36 research outputs found

    Electron Tomography Reveals Novel Microtubule Lattice and Microtubule Organizing Centre Defects in +TIP Mutants

    Get PDF
    <div><p>Mal3p and Tip1p are the fission yeast (<i>Schizosaccharomyces pombe</i>) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by <i>mal3</i> and <i>tip1</i> deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families.</p></div

    Mal3p and Tip1p are both involved in MT bundle anchoring to the NE/SPB.

    No full text
    <p><b>A)</b> Fluorescence microscopy of cells expressing Sid4-CFP and GFP-tubulin show a disruption in SPB-MT interaction in mutant cells. Scale bar 2 µm <b>B)</b> Slices from a tomogram reveal relatively normal SPB morphology in <i>tip1Δ</i> mutants. <b>C)</b> 3D models of MT bundle association with the SPB in four different <i>tip1Δ</i> cells. <b>D)</b> Tomographic slice showing a duplicated SPB in a <i>mal3Δ</i> mutant. SPB1 is in close association with a MT, whereas the MT bends away from SPB2. <b>E)</b> A 3D model of the entire SPB of which a slice was shown in D). Note that no MTs are associated with the right SPB. <b>F)</b> A large percentage of 3D models of bundles in <i>tip1Δ</i> and <i>mal3Δ</i> mutants were not associated with the NE. <b>G)</b> Green channel red channel, and merged image of <i>mto1Δ</i> cells expressing Cut12-GFP and either mCherry-Atb2 (left panels) or Mal3-tagRFP (right panels). The centre cell of the left panel shows an SPB without attached MTs. The two cells of the right panels have SPBs without MTs but a punctate Mal3p signal co-localizes with both SPBs. Scale bar 2 µm.</p

    mal3Δ cells show altered SPB morphology and size.

    No full text
    <p><b>A)</b> 1 nm thick section from a tomogram of a duplicated SPB. Central bridge (CB), central plaques (CP), microtubule (MT), nuclear envelope (NE). <b>B)</b> A 3D model of the duplicated WT SPB shown in A), amorphous SPB electron density represented in transparent gold, central bridge in yellow and the central plaques in red. The model is displayed with a slice from the electron tomogram, in which the NE and an associated MT shows in the front view. <b>C)</b> The same 3D model without the tomographic slice. <b>D)</b> The 3D model of the oblique central bridge and the central plaques only. <b>E)</b> The lengths of cells in which the SPBs were examined. <b>F–H)</b> All these measurements come from 3D models (made in IMOD) from serial thin section reconstructions of entire SPBs. Graphs show a comparison of the length, length and volume of the SPB 3D models. The black line across the boxes is the average, the white line the median, the box show the 25<sup>th</sup> to the 75<sup>th</sup> percentile. Error bars show the 5<sup>th</sup> and 95<sup>th</sup> percentile. <b>I)</b> A mixture of single and duplicated WT SPBs was found in early G2 cells, and their volume were measured and plotted against the cell length. No increase in volume is seen as the SPB duplicates. <b>J)</b> Thin section electron micrograph of WT and <i>mal3Δ</i> SPBs. The <i>mal3Δ</i> SPBs show abnormal morphology with unclear central plaques. <b>K)</b> Normalized fluorescence of the +TIP mutants and MBC treated cells compared to WT cells expressing the same fluorescent marker. The graph shows an increase in fluorescence for <i>mal3Δ</i> and <i>tip1Δ</i> mutants, not found in MBC treated cells. Error bars denote SEM, 90 or more cells/strain were analyzed.</p

    tip1Δ has an increased proportion of thin filaments.

    No full text
    <p><b>A)</b> A wild type MT bundle (green), containing two thin filaments (turquoise). The white arrowhead points to the filament shown in B. <b>B)</b> Slices from the tomogram (every 3 nm) showing a thin hollow filament next to a normal MT. The insert is a snapshot of the marked position (turquoise arrowhead) rotated 90 degrees in the x-axis, so that the filaments are visible in cross-section. This clearly shows that both filaments are hollow and that they have different diameters. <b>C–D)</b> The lengths and widths of individual thin filaments <b>E)</b> The proportion of the total polymerized tubulin incorporated into thin filaments in WT, <i>mal3Δ</i> and <i>tip1Δ</i> clearly shows an increase in these filaments in <i>tip1Δ.</i></p

    MT bundle organization in <i>mal3Δ</i> is similar to WT but disturbed in <i>tip1Δ</i>.

    No full text
    <p>Using serial section electron tomograms 3D model of cells and individual MT bundles was made. <b>A–C)</b> 3D model of <i>mal3Δ</i> cells, and <b>D–F)</b><i>tip1Δ</i> cells. In the model, plasma membrane is colored transparent green, the nuclear envelope transparent pink, SPBs in yellow, MTs in green and in their ends colored balls that represent their end structure. <b>G)</b> A line drawing of the different MT ends structures found and their color code in the model. <b>H)</b> Distribution of MT end structures, both on minus and plus ends. I) The distribution of end structures at MT plus ends. <b>J)</b> MTs with both open ends were found in all three cell strains, but they were more common in cells lacking Tip1p.</p

    Nuclear envelope budding and its cellular functions

    No full text
    The nuclear pore complex (NPC) has long been assumed to be the sole route across the nuclear envelope, and under normal homeostatic conditions it is indeed the main mechanism of nucleo-cytoplasmic transport. However, it has also been known that e.g. herpesviruses cross the nuclear envelope utilizing a pathway entitled nuclear egress or envelopment/de-envelopment. Despite this, a thread of observations suggests that mechanisms similar to viral egress may be transiently used also in healthy cells. It has since been proposed that mechanisms like nuclear envelope budding (NEB) can facilitate the transport of RNA granules, aggregated proteins, inner nuclear membrane proteins, and mis-assembled NPCs. Herein, we will summarize the known roles of NEB as a physiological and intrinsic cellular feature and highlight the many unanswered questions surrounding these intriguing nuclear events
    corecore