SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients

A Novel Mouse Synaptonemal Complex Protein Is Essential for Loading of Central Element Proteins, Recombination, and Fertility

The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes

Newly imported proteins in mitochondria are particularly sensitive to aggregation

Aim: A functional proteome is essential for life and maintained by protein quality control (PQC) systems in the cytosol and organelles. Protein aggregation is an indicator of a decline of PQC linked to aging and disease. Mitochondrial PQC is critical to maintain mitochondrial function and thus cellular fitness. How mitochondria handle aggregated proteins is not well understood. Here we tested how the metabolic status impacts on formation and clearance of aggregates within yeast mitochondria and assessed which proteins are particularly sensitive to denaturation. Methods: Confocal microscopy, electron microscopy, immunoblotting and genetics were applied to assess mitochondrial aggregate handling in response to heat shock and ethanol using the mitochondrial disaggregase Hsp78 as a marker for protein aggregates. Results: We show that aggregates formed upon heat or ethanol stress with different dynamics depending on the metabolic state. While fermenting cells displayed numerous small aggregates that coalesced into one large foci that was resistant to clearance, respiring cells showed less aggregates and cleared these aggregates more efficiently. Acute inhibition of mitochondrial translation had no effect, while preventing protein import into mitochondria by inhibition of cytosolic translation prevented aggregate formation. Conclusion: Collectively, our data show that the metabolic state of the cells impacts the dynamics of aggregate formation and clearance, and that mainly newly imported and not yet assembled proteins are prone to form aggregates. Because mitochondrial functionality is crucial for cellular metabolism, these results highlight the importance of efficient protein biogenesis to maintain the mitochondrial proteome operational during metabolic adaptations and cellular stress

Exosomes purified from a single cell type have diverse morphology

Extracellular vesicles (EVs) are produced by all known organisms and are important for cell communication and physiology. Great morphological diversity has been described regarding EVs found in body fluids such as blood plasma, breast milk, and ejaculate. However, a detailed morphological analysis has never been performed on exosomes when purified from a single cell type. In this study we analysed and quantified, via multiple electron microscopy techniques, the morphology of exosomes purified from the human mast cell line HMC-1. The results revealed a wide diversity in exosome morphology, suggesting that subpopulations of exosomes with different and specific functions may exist. Our findings imply that a new, more efficient way of defining exosome subpopulations is necessary. A system was proposed where exosomes were classified into nine different categories according to their size and shape. Three additional morphological features were also found in exosomes regardless of their morphological classification. These findings show that exosomes purified from a single cell line are also morphologically diverse, similar to previous observations for EVs in body fluids. This knowledge can help to improve the interpretation of experimental results and widen our general understanding of the biological functions of exosomes

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

<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.

<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.

<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^th to the 75^th percentile. Error bars show the 5^th and 95^th 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

Microtubules lacking Mal3p have ‘kinks’.

<p><b>A)</b> Interphase microtubules with ‘kinked’ lattice, which were never observed in WT. <b>B)</b> line drawing of a kinked microtubule.</p

tip1Δ has an increased proportion of thin filaments.

<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