Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy.

Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.

Cep78 is a new centriolar protein involved in Plk4-induced centriole overduplication.

Centrioles are core components of centrosomes, the major microtubule-organizing centers of animal cells, and act as basal bodies for cilia formation. Control of centriole number is therefore crucial for genome stability and embryogenesis. Centriole duplication requires the serine/threonine protein kinase Plk4. Here, we identify Cep78 as a human centrosomal protein and a new interaction partner of Plk4. Cep78 is mainly a centriolar protein that localizes to the centriolar wall. Furthermore, we find that Plk4 binds to Cep78 through its N-terminal domain but that Cep78 is not an in vitro Plk4 substrate. Cep78 colocalizes with Plk4 at centrioles and is required for Plk4-induced centriole overduplication. Interestingly, upon depletion of Cep78, newly synthesized Plk4 is not localized to centrosomes. Our results suggest that the interaction between Cep78 and the N-terminal catalytic domain of Plk4 is a new and important element in the centrosome overduplication process.

Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing.

Repetitive-element-derived Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi2 (also known as Piwil4) in a genome defence mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility. A third Piwi family member, Miwi (also known as Piwil1), is expressed in specific postnatal germ cells and associates with a unique set of piRNAs of unknown function. Here we show that Miwi is a small RNA-guided RNase (slicer) that requires extensive complementarity for target cleavage in vitro. Disruption of its catalytic activity in mice by a single point mutation causes male infertility, and mutant germ cells show increased accumulation of LINE1 retrotransposon transcripts. We provide evidence for Miwi slicer activity directly cleaving transposon messenger RNAs, offering an explanation for the continued maintenance of repeat-derived piRNAs long after transposon silencing is established in germline stem cells. Furthermore, our study supports a slicer-dependent silencing mechanism that functions without piRNA amplification. Thus, Piwi proteins seem to act in a two-pronged mammalian transposon silencing strategy: one promotes transcriptional repression in the embryo, the other reinforces silencing at the post-transcriptional level after birth.

The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements.

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

The CryoCapsule: simplifying correlative light to electron microscopy.

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells.

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells.

Electron tomography of the microtubule cytoskeleton in multinucleated hyphae of Ashbya gossypii.

We report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

GCP6 is a substrate of Plk4 and required for centriole duplication.

Centriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the γ-tubulin ring complex (γ-TuRC) that nucleates microtubules. GCP6 is a member of the γ-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the γ-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal γ-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication.

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Gamma-tubulin complex-mediated anchoring of spindle microtubules to spindle-pole bodies requires Msd1 in fission yeast.

The anchoring of microtubules to subcellular structures is critical for cell polarity and motility. Although the process of anchoring cytoplasmic microtubules to the centrosome has been studied in some detail, it is not known how spindle microtubules are anchored to the mitotic centrosome and, particularly, whether anchoring and nucleation of mitotic spindles are functionally separate. Here, we show that a fission yeast coiled-coil protein, Msd1, is required for anchoring the minus end of spindle microtubules to the centrosome equivalent, the spindle-pole body (SPB). msd1 deletion causes spindle microtubules to abnormally extend beyond SPBs, which results in chromosome missegregation. Importantly, this protruding spindle is phenocopied by the amino-terminal deletion mutant of Alp4, a component of the gamma-tubulin complex (gamma-TuC), which lacks the potential Msd1-interacting domain. We propose that Msd1 interacts with gamma-TuC, thereby specifically anchoring the minus end of microtubules to SPBs without affecting microtubule nucleation.

Organization of organelles within hyphae of Ashbya gossypii revealed by electron tomography.

Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.

Electron tomography reveals a flared morphology on growing microtubule ends.

Microtubules (MTs) exhibit dynamic instability, alternating between phases of growth and shortening, mostly at their uncapped plus ends. Based on results from cryo-electron microscopy it was proposed that growing MTs display mainly curved sheets and blunt ends; during depolymerisation curled 'ramshorns' predominate. Observations of MTs in mitotic cells have suggested that the situation in vivo differs from that in vitro, but so far, a clear comparison between in vivo and in vitro results has not been possible because MT end structures could not be correlated directly with the dynamic state of that particular MT. Here we combine light microscopy and electron tomography (ET) to show that growing MT plus ends in the fission yeast Schizosaccharomyces pombe display predominantly a flared morphology. This indicates that MT polymerisation in vivo and in vitro can follow different paths.

A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells.

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the 'Golgi-depleted' karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

Organization of interphase microtubules in fission yeast analyzed by electron tomography.

Polarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.

Internal structure of an intact Convallaria majalis pollen grain observed with X-ray Fresnel coherent diffractive imaging.

We have applied Fresnel Coherent Diffractive Imaging (FCDI) to image an intact pollen grain from Convallaria majalis. This approach allows us to resolve internal structures without the requirement to chemically treat or slice the sample into thin sections. Coherent X-ray diffraction data from this pollen grain-composed of a hologram and higher resolution scattering information-was collected at a photon energy of 1820 eV and reconstructed using an iterative algorithm. A comparison with images recorded using transmission electron microscopy demonstrates that, while the resolution of these images is limited by the available flux and mechanical stability, we observed structures internal to the pollen grain-the intine/exine separations and pore dimensions-finer than 60 nm. The potential of this technique for further biological imaging applications is discussed.

An amyloidogenic determinant in N-terminal pro-brain natriuretic peptide (NT-proBNP): Implications for cardiac amyloidoses.

Deposition of amyloid in the atria (isolated atrial/cardiac amyloid) is fairly common in the aging heart. It consists of amyloid fibrils, formed both by atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and the precursor molecule of ANP, proANP. This study examines whether amyloidogenic determinants (short peptides/amyloid forming favoring regions) exist in the sequence of NT-proBNP, the N-terminal part of proBNP, and if these determinants form amyloid-like fibrils in vitro. We have predicted a possible amyloidogenic determinant in the sequence of the NT-proBNP, and we conclusively show, after its synthesis, that it forms amyloid-like fibrils in vitro, utilizing transmission electron microscopy, X-ray diffraction, attenuated total reflectance Fourier-transform infrared spectroscopy, and polarizing microscopy. Thus, for the first time, in this study, a possible biological role is attributed to a certain, specific part of this important cardiac prohormone/natriuretic peptide, which acts as an important biomarker indicative of heart failure. Its possible direct involvement in isolated cardiac amyloidosis, atrial fibrillation, and other types of cardiac amyloidoses is indicated and discussed. Since these cardiac hormones and their prohormones play key roles in cardiovascular homeostasis through natriuresis, diuresis, vasorelaxation, and inhibition of renin and aldosterone secretion (pathophysiology of hypertension and cardiovascular regulation), we also try to suggest these specific, short peptides as possible future structural targets of efforts toward inhibiting formation of natriuretic peptide(s) amyloid.

Molecular mechanisms of kinetochore capture by spindle microtubules.

For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial kinetochore capture have remained elusive. Here we visualized individual kinetochore-microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct kinetochore components are used for kinetochore capture by microtubules and for ensuring subsequent sister kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured kinetochores along microtubules. During such transport, kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured kinetochores to the plus ends of microtubules.

Differential Nanoparticle Sequestration by Macrophages and Scavenger Endothelial Cells Visualized in Vivo in Real-Time and at Ultrastructural Resolution.

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes.

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.

The molecular mechanisms underlying early/recycling endosomes-to-TGN transport are still not understood. We identified interactions between the TGN-localized putative t-SNAREs syntaxin 6, syntaxin 16, and Vti1a, and two early/recycling endosomal v-SNAREs, VAMP3/cellubrevin, and VAMP4. Using a novel permeabilized cell system, these proteins were functionally implicated in the post-Golgi retrograde transport step. The function of Rab6a' was also required, whereas its closely related isoform, Rab6a, has previously been implicated in Golgi-to-endoplasmic reticulum transport. Thus, our study shows that membrane exchange between the early endocytic and the biosynthetic/secretory pathways involves specific components of the Rab and SNARE machinery, and suggests that retrograde transport between early/recycling endosomes and the endoplasmic reticulum is critically dependent on the sequential action of two members of the Rab6 subfamily.