 and KIF4A organize microtubules into bundles with compacted antiparallel overlaps at the center. PRC1 crosslinks antiparallel microtubules and recruits KIF4A to the antiparallel overlap zone. In turn, KIF4A walks towards the microtubule plus-end pulling on PRC1, generating antiparallel microtubule sliding, which compacts the antiparallel overlap at the bundle center. Parallel microtubule minus segments grow outward, gnerating an organization similar to anaphase midzone bundles. B TIRF microscopy images taken ~20 min after self-organization of microtubule bundles in the presence of 20 nM unpho- sphorylated (top) or fully CDK1-phosphorylated (bottom) PRC1-Alexa546 (green), 5−50 nM KIF4A-mGFP (blue), and 18 µM Alexa647-tubulin (red). See Suppl. Figure 4 for same data displaying the individual channels separately. Scale bar as indicated.

Article Snippet: Nikon objective lens and rotating 360° TIRF illumination (iLas2, Gattaca).

Techniques: Phospho-proteomics, Microscopy

Journal: bioRxiv

Article Title: Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

doi: 10.1101/2025.11.21.689671

Figure Lengend Snippet: Saccharomyces cerevisiae and Schizosaccharomyces pombe actins exhibit indistinguishable ATP-actin dynamics from rabbit actin but faster ADP-actin depolymerization rates. A. Phylogenetic tree of the 3 actins used in this study, Saccharomyces cerevisiae actin Sc Act1 (Uniprot P60010), Schizosaccharomyces pombe actin Sp Act1 (Uniprot P10989) and Oryctolagus cuniculu s (rabbit) alpha skeletal muscle actin Oc ACTA1 (Uniprot P68135). Arabidopsis thaliana actin 1 At ACT1 (Uniprot P0CJ46) was used as an external group. Scale bar unit is the average number of substitutions per amino-acid. B. Method used to study the dynamics of actin filaments in TIRF-assisted microfluidics experiments. ATP-atto488 G-actin (green) is flowed into a PDMS microfluidic chamber and polymerized in F-buffer (yellow) onto spectrin-actin seeds (grey) adsorbed onto the surface. This results in filaments anchored to the surface by their pointed end and whose dynamic barbed end can be monitored using TIRF microscopy. C. Example of a field of view obtained with the method sketched in B using 0.5 µM Sp Act1 and 0.25 µM ATP-atto488. D. Kymograph of the filament outlined in C (yellow line) over the course of the acquisition. The elongation rate is obtained from the angle formed by the line following the filament barbed end over time (yellow line) and the abscissa. E. Plot of the barbed end polymerization rate as a function of the actin concentration for both Sp Act1 (blue) and Sc Act1 (magenta). Each point is the mean of at least 4 independent experiments where the elongation rate of 30 filaments was quantified as in D, and error bars are standard deviations. A linear regression for points from 0.25 µM to 1.2 µM actin was used to extract k on ATP as the slope and k off ATP as the y-intercept . F. The rates k off ADP correspond to the values at 0 µM actin in E . Each point is the mean of an independent replicate, and black bars are means of the replicates for a given condition. Dotted line is the value obtained for Oc ACTA1 in , for reference. Indicated significance is from an unpaired student t-test. (** : p≤0.01). G. Method used to study the compatibility of actins from different species. Pure actin solutions of 0.6µM Oc ACTA1, Sp Act1 and Sc Act1, labelled with a half molar ratio of ATP-atto488, are prepared and used to obtain 50/50 mixes containing two actin types. For one given experiment, two pure solutions in F-buffer (yellow) and the resulting mix are simultaneously injected in a different channel of a microfluidics chamber and their elongation rate is recorded and averaged over 30 filaments. An expected rate is calculated based on the mean rates of the two pure solutions and used to calculate the observed-to-expected ratio. H. Plot of the observed-to-expected ratio of the barbed end polymerization rate for each of the different mixes. Each point is the ratio extracted from a replicate, and black lines are means of the replicates for a given condition.

Article Snippet: The microfluidic chamber (or the open chamber or the coverslip) was attached on a motorized stage (Marzhauser) onto a Nikon Ti2 inverted microscope, equipped with a TIRF 1.49 NA 100 x oil-immersion objective, a sCMOS-kinetix camera (photometrics), a dichroic Quad band and emission filter ZET405/488/561/647 cube, tunable 488, 561 and 642 nm 100 mW lasers, which were controlled using a Total Internal Reflection Fluorescence (TIRF) set-up (iLAS2, Gataca Systems) through the micromanager software ( ; D. ).

Techniques: Microscopy, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

doi: 10.1101/2025.11.21.689671

Figure Lengend Snippet: Saccharomyces cerevisiae and Schizosaccharomyces pombe ADP·Pi-actin filaments depolymerize fast. A. Method used to study the depolymerization of ADP·Pi-actin filaments in TIRF-assisted microfluidics experiments. ATP-atto488 G-actin (green) is flowed into a PDMS microfluidic chamber and polymerized in phosphate-buffer (orange) onto spectrin actin seeds (grey). The filaments are then exposed to the same phosphate buffer without actin and their depolymerization is monitored. B. Example of a kymograph of an actin filament over the course of an experiment as in A using 1µM Sp Act1, 0.5µM ATP-atto488 and 25mM phosphate. C. Plot of the barbed end depolymerization rate as a function of the phosphate concentration for both Sp Act1 (blue) and Sc Act1 (Magenta). Each point is the mean of at least 4 independent experiments where the elongation rate of at least 15 filaments was quantified as in B and error bars are standard deviations. D. Depolymerization rates k ADP·Pi correspond to data at 200µM phosphate in C . Each point is the mean of an independent replicate, and black bars are means of the replicates for a given condition. Dotted line is the value obtained for Oc ACTA1 in , for reference. Indicated significance is from an unpaired student t-test (*** : p≤0.001)

Article Snippet: The microfluidic chamber (or the open chamber or the coverslip) was attached on a motorized stage (Marzhauser) onto a Nikon Ti2 inverted microscope, equipped with a TIRF 1.49 NA 100 x oil-immersion objective, a sCMOS-kinetix camera (photometrics), a dichroic Quad band and emission filter ZET405/488/561/647 cube, tunable 488, 561 and 642 nm 100 mW lasers, which were controlled using a Total Internal Reflection Fluorescence (TIRF) set-up (iLAS2, Gataca Systems) through the micromanager software ( ; D. ).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

doi: 10.1101/2025.11.21.689671

Figure Lengend Snippet: Saccharomyces cerevisiae and Schizosaccharomyces pombe actin filaments release inorganic phosphate faster than rabbit alpha-skeletal actin filaments. used to study the phosphate release rate of actin filaments in TIRF-assisted microfluidics experiments. ATP-atto488 G-actin (green) is flowed into a PDMS microfluidic chamber and polymerized in phosphate-buffer (orange) onto spectrin actin seeds (grey). The filaments are then exposed to F-buffer (yellow) without actin and depolymerization is monitored over time. B. Example of a kymograph of an actin filament over the course of an experiment as in A using 0.8µM Sp Act1, 0.4µM ATP-atto488 and 50mM phosphate. C. Scheme explaining the different rates measured in this experiment. The depolymerization rate V ADP·Pi is in subunits per second. D. Plot of the phosphate release rate k r for Sp Act1 (blue) and Sc Act1 (magenta) following a polymerization at 50 and 200mM phosphate. E. Plot of the depolymerization rate V ADP at the end of the experiment for Sp Act1 (blue) and Sc Act1 (magenta) following a polymerization at 50 and 200mM phosphate. F. Plot of the depolymerization rate V ADP·Pi at the beginning of the experiment for Sp Act1 (blue) and Sc Act1 (magenta) following a polymerization at 50 and 200mM phosphate. G. Plot of the barbed end phosphate release rate k BE for Sp Act1 (blue) and Sc Act1 (magenta) following a polymerization at 50 and 200mM phosphate, calculated from V ADP·Pi (F), k ADP·Pi and k ADP using the equation in C. Dotted line is the value obtained for Oc ACTA1 in (D, F, G) or (D), for reference. Each point is the result of the fit of the depolymerization rate curve extracted from at least 40 filaments. Black bars are means of the replicates for a given condition. Indicated significances are from unpaired student t-tests (ns : p>0.05 ; ** : p≤0.01 ; *** : p≤0.001 ; **** : p≤0.0001).

Article Snippet: The microfluidic chamber (or the open chamber or the coverslip) was attached on a motorized stage (Marzhauser) onto a Nikon Ti2 inverted microscope, equipped with a TIRF 1.49 NA 100 x oil-immersion objective, a sCMOS-kinetix camera (photometrics), a dichroic Quad band and emission filter ZET405/488/561/647 cube, tunable 488, 561 and 642 nm 100 mW lasers, which were controlled using a Total Internal Reflection Fluorescence (TIRF) set-up (iLAS2, Gataca Systems) through the micromanager software ( ; D. ).

Techniques:

Journal: bioRxiv

Article Title: Rapid aging and disassembly of actin filaments from two evolutionary distant yeasts

doi: 10.1101/2025.11.21.689671

Figure Lengend Snippet: The nucleotidic exchange is faster in Schizosaccharomyces pombe than in rabbit monomeric actin. A. Method used to study the exchange rate of the nucleotide inside of monomeric actin. G-Actin was mixed with ATP-atto488 and immediately injected in polymerizing conditions into an open chamber canal made with coverslips and parafilm strips. Filaments were maintained close to the surface by using methylcellulose. Movies of the polymerizing filaments were acquired by TIRF microscopy. If the exchange inside of the monomer is slow and polymerization fast, the labelling fraction will increase over time and hence the intensity along the filament will increase from pointed end to barbed end. Instead, if the exchange is fast, the intensity will be homogenous. B. Example of a field of view using the method as in A with 0.35µM Oc ACTA1 and 0.175µM ATP-atto488 in F-buffer without ATP supplemented with 0.18% methylcellulose. C. Example of a field of view using the method as in A with 0.35µM Sp Act1 and, 0.175µM ATP-atto488 in F-buffer without ATP supplemented with 0.18% methylcellulose. D. Plot of the normalized mean intensity profile (see Methods) of 20 filaments from an experiment as in A for Oc ACTA1 (black) and as in B for Sp Act1 (blue). Filaments were registered by their brighter end, defined as the origin on the graph. Error bars are standard deviations.

Article Snippet: The microfluidic chamber (or the open chamber or the coverslip) was attached on a motorized stage (Marzhauser) onto a Nikon Ti2 inverted microscope, equipped with a TIRF 1.49 NA 100 x oil-immersion objective, a sCMOS-kinetix camera (photometrics), a dichroic Quad band and emission filter ZET405/488/561/647 cube, tunable 488, 561 and 642 nm 100 mW lasers, which were controlled using a Total Internal Reflection Fluorescence (TIRF) set-up (iLAS2, Gataca Systems) through the micromanager software ( ; D. ).

Techniques: Injection, Microscopy

Journal: eLife

Article Title: Synergistic stabilization of microtubules by BUB-1, HCP-1, and CLS-2 controls microtubule pausing and meiotic spindle assembly

doi: 10.7554/eLife.82579

Figure Lengend Snippet: ( A ) Coomassie-stained gels of purified proteins used for in vitro assays. Arrowheads indicate the protein of interest. ( B ) Microtubule pelleting assay in the presence of 1 µM purified BUB-1 protein. Schematic of the experiment principle (left) and Coomassie staining of supernatant and pellet fractions (right) with indicated concentrations of microtubules (MTs). ( C ) Gel filtration assay of tubulin in the presence of indicated CLS-2 TOGL domains. Coomassie staining of the fractions of interest are shown on the right. ( D ) Embryonic viability assay of worms carrying cls-2::gfp transgenes with indicated truncations in the S/R-rich region, and upon depletion of endogenous cls-2 . Position of the divergent LxxPTPh motif is indicated on the protein fusion diagram. ( E–F ) TIRF-microscopy localization of 100 nM purified CLS-2::GFP protein (green) on microtubules (magenta). Stills from imaging in indicated conditions ( E ) and Venn diagram of CLS-2::GFP foci associated with microtubule rescue and pause events ( F ). Scale bar 10 µm. Figure 6—figure supplement 1—source data 1. Panels A-B source data. Raw images and uncropped annotated image of Coomassie-stained gels for purification of BUB-1, HCP-1, CLS-2::GFP and CLS-2 R970A ::GFP proteins, and of microtubule/BUB-1 pelleting assay. Figure 6—figure supplement 1—source data 2. Panel C source data. Raw images and uncropped annotated image of Coomassie-stained gels for protein fractions of gel filtration assay. Figure 6—figure supplement 1—source data 3. Panel D source data. Embryonic viability assay of worms carrying a cls-2::gfp transgene with mutations in the S/R-rich domain, upon depletion of cls-2 compared to non-depleted controls.

Article Snippet: Microtubule dynamics was monitored between 22.5°C and 23°C on an azimuthal TIRF microscope (Nikon Eclipse Ti2 equipped with the Ilas2 module, Gataca systems) using a 60 x, 1.47NA oil immersion TIRF objective and a Photometrics Prime BSI sCMOS camera.

Techniques: Staining, Purification, In Vitro, Filtration, Viability Assay, Microscopy, Imaging

Journal: eLife

Article Title: Synergistic stabilization of microtubules by BUB-1, HCP-1, and CLS-2 controls microtubule pausing and meiotic spindle assembly

doi: 10.7554/eLife.82579

Figure Lengend Snippet: ( A ) Schematic of the TIRF microscopy-based microtubule assay. Labeled tubulin (ATTO-565, magenta) fluoresces only when close to the surface of the coverslip. Microtubules polymerize from biotinylated GMPCPP seeds (tubulin-ATTO-488, cyan) bound to a Neutravidine-coated glass coverslip. ( B ) Representative kymographs of microtubules (magenta) growing from GMPCPP seeds (cyan) in the presence or absence of BUB-1, HCP-1, CLS-2-GFP and/or CLS-2 R970A -GFP (100 nM each). Schematics on the left highlights the different microtubule dynamics events observed. ( C–F ) Dot plot showing the quantification of growth rate ( C ), and histograms showing the average catastrophe ( D ), rescue ( F ), and pause ( D ) events per microtubule. Dunnett’s multiple comparison tests, alpha = 0.01, **p<0.01, ****p<0.0001, n.s . not significant. Error bars, Mean and standard deviation ( C ) or standard error of the mean ( D–F ). ( G ) Microtubule bundling assay. Organization of microtubules (magenta) observed in indicated conditions. Figure 6—source data 1. Panel B-F source data. Raw measurements and calculations for quantification of microtubule dynamics. Parameters were extracted from kymographs using the ImageJ software ( https://imagej.nih.gov/ij/index.html ). Each sheet corresponds to an experiment. The first sheet (read me) provides details about data extraction. Figure 6—source data 2. B-F statistics source data. Processed data for the analysis of microtubule dynamics. Details of statistical analyses are provided.

Article Snippet: Microtubule dynamics was monitored between 22.5°C and 23°C on an azimuthal TIRF microscope (Nikon Eclipse Ti2 equipped with the Ilas2 module, Gataca systems) using a 60 x, 1.47NA oil immersion TIRF objective and a Photometrics Prime BSI sCMOS camera.

Techniques: Microscopy, Labeling, Comparison, Standard Deviation, Software, Extraction