Journal: The EMBO Journal

Article Title: The Nedd4L ubiquitin ligase is activated by FCHO2-generated membrane curvature

doi: 10.1038/s44318-024-00268-1

Figure Lengend Snippet: DsRed-CLC were co-expressed with Nedd4L-GFP in wild-type HeLa cells. Images were acquired at 1 s intervals through TIRF microscopy. Upper panel, TIRF images of HeLa cells. Insets represent enlarged images of the dashed boxes. Bottom panel, selected snapshots (every 10 s) from a time series were obtained from a single CCP indicated by the arrowhead. The time point at which Nedd4L started to accumulate was set 0. Scale bar, 2 μm. .

Article Snippet: Images were acquired for 5 min at 1 s intervals with a TIRF microscopy system (Olympus), a 100× oil immersion objective NA = 1.45 (Olympus) and MetaMorph Imaging System Software.

Techniques: Microscopy

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Activity-dependent filopodial extension drives the increase in footprint surface area in neurosecretory cells. A, B, Bovine chromaffin (A) and PC12 (B) cells expressing GFP-GPI were examined by TIRF microscopy, imaged at 2 Hz and stimulated with Ba2+ (2 mm) or vehicle treated as indicated. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the pretreatment area (white with red outline). Scale bars, 5 μm. Arrowheads indicate outgrowing filopodia. C, Changes in surface area over time were plotted as indicated. D, Average change in footprint area (normalized to initial area before treatment) for stimulated chromaffin and PC12 cells and vehicle-treated controls 400 s after stimulation (n = 6/10/8/5). E, Bovine chromaffin cells were treated with vehicle or 2 mm BaCl2 for 8 min and the region of the cell containing the cell footprint was analyzed by electron microscopy. Note the numerous filopodia emerging from the footprint in stimulated cells. The inset shows a region of membrane extension containing SGs (arrowheads). Proximity to the plasma membrane is confirmed by the profiles of clathrin-coated pits containing ruthenium red stain, indicative of continuity with the plasma membrane (arrows). Bottom, Outlines of the cells in the top. Left: Ratio of outline length to visible surface before and stimulation. Right: Counts of objects outside the cell surface in unstimulated and stimulated conditions (n = 6).

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Activity Assay, Expressing, Microscopy, Fluorescence, Electron Microscopy, Staining

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Vesicular fusion does not account for the entire footprint enlargement. SG granule fusion was measured in bovine chromaffin cells expressing NPY-mCherry. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation (black arrow) with Ba2+ (2 mm). A, Individual fusion events were defined as the disappearance of NPY-positive SGs, followed by a cloud-like dispersal of NPY-mCherry to the extracellular space, corresponding to a spike in overall NPY-mCherry intensity. B, SG fusion was measured in bovine chromaffin cells expressing NPY-mCherry and VAMP2-pHluorin. Cells were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). C, Validation of the NPY-mCherry assay described in A by comparison with VAMP2-pHluorin overall fluorescence intensity change. To count fusion events with VAMP2-pHluorin, we determined the average fluorescence intensity change due to alkalinization of the pHluorin moiety during a fusion event by calculating the difference in fluorescence intensity between two adjacent frames before and during a fusion event and averaging over a number of events. We then assigned this value to each individual fusion event detected using the NPY-mCherry assay (orange). Note that these results do not significantly differ from the direct VAMP2-pHluorin fluorescence measurement (green). D, Chromaffin and PC12 cells were cotransfected with NPY-mCherry and VAMP2-pHluorin and analyzed by TIRF microscopy. Arrowheads in the inset indicate vesicles where both markers colocalize. The graph depicts the percentage of VAMP2-pHluorin-positive vesicles that also contain NPY-mCherry and vice versa (n = 7). E, Chromaffin and PC12 cells cotransfected with GFP-GPI and NPY-mCherry and stimulated with Ba2+ (2 mm). The extent of footprint enlargement based on the actual GFP-GPI measurements (green) was compared with the estimated membrane increase based on the measured number of SG fusion events (NPY-mCherry assay) multiplied by the predicted membrane surface area of a 300 nm SG (orange; n = 4–6). Note that the actual footprint increase is significantly larger than that of the estimate based on fusion alone.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Expressing, Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Footprint expansion does not depend on regulated exocytosis. NPY-mCherry was expressed in PC12 cells, DKD-PC12 cells, and DKD-PC12 cotransfected with Munc18–1-GFP. A, Cells were imaged at 2 Hz by TIRF microscopy before and for 7 min after 2 mm Ba2+ stimulation and fusion numbers were counted and normalized to footprint area. Note the rescue of exocytosis elicited by Munc18–1-GFP reexpression in DKD-PC12 cells. B, GFP-GPI was expressed in DKD-PC12 cells and DKD-PC12 cotransfected with (untagged) Munc18–1. Cells were examined by TIRF microscopy and imaged at 2 Hz before and during Ba2+ (2 mm) stimulation. Top, Time-lapse of the GFP-GPI fluorescence in the footprint. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. C, Footprint area change for GFP-GPI-transfected DKD PC12 cells expressing empty vector or Munc18–1 and wild-type (wt)-PC12 cells after Ba2+ (2 mm) stimulation (400 s). Note that all three populations exhibit similar increases in footprint area. The numbers in the bars indicate the number of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Transfection, Expressing, Plasmid Preparation

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Interfering with actin polymerization and myosin II function reduces footprint enlargement. Bovine chromaffin (A, B) and PC12 (C, D) cells coexpressing GFP-GPI and NPY-mCherry were treated with cytochalasin D (CytoD) or blebbistatin (Blebbi) for 20 min before examination by time-lapse TIRF microscopy. Cells were then stimulated with Ba2+ and imaged at 2 Hz. A, B, Time-lapse profile of the GFP-GPI fluorescence in the footprint of chromaffin (A) or PC12 (B) cells treated as indicated. Bottom, GPI-GFP footprint area (green) with an overlay of the prestimulation area (white with red outline). Scale bars, 5 μm. B, D, Footprint area change under indicated conditions 400 s after Ba2+ stimulation or vehicle treatment for chromaffin (B) and PC12 cells (D) based on either the actual GFP-GPI measurements (black bars) or the corresponding estimated (est.) footprint increase based on fusion events (white bars). Note that for both blebbistatin and cytochalasin D treatments in chromaffin cells, the measured and estimated footprint area changes do not differ significantly. The numbers inside the bars indicate the numbers of cultures analyzed.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: Newly added footprint area contains high levels of F-actin. A, PC12 cells cotransfected with Lifeact-RFP (LA-RFP; red) and GFP-GPI (green) were examined by TIRF microscopy and imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Bottom, Ratio of Lifeact-RFP:GFP-GPI fluorescence in pseudocolor to indicate changes in F-actin concentration. B, 3D surface intensity plot of Lifeact-RFP:GFP-GPI ratio before and 400 s after stimulation, highlighting the increased F-actin concentration (red) at the edge of the footprint and the decrease in F-actin in the center of the footprint. C, Quantification of Lifeact-RFP and GFP-GPI ratios using regions of interest from the center or the expanding edge of PC12 cell footprints (n = 3). After stimulation, the Lifeact-RFP:GFP-GPI ratio in the initial footprint area decreases while simultaneously increasing in the added footprint area.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Fluorescence, Concentration Assay

Journal: The Journal of Neuroscience

Article Title: Secretagogue Stimulation of Neurosecretory Cells Elicits Filopodial Extensions Uncovering New Functional Release Sites

doi: 10.1523/JNEUROSCI.2634-13.2013

Figure Lengend Snippet: SGs move into the newly added footprint area. A, Time-lapse of PC12 cells cotransfected with NPY-mCherry(NPY-Ch) and GFP-GPI, imaged at 2 Hz before and for 7 min after stimulation with Ba2+ (2 mm). Top, NPY-mCherry-positive SGs appear outside the initial area (green dashed line) and are evenly distributed across the entire footprint (green outline). Bottom and side, x-y-t reconstruction of NPY-mCherry-positive SGs, with new SGs appearing in both the initial and the newly added area after stimulation. Note that the SG density (number of granules per unit area) remains constant over time (small inset) in all areas. B, Time-lapse TIRF microscopy of a bovine chromaffin cell cotransfected with Lifeact-GFP (LA-GFP) and NPY-mCherry showing the distribution of F-actin and SGs before and 400 s after stimulation. Right, Trajectories of SGs in the added footprint membrane area show that they are entering the newly forming area that also contains Lifeact-GFP positive structures (bottom). Scale bar, 5 μm. C,D, Maps of NPY-mCherry positive SGs trajectories in a Ba2+-stimulated chromaffin cell color coded for time after stimulation (C) and square displacement (D), as indicated. The white outline indicates the footprint area before stimulation. E, Mean square displacement (MSD) analysis of SGs in the original footprint and added areas. Note that there is no significant difference in mobility between either SG populations. F, SGs undergo regulated exocytosis in the newly added area. Time-lapse profile of a bovine chromaffin cell transfected with Lifeact-GFP (LA-GFP) and NPY-mCherry (red outline of the initial footprint area) stimulated with Ba2+ at t = 0 and imaged at 2 Hz. Arrowheads depict a SG entering an added area and undergoing fusion (pseudocolored in the inset). In the merged insets, the GFP channel is replaced with an outline of the limit of the footprint. Scale bar, 5 μm. G, Quantification of fusion events per area in both the original footprint and the added area. During footprint expansion, starting 120 s after stimulation, the number of fusions/area in the new area does not differ significantly from those in the initial footprint. Scale bar, 5 μm.

Article Snippet: Transfected cells on glass-bottomed culture dishes (MatTek) were visualized with a total internal reflection fluorescence (TIRF) microscope (Marianas, SDC Everest; Intelligent Imaging Innovations) fitted with a 100× oil-immersion objective (numerical aperture = 1.46, Carl Zeiss Pty Ltd) using an EMCCD camera (QuantEM 512sc) and Slidebook software (version 4.2.0.27/28).

Techniques: Microscopy, Transfection

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA is a dynein adaptor that requires dynactin and Lis1 to activate dynein motility. (A) Schematic of mScarlet-tagged NuMA FL and SNAP-tagged NuMA N-term constructs, indicating the main structural parts of NuMA (N-terminal region, predicted coiled-coil, C-terminal tail) and the domains or motifs implicated in dynein/dynactin-binding ( ; ). (B) Schematic of microscopy flow chambers (left), with functionalized glass surface and components of dynein motility assays (right). (C) Representative TIRF microscopy kymographs showing the motility of mEGFP-dynein in the presence of dynactin and AF546-NuMA N-term at different mCherry-Lis1 concentrations. Concentrations as indicated. (D) Processive run frequency of mEGFP-dynein (median ± 95% CI, estimated by bootstrapping). Each distinct color refers to a replicate; each data point represents the number of events in one field of view of one replicate; n = 99, 101, 27, 100, 29, 26, 103 microtubules. The grey curve represents a hyperbolic fit; apparent K d and goodness-of-fit (expressed by R 2 ) are indicated; dashed lines: 95% CI of the fit (estimated by bootstrapping). (E) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Each color refers to a replicate; each circle represents the median velocity of one replicate; n = 760, 3,751, 7,128, velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.6121 (10 versus 100 nM), 0.6121 (10 versus 5,000 nM), and 0.6593 (100 versus 5,000 nM). Protein combination and concentrations in D and E as in C, and as indicated. (F) Representative kymographs showing the motility of mEGFP-dynein in the presence of dynactin and mCherry-Lis1 at different AF546-NuMA N-term concentrations. Concentrations as indicated. (G) Processive run frequency of mEGFP-dynein (median ± 95% CI). Symbols and curve as in D; n = 103, 70, 100, 70 microtubules. (H) Velocity distribution of processive mEGFP-dynein runs (mean of medians ± SEM). Circles as in E; n = 1,585, 3,482, 2,649 velocities; adjusted P values by Welch’s ANOVA with Holm-Sidak’s post-hoc test for multiple comparisons: 0.7632 (100 versus 200 nM), 0.7084 (10 versus 500 nM), and 0.9235 (200 versus 500 nM). Protein combinations and concentrations in G, H as in F, and as indicated. (I–K) Representative kymographs showing the motility of (I) mEGFP-dynein (left) and AF647-NuMA N-term (right) in the presence of dynactin and Lis1, (J) mEGFP-dynein in the presence of dynactin, Lis1, and mScarlet-NuMA FL , (K) mEGFP-dynein (left) and mScarlet-NuMA FL (right) in the presence of dynactin and Lis1. Arrowheads of the same color indicate co-localization in the same processive events. All data refer to motility on surface-immobilized Atto647N-labeled GMPCPP-microtubules (MTs) in dynein microscopy buffer. All data are from at least three biological replicates. Experiments shown in C‒H and J were carried out at 30°C, and those shown in I and K at 18°C.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Construct, Binding Assay, Microscopy, Labeling

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: Effect of Lis1, temperature, and adaptor identity on dynein motility. (A) Representative kymographs showing the motility of mEGFP-dynein on surface-immobilized Atto647N-labeled GMPCPP-microtubules in the presence of dynactin and BicD2N 1–400 at different mCherry-Lis1 concentrations. Concentrations as indicated. Lis1 stimulates BicD2N 1–400 -induced processive motility in a dose-dependent manner. (B) Velocity distributions of mEGFP-dynein processive runs (mean ± SEM) in the presence of either mScarlet-NuMA N-term , mScarlet-NuMA FL or BicD2N 1–400 , as indicated. Each circle corresponds to the velocity of a single processive segment of a run; n = 49, 55, 50, 72, 166 velocities; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.8246 (N-term versus FL, 30°C), 0.8429 (N-term versus BicD2N 1–400 , 30°C), 0.7750 (FL versus BicD2N 1–400 , 30°C); P value by Welch’s t test: 0.7228 (N-term versus FL, 18°C). Experiments performed with NuMA contained 3 nM mEGFP-dynein, 7 nM dynactin, 50 nM NuMA, 650 nM Lis1; experiments with BicD2N 1–400 contained 7 nM mEGFP-dynein, 14 nM dynactin, 200 nM BicD2N 1–400 , 100 nM Lis1. Velocities measured at 18°C were approximately threefold lower than those measured at 30°C, consistent with the temperature effect previously reported ( ; ). (C and D) Comparison of run lengths for dynein activated by NuMA N-term versus BicD2N 1–400 (C) and by NuMA N-term versus NuMA FL (D). Left: survival probability (1-CDF) of all measured run lengths for NuMA N-term ( n = 204) and BicD2N 1‒400 -activated ( n = 206) complexes. A large fraction of these processive events reach the microtubule end (NuMA N-term n end = 57; BicD2N 1–400 n end = 72); center: ratio of end-reaching events to total number of processive events per microtubule versus microtubule length; right: corrected survival probability of run length using the Kaplan-Meier estimator with end events being treated as right censored data points . Survival probabilities are fitted with exponential function to estimate the run length; errors are approximated by ∆ R = 2 R / N ; dotted lines indicate the 95% CI of the survival probability. Experiments in C were performed at 7 nM mEGFP dynein, 14 nM dynactin, 100 nM mCherry-Lis1, and 200 nM AF546-NuMA N-term or BicD2N 1‒400 . Experiments in D were performed at 3 nM mEGFP dynein, 7 nM dynactin, 650 nM Lis1, and 50 nM AF546-NuMA N-term or mScarlet-NuMA FL . All experiments were carried out in dynein microscopy buffer. (E) Representative TIRF microscopy images showing the binding of 50 nM mScarlet-NuMA FL to surface-immobilized Atto647N-labeled GMPCPP-microtubules in BRB80 (containing 80 mM Pipes, as in NuMA microscopy buffer) and BRB20 (containing 20 mM Pipes, as in dynein microscopy buffer), supplemented by 60 mM KCl. At 80 mM Pipes, NuMA binds all along the GMPCPP microtubules, as shown in . At 20 mM Pipes, NuMA’s solubility is reduced, as shown by numerous NuMA aggregates in solution, which impacts its ability to bind microtubules.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Labeling, Comparison, Microscopy, Binding Assay, Solubility

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA’s microtubule-binding region is located close to its C-terminus. (A) Schematic of mScarlet-tagged NuMA FL and C-terminal fragments, indicating the main structural parts of NuMA and functional domains identified in the C-terminal region (microtubule-binding domain [MTBD] 1 and 2 [ ; ], clustering domain , LGN oligomerization domain , LGN binding domain [LGNBD] , nuclear localization signal [NLS] ). (B) Coomassie Blue-stained SDS-PAGE showing the pellet (P) and supernatant (S) fractions of microtubule co-sedimentation assays. 1 µM mScarlet-tagged NuMA FL or C-terminal fragments were incubated alone or with Atto647N-labeled GMPCPP-microtubules (0.5 µM polymerized tubulin) at 30°C for 15 min. Upon centrifugation, in the absence of microtubules, all proteins remained in the supernatant (left gel). When incubated with microtubules, they co-sedimented with the microtubule pellet (green arrow) at different degrees. NuMA C-term L was found exclusively in the pellet; NuMA FL predominantly in the pellet; NuMA C-term S2 partially in the pellet; NuMA C-term S1 entirely in the supernatant. Arrows indicate the expected molecular weight for each NuMA construct. (C) Representative TIRF microscopy images of 40 nM mScarlet-tagged NuMA constructs binding to surface-immobilized Atto647N-labeled GMPCPP-microtubules. (D) Background-corrected and microtubule length-corrected mScarlet intensity of the different NuMA constructs binding to microtubules as shown in C (mean of medians ± SEM). Each big circle represents the median value of one replicate; each small circle represents the intensity on a single microtubule; n = 45 microtubules for all conditions; P values by Welch’s t test: 0.0364 (FL versus C-term L), 0.1346 (FL versus C-term S1), 0.0530 (FL versus C-term S2). (E) Clustering propensity: sums of the background-corrected intensities of all outliers of the mScarlet NuMA intensity distributions for the different NuMA constructs shown in C nonspecifically bound to the glass surface (mean of medians ± SEM). Each big circle represents the median value of one replicate; each small circle represents the clustering at one surface area of one replicate; n = 9 areas for all conditions; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.0297 (FL versus C-term L), 0.2367 (FL versus C-term S2), 0.0358 (C-term L versus C-term S2). (F) Representative TIRF microscopy images of 40 nM mScarlet-tagged NuMA constructs binding to surface-immobilized Atto647N-labeled GMPCPP-microtubules in the presence of 10 µM Atto647N-tubulin. Red arrowheads indicate selective end binding; yellow arrowheads indicate examples of NuMA C-term L clusters nonspecifically adsorbed to the surface. (G) Background-corrected and microtubule length-corrected mScarlet intensity of the different NuMA constructs binding to microtubules in the presence of 10 µM Atto647N-tubulin as shown in E (mean of medians ± SEM). Symbols as in D; n = 45 microtubules for all conditions; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.2825 (FL versus C-term L), 0.0143 (FL versus C-term S2), 0.0184 (C-term L versus C-term S2) (H) Microtubule end selectivity: percentage of microtubules showing mScarlet signal exclusively at one end, of all microtubules with an mScarlet signal, for mScarlet-NuMA constructs binding to microtubules in the presence of tubulin as shown in C (mean ± SEM). Each color represents a replicate; n = 126, 71, 161 microtubules; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.1339 (FL versus C-term L), 0.0028 (FL versus C-term S2), 0.1732 (C-term L versus C-term S2). All data are from three biological replicates. All assays were executed in NuMA microscopy buffer. Source data are available for this figure: .

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Binding Assay, Functional Assay, Staining, SDS Page, Sedimentation, Incubation, Labeling, Centrifugation, Molecular Weight, Construct, Microscopy

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA’s microtubule-binding region preferentially binds to microtubule minus ends and prevents their growth. (A) Schematic of a TIRF microscopy assay with fluorescent NuMA constructs and tubulin being flowed simultaneously into a channel containing surface-immobilized GMPCPP-microtubule seeds. (B) Representative TIRF microscopy image (left) and kymograph (right) showing minus and plus ends (dim magenta) dynamically elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (bright magenta) in the presence of Atto647N-tubulin. (C, E, and G) Representative TIRF microscopy images (top) and kymographs (bottom) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin and various concentrations of different mScarlet-tagged NuMA constructs (green). (D, F, and H) Growth velocity distributions of microtubule minus and plus ends related to the experiments shown in C, E, and G (mean of medians ± SEM). Each big circle represents the median velocity of one replicate; each small circle represents the growth velocity of one microtubule end segment; D: n = 109, 60, 69, 71 (left plot) and 109, 59, 68, 70 (right plot); adjusted P values: 0.3126, 0.0030, 0.0027 (left plot) and 0.8469, 0.8469, 0.6588 (right plot). F: n = 109, 72, 66, 67 (left plot) and 109, 72, 68, 65 (right plot); adjusted P values: 0.1334, 0.0188, 0.0041 (left plot) and 0.9642, 0.9642, 0.9642 (right plot). H: n = 109, 74, 70, 66 (left plot) and 109, 74, 70, 75 (right plot); adjusted P values: 0.0785, 0.0042, 0.0031 (left plot) and 0.6572, 0.8526, 0.8526 (right plot). All data are from at least three biological replicates; adjusted P values were calculated by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons; each condition was compared to the control at 0 nM NuMA. All experiments were performed in a NuMA microscopy buffer. All data are from three biological replicates.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Binding Assay, Microscopy, Construct, Labeling, Control

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: Effect of NuMA C-term S1 at a high concentration on microtubule dynamics. (A) Representative TIRF microscopy image (top) and kymograph (bottom) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin and mScarlet-NuMA C-term S1 (green). (B) Growth velocity distributions of minus and plus ends of microtubules under conditions explained in A (mean of medians ± SEM). Each big circle represents the median velocity of one replicate; each small circle represents the growth velocity of one microtubule end segment; n = 101, 58 (left plot) and 109, 63 (right plot); P values by Welch’s t test: 0.2241 (left plot), 0.5035 (right plot). (C) Representative TIRF microscopy image (left) and kymograph (right) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin, before (above the dashed line) or after (below the dashed line) the addition of mScarlet-NuMA C-term S1 (green).

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Concentration Assay, Microscopy, Labeling

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA caps and stabilizes dynamic microtubule minus ends. (A) Schematic of a two-flush TIRF microscopy assay: fluorescent tubulin is flowed into a channel containing surface-immobilized GMPCPP-seeds (first flush); microtubules are allowed to elongate from GMPCPP-seeds for ≈10 min; and tubulin is re-added together with fluorescent NuMA constructs (second flush). (B) Representative kymograph showing a control experiment with microtubule minus and plus ends (dim magenta) dynamically elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (bright magenta) in the presence of 10 µM Atto647N-tubulin; the dashed line marks the second flush of tubulin. (C, E, and G) Representative TIRF microscopy images (top) and kymographs (bottom) showing the growth behavior of microtubule ends (dim magenta) elongating from surface-immobilized Atto647N-labeled GMPCPP-seeds (bright magenta) in the presence of Atto647N-tubulin, before (above the dashed line) or after (below the dashed line) the addition of different mScarlet-tagged NuMA constructs (green) at different concentrations. Related to . (D, F, and H) Growth velocity distributions of minus and plus ends elongating from GMPCPP-seeds in the presence of 10 µM Atto647N-tubulin before (circles) and after (triangles) the addition of different mScarlet-tagged NuMA constructs at different concentrations (mean of medians ± SEM). Each big symbol represents the median velocity of one replicate and each small symbol represents the growth velocity of one microtubule end segment; D: n = 72, 73, 67; P values: 0.0572, 0.0111, 0.0012 (left plot) and 0.1221, 0.4972, 0.0008 (right plot). F: n = 72, 51, 66 (left plot) and 72, 53, 66 (right plot); P values: 0.0572, 0.0196, 0.0027 (left plot) and 0.1221, 0.9029, 0.4875 (right plot). H: n = 72, 57, 72 (left and right plots); P values: 0.0572, 0.0038, 0.0175 (left plot) and 0.1221, 0.6881, 0.1384 (right plot). All data are from three biological replicates; P values were calculated by paired t test comparing the “pre-NuMA addition” and “post-NuMA addition” for each condition. All experiments were performed in NuMA microscopy buffer.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy, Construct, Control, Labeling

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: γTuRC prevents NuMA accumulation at microtubule minus ends. (A) Schematic of a γTuRC nucleation assay performed in the presence of mScarlet-NuMA FL . (B) Representative TIRF microscopy kymographs showing microtubules nucleated by surface-immobilized mBFP-γTuRC, and spontaneously nucleated microtubules in solution in the presence of 10 µM Atto647-tubulin and 5 nM mScarlet-NuMA FL . γTuRC and solution-nucleated microtubules can be distinguished because the latter diffuse on the surface. γTuRC-nucleated microtubules only occasionally display weak NuMA fluorescence at their minus ends (second kymograph), whereas the minus ends of solution-nucleated microtubules frequently show intense NuMA signals (third and fourth kymographs). (C) Frequency of microtubule minus end localization of NuMA on γTuRC-nucleated and spontaneously nucleated microtubules in the presence of 10 µM Atto647-tubulin and 5, 15, and 30 nM mScarlet-NuMA FL (mean ± SEM); each diamond represents the mean frequency of each condition, each distinct small symbol represents the frequency of a single replicate within a condition; bottom n = 80, 101, 104 microtubules; top n = 21, 54, 52 microtubules. For γTuRC-nucleated microtubules, cases in which NuMA localization at the minus end coincided with NuMA/γTuRC co-localization prior to nucleation were excluded from the analysis. (D) Background-corrected maximum intensity of mScarlet-NuMA FL at 30 nM localizing to either γTuRC-nucleated microtubule minus ends, spontaneously nucleated microtubule minus ends, or at randomly chosen microtubule-free surface areas (mean of medians ± SEM), in the presence of 10 µM Atto647-tubulin. Each big symbol represents the median of one replicate; each small symbol represents the intensity at one microtubule minus end or surface area; each replicate is represented by a distinct symbol shape (circle, diamond, triangle, square); n = 68, 18, 12 intensities; adjusted P values by Welch’s ANOVA test with Holm-Sidak’s post-hoc test for multiple comparisons: 0.0295 (solution versus γTuRC), 0.0295 (solution versus surface), 0.3793 (γTuRC versus surface). (E and F) Plots showing an increase of the solution-nucleated (E) and γTuRC-nucleated (F) microtubule number over time, in the presence of 0–30 nM mScarlet-NuMA FL (mean ± SEM). NuMA promotes spontaneous nucleation of microtubules in solution in a dose-dependent manner (E), without exerting any effect on γTuRC-mediated nucleation (F). Experiments were performed in γTuRC microscopy buffer. All data are from at least three biological replicates.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy, Fluorescence

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA gradually accumulates at the minus ends of enzymatically γTuRC-uncapped microtubule s . (A) Schematic of a γTuRC nucleation assay performed in the presence of KIF2A, spastin, and mScarlet-NuMA FL . The combined action of spastin and KIF2A triggers γTuRC release, which is followed by microtubule treadmilling and minus end capping by NuMA. (B) Representative TIRF microscopy kymographs showing microtubules nucleated by surface-immobilized mBFP-γTuRC in the presence of 11 µM Atto647-tubulin, 20 nM KIF2A, 10 nM spastin, and 30 nM mScarlet-NuMA FL . γTuRC-capped microtubule minus ends are stabilized, while released microtubule minus ends typically depolymerize under the action of KIF2A, which also increases the catastrophe frequency at the plus end, often resulting in early microtubule disappearance. After some time, NuMA can cap the minus ends of released microtubules, arresting treadmilling and increasing their lifetimes. (C) Representative TIRF microscopy time course images of microtubule minus ends stabilized by γTuRC (yellow circles) undergoing γTuRC release, consequential treadmilling (white dashed circles), and eventually NuMA capping (green circles). Protein concentrations as in B; timestamps refer to mm:ss; related to . (D) Frequency of different γTuRC-nucleated microtubule populations (mean ± SEM). Each distinct symbol represents a replicate; protein concentrations as in B; n = 349 γTuRC-nucleated microtubules. (E) Lifetime of γTuRC-nucleated microtubules following their release, in the absence or presence of NuMA capping their minus ends (means of medians ± SEM). Each big circle represents the median value of one replicate, each small symbol represents the lifetime of one microtubule of one replicate; n = 133 γTuRC released microtubules; P value by Welch’s t test: 0.0030. Experiments were performed in γTuRC release buffer. All data are from three biological replicates.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: NuMA binds to the minus ends of laser-ablated microtubules. (A) Schematic of a TIRF microscopy assay with microtubules elongating from surface-immobilized GMPCPP-seeds being severed by laser ablation in the presence of mScarlet-NuMA FL . (B and C) Representative TIRF microscopy time course images of a microtubule elongating from a surface-immobilized Atto647N-labeled GMPCPP-seed (magenta) in the presence of 40 nM mScarlet-NuMA FL (green) and 10 µM Atto647N-tubulin (magenta). Upon severing of the microtubule by laser ablation (asterisks), NuMA binds selectively to the newly generated minus end (arrowheads). Timestamps refer to mm:ss. (B) Ablation performed at one (top panel) or two (bottom panel) sites close to the microtubule minus end generates short segments that diffuse away in solution, thus only the minus end of the longest fragment which stays anchored to the surface can be observed. (C) Ablation performed farther from the microtubule minus end produces longer fragments that remain attached to the surface, allowing visualization of both new plus and minus ends. Related to . (D) Frequency of NuMA localization to freshly generated plus and minus ends after laser ablation in the presence of 40 nM mScarlet-NuMA FL and 10 µM Atto647N-tubulin (mean ± SEM, five biological replicates). Each color represents a replicate; n = 54 ablation sites. All experiments were performed in NuMA microscopy buffer.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy, Labeling, Generated

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: Full-length NuMA can mediate dynein/dynactin-driven microtubule transport. (A) Schematic (top) of the polymerization protocol to obtain microtubule “lollipops” with representative TIRF microscopy image (bottom) of lollipops obtained from a mixture of 0.8 µM Atto647N-tubulin (magenta), 40 nM mScarlet-NuMA FL (green), and 1 mM GMPCPP. Minus ends can be identified by the selective presence of mScarlet-NuMA FL . (B) Schematic of a two-flush TIRF microscopy dynein-driven microtubule transport assay: first, dynein, dynactin, and Lis1 are flowed into a channel containing long surface-immobilized GMPCPP-microtubules; dynein is allowed to accumulate on microtubules for ≈3 min; in a second step, lollipops are introduced into the channel, with dynactin and Lis1, leading to dynein/dynactin/lollipop-bound NuMA transporting lollipop microtubules. (C and D) Representative time course TIRF microscopy images (C) and related kymograph (D) of transport events performed in the presence of 14 nM mEGFP-dynein (pre-bound to immobilized GMPCPP-microtubules), 28 nM dynactin and 1,000 nM Lis1. For both the faster (red arrowheads) and slower (yellow arrowheads) transport events, the lollipop is bound to the immobilized microtubule through a single anchoring point, corresponding to co-localizing mEGFP-dynein and mScarlet-NuMA FL (arrowheads), resulting in it to dangle while being transported. Related to . (E) Left: frequency of lollipop microtubule behaviors on immobilized microtubules (mean ± SEM, three biological replicates); each distinct symbol represents a replicate; protein concentrations as in C and D; n = 82 landed lollipops. Right: cartoons representing the three categories of transported lollipops; dynein and NuMA are depicted in cyan and green, respectively, the lollipop microtubule is shorter and brighter; the polarity of both lollipop and immobilized microtubule are indicated; arrow points to the direction of dynein motility (blue) and microtubule transport (magenta). Experiments were performed in dynein microscopy buffer with the omission of methylcellulose.

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy, Transport Assay

Journal: The Journal of Cell Biology

Article Title: NuMA is a mitotic adaptor protein that activates dynein and connects it to microtubule minus ends

doi: 10.1083/jcb.202408118

Figure Lengend Snippet: Different modes of dynein/dynactin/NuMA FL -mediated microtubule transport. (A–E) Representative time course TIRF microscopy images with schematics illustrating the type of transport (A, C, and E) and related kymographs (B and D) of microtubule transport experiments performed in the presence of 14 nM mEGFP-dynein (prebound to surface-immobilized Atto647N-labeled GMPCPP-microtubules), 28 nM dynactin, 1,000 nM Lis1, and “lollipop” microtubule minus-end bound mScarlet-NuMA FL . (A and B) A lollipop gets aligned to the immobilized microtubule, apparently through some crosslinking dynein bound at a distance from the lollipop minus end (red arrowhead, strongest NuMA signal). The orientation is parallel, as the lollipop minus end is facing the immobilized microtubule minus end (recognized by the direction of dynein-driven transport, and accumulated dynein). (C and D) Similarly to A and B, a lollipop is aligned to the immobilized microtubule, however, in antiparallel orientation, which results in the plus end of the lollipop being slid toward the minus end of the immobilized microtubule. (E) A lollipop-bound NuMA (red arrowhead) in solution lands directly on the minus end of the immobilized microtubule, where dynein has accumulated, resulting in transport-independent gathering of minus ends. The timestamps refer to mm:ss. Schematics as described in .

Article Snippet: The final assay mix (15 μl) was flushed all at once in a channel containing immobilized long microtubules at 30°C in the TIRF microscope incubator (OkoLab).

Techniques: Microscopy, Labeling

Journal: iScience

Article Title: ARHGEF9 regulates melanoma morphogenesis in environments with diverse geometry and elasticity by promoting filopodial-driven adhesion

doi: 10.1016/j.isci.2022.104795

Figure Lengend Snippet: ARHGEF9 regulates adhesion dynamics in WM266-4 cells (A and B) Representative total internal reflection fluorescence microscopy images of a WM266-4 cell and ARHGEF9 depleted cell expressing eGFP-Paxillin and LifeAct-Mars for actin. (C) Single lineage features output from the focal adhesion analysis server, wild-type (n = 487), ARHGEF9 OTP siRNA (n = 165), Y-27632 (n = 410), Blebbistatin (n = 622) and PF573288 (n = 554). Paxillin dynamics visualizations showing adhesion dynamics over time in wild-type and ARHGEF9. (D) Paxillin dynamics visualizations showing adhesion dynamics over time in WT and OTP ARHGEF9 KD cells. (E) Adhesion kinetics data measuring assembly and disassembly rates as well as the length of each phase, calculated using the FAAS. Assembling adhesions that were detected were as follows, Wild-type (n = 119), ARHGEF9 OTP siRNA (n = 56), Y-27632 (n = 142), Blebbistatin (n = 161) and PF-573288 (n = 217). Disassembling adhesions that were detected were as follows, Wild-type (n = 216), ARHGEF9 OTP siRNA (n = 94), Y-27632 (n = 95), Blebbistatin (n = 182) and PF-573288 (n = 216).

Article Snippet: Cells were imaged on a 3i microscopy vector total internal reflection (TIRF) microscope system (3i Intelligent Imaging).

Techniques: Fluorescence, Microscopy, Expressing