[Show abstract][Hide abstract]ABSTRACT: SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.
Full-text Article · Mar 2014 · The Journal of Cell Biology
[Show abstract][Hide abstract]ABSTRACT: Influence of the pH on the mean number of photons emitted and experimental considerations. (a) Mean photon count vs. pH for STORM buffer #3 (“BME+MEA”, see Table S1) with (red curve) and without (black curve) COT; three different regions are highlighted: blue (pH >8), green (8>pH >6.75), and red (pH <6.75) - corresponding to different average ON times (τon), which were determined by calculating the average number of frames a given single molecule was on. (b) Representative single molecule traces over 5 frames (1 frame = 40 ms, laser intensity ∼4 kW/cm2). As the pH decreases, the average ON time of Alexa-647 increases, and while this is at first advantageous because of the increased brightness (compare brightness of peaks in the green box versus the blue box), it becomes problematic at pH lower than ∼6.5 since the ON time becomes very large, leading to a higher probability of overlapping peaks. At low pH values, one way to decrease τon would be to increase the laser power, but laser powers are limited: on the commercial microscope used for 3D imaging, the maximum intensity at the focus is equal to 4.5 kW.cm−2, thus imaging was typically performed in the pH range from ∼7–8. The OFF time were also probably affected, but STORM images only provide us with a direct measurement of the ON times. This behaviour is consistent with single molecule measurements previously reported .
[Show abstract][Hide abstract]ABSTRACT: Blinking in the presence of COT. Analyses performed on an immunostained tubulin sample showing (a) Number of detected molecules in a 128×128 pixel region (pixel size 100 nm) over 64.000 frames with only the 641 nm laser ON (∼4 kW/cm2) using buffer #4 (see Table S1); (b) Number of detected molecules in a 128×128 pixel region over 64.000 frames with both 641 nm and 405 nm lasers ON (∼4 kW/cm2 and ∼1 W/cm2) using buffer # 4; (c) Number of detected molecules in a 64×64 pixels region over 40.000 frames with 641 nm laser ON, and adding the 405 nm laser after 14.000 frames. (∼4 kW/cm2) using buffer #4.
[Show abstract][Hide abstract]ABSTRACT: Astigmatism z-calibrations. (a) Estimated z-localization in nm for each couple of (width: wx, height: wy) expressed in pixels between a minimum and a maximum value for the 100× objective obtained from the calibration data (see Data Analysis for more details). (b) Estimated error in the localization associated to the matrix shown in (a). (c) Measured z position vs. measured width and height for the dataset shown in Figure 4a (corresponding region in (a) illustrated by the dotted square). (d) Estimated z localization in nm for each couple of (width: wx, height: wy) expressed in pixels between a minimum and a maximum value for the 60x water objective obtained from the calibration data. (e) Estimated error in the localization associated to the matrix shown in (d). (f) Measured z position vs. measured width and height for the dataset shown in Figure 4e (corresponding region in (d) illustrated by the dotted square).
[Show abstract][Hide abstract]ABSTRACT: Mechanism of Alexa-647 blinking. Energy diagram of Alexa-647 under the influence of the different chemicals used in this article. STORM blinking relies on the cycling between the ON state, and the reduced OFF state (long-lived dark states), and is achieved by adding a reducing agent in an oxygen-depleted environment . Adding COT to this buffer improves the lifetime of the ON time through direct energy exchange with the triplet state , while adding Trolox acts on both the ON state through its reduced form (TX), and on the dark state trough its oxidized form (TQ) , thereby interfering with the proper cycling of the dye. Adapted from .
[Show abstract][Hide abstract]ABSTRACT: Super-resolution imaging methods have revolutionized fluorescence microscopy by revealing the nanoscale organization of labeled proteins. In particular, single-molecule methods such as Stochastic Optical Reconstruction Microscopy (STORM) provide resolutions down to a few tens of nanometers by exploiting the cycling of dyes between fluorescent and non-fluorescent states to obtain a sparse population of emitters and precisely localizing them individually. This cycling of dyes is commonly induced by adding different chemicals, which are combined to create a STORM buffer. Despite their importance, the composition of these buffers has scarcely evolved since they were first introduced, fundamentally limiting what can be resolved with STORM. By identifying a new chemical suitable for STORM and optimizing the buffer composition for Alexa-647, we significantly increased the number of photons emitted per cycle by each dye, providing a simple means to enhance the resolution of STORM independently of the optical setup used. Using this buffer to perform 3D-STORM on biological samples, we obtained images with better than 10 nanometer lateral and 30 nanometer axial resolution.
[Show abstract][Hide abstract]ABSTRACT: List of the localized peaks after grouping used to build figure S5. The first column correspond to the molecule index, the second one correspond to the last frame in which a molecule appears, and the 3rd and 4th column correspond to the x and y localization, in nanometer.
[Show abstract][Hide abstract]ABSTRACT: Photon counts & localization precision. (a) Normalized frame and molecule photon counts distribution extracted from the dataset shown in Figure 4a–c (Buffer #4 in Table S1), mean values are indicated in the top right corner. (b) Frame localization precision in both axial and lateral directions estimated from the dataset shown in Figure 4a–c by calculating the standard deviation of repeated localizations (threshold of 5 localizations, see Notes S1 for more details). (c) Frame and molecule photon counts distribution extracted from the dataset shown in Figure 4d–f (Buffer #5 in Table S1), mean values are indicated in the top right corner. (d) Frame localization precision in both axial and lateral directions estimated from the dataset shown in Figure 4d–f by calculating the standard deviation of repeated localizations (threshold of 5 localizations).
[Show abstract][Hide abstract]ABSTRACT: Imaging properties using Pyranose Oxidase. (a) 2D STORM image of a COS-7 cell stained with alpha-tubulin primary and Alexa-647 F(ab’)2 secondary antibodies in “Pyranose Oxidase  Buffer” (Buffer #6 in Table S1), and (b) Zoom on the boxed region defined in (a) showing the hollowness of microtubules. (c) Lateral profile measured and averaged over a 500 nm-long microtubule region (green box in (b)) and indicated FWHM values as well as distance between two peaks, showing distances consistent with Figure 3e. (d) Normalized frame (red) and grouped molecules (black) photon count distribution, as well as average values given in the top right corner. (e) Normalized frame localization precision in the lateral direction estimated from the dataset shown in (a) by calculating the standard deviation of repeated localizations (threshold of 5 localizations). The actual localization precision is expected to be closer to the standard error of the mean, but using only peaks on for more than 5 frames would skew the value.
[Show abstract][Hide abstract]ABSTRACT: Model of microtubules nanostructure. (a) Schematics of the cross-section of an immuno-labeled microtubule. The epitope of the alpha-tubulin antibody used is located towards the C-terminal end of the protein (manufacturer's datasheet), which was shown by various electron-microscopy studies to be located outside of the tube (reviewed in ). We then assume that the primary antibodies are decorated isotropically with smaller secondary antibody fragments, resulting in a hollow labeled tubular structure with respective inner and outer diameter of ∼25 nm, and ∼50 nm. (b) Expected signal from the projection of this 25–50 nm tube assuming different “resolution” (Gaussian blurring of 1–15 nm indicated in sigma). (c) Lateral profile from Figure 3d (black line) compared with the modeled structure assuming a blurring factor of 4 nm (red line) (d) axial profile from Figure 4e (black line) compared with the modeled structure assuming a blurring factor of 9 nm (red line).
[Show abstract][Hide abstract]ABSTRACT: High resolution STORM image of microtubules. STORM image of the data used for Figure 3a–f is displayed by binning the grouped localizations into a regular grid of 3×3 nm. A value of n corresponds to n molecules per pixel.
[Show abstract][Hide abstract]ABSTRACT: 3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.
Full-text Article · Jun 2013 · Biomedical Optics Express
[Show abstract][Hide abstract]ABSTRACT: Patients with MCPH (autosomal recessive primary microcephaly) exhibit impaired brain development, presumably due to the compromised function of neuronal progenitors. Seven MCPH loci have been identified, including one that encodes centrosome protein 4.1 associated protein (CPAP; also known as centromere protein J, CENPJ). CPAP is a large coiled-coil protein enriched at the centrosome, a structure that comprises two centrioles and surrounding pericentriolar material (PCM). CPAP depletion impairs centriole formation, whereas CPAP overexpression results in overly long centrioles. The mechanisms by which CPAP MCPH patient mutations affect brain development are not clear. Here, we identify CPAP protein domains crucial for its centriolar localization, as well as for the elongation and the formation of centrioles. Furthermore, we demonstrate that conditions that resemble CPAP MCPH patient mutations compromise centriole formation in tissue culture cells. Using adhesive micropatterns, we reveal that such defects correlate with a randomization of spindle position. Moreover, we demonstrate that the MCPH protein SCL/TAL1 interrupting locus (STIL) is also essential for centriole formation and for proper spindle position. Our findings are compatible with the notion that mutations in CPAP and STIL cause MCPH because of aberrant spindle positioning in progenitor cells during brain development.
Full-text Article · Nov 2011 · Journal of Cell Science
[Show abstract][Hide abstract]ABSTRACT: Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.
[Show abstract][Hide abstract]ABSTRACT: Centrosome duplication occurs once per cell cycle and ensures that the two resulting centrosomes assemble a bipolar mitotic spindle. Centriole formation is fundamental for centrosome duplication. In Caenorhabditis elegans, the evolutionarily conserved proteins SPD-2, ZYG-1, SAS-6, SAS-5, and SAS-4 are essential for centriole formation, but how they function is not fully understood. Here, we demonstrate that Protein Phosphatase 2A (PP2A) is also critical for centriole formation in C. elegans embryos. We find that PP2A subunits genetically and physically interact with the SAS-5/SAS-6 complex. Furthermore, we show that PP2A-mediated dephosphorylation promotes centriolar targeting of SAS-5 and ensures SAS-6 delivery to the site of centriole assembly. We find that PP2A is similarly needed for the presence of HsSAS-6 at centrioles and for centriole formation in human cells. These findings lead us to propose that PP2A-mediated loading of SAS-6 proteins is critical at the onset of centriole formation.
[Show abstract][Hide abstract]ABSTRACT: Monitored from the one-cell stage until the end of the second cell cycle. Images were captured every 5 s, and the movie is played at 10 frames per second. Embryos are ∼50 μm long and oriented with anterior to the left; time is indicated in minutes:seconds.