Chloroplast Sec14-like 1 (CPSFL1) is essential for normal chloroplast development and affects carotenoid accumulation in Chlamydomonas.

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of Chlamydomonas reinhardtii and isolated cpsfl1 The cpsfl1 mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The CPSFL1 gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The cpsfl1 mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and ß-carotene when expressed in Escherichia coli and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II.

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559 Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

Listeria monocytogenes triggers noncanonical autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy.

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenesL. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.

Genetic analysis of a novel tubulin mutation that redirects synaptic vesicle targeting and causes neurite degeneration in C. elegans.

Neuronal cargos are differentially targeted to either axons or dendrites, and this polarized cargo targeting critically depends on the interaction between microtubules and molecular motors. From a forward mutagenesis screen, we identified a gain-of-function mutation in the C. elegans α-tubulin gene mec-12 that triggered synaptic vesicle mistargeting, neurite swelling and neurodegeneration in the touch receptor neurons. This missense mutation replaced an absolutely conserved glycine in the H12 helix with glutamic acid, resulting in increased negative charges at the C-terminus of α-tubulin. Synaptic vesicle mistargeting in the mutant neurons was suppressed by reducing dynein function, suggesting that aberrantly high dynein activity mistargeted synaptic vesicles. We demonstrated that dynein showed preference towards binding mutant microtubules over wild-type in microtubule sedimentation assay. By contrast, neurite swelling and neurodegeneration were independent of dynein and could be ameliorated by genetic paralysis of the animal. This suggests that mutant microtubules render the neurons susceptible to recurrent mechanical stress induced by muscle activity, which is consistent with the observation that microtubule network was disorganized under electron microscopy. Our work provides insights into how microtubule-dynein interaction instructs synaptic vesicle targeting and the importance of microtubule in the maintenance of neuronal structures against constant mechanical stress.

Characterization of a planctomycetal organelle: a novel bacterial microcompartment for the aerobic degradation of plant saccharides.

Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae.

Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens.

A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions.

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans.

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a "canonical" IFT motor, whereas OSM-3 is an "accessory" IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motility.

Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.

Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography.

Despite the fact that most bacteria grow in biofilms in natural and pathogenic ecosystems, very little is known about the ultrastructure of their component cells or about the details of their community architecture. We used high-pressure freezing and freeze-substitution to minimize the artifacts of chemical fixation, sample aggregation, and sample extraction. As a further innovation we have, for the first time in biofilm research, used electron tomography and three-dimensional (3D) visualization to better resolve the macromolecular 3D ultrastructure of a biofilm. This combination of superb specimen preparation and greatly improved resolution in the z axis has opened a window in studies of Myxococcus xanthus cell ultrastructure and biofilm community architecture. New structural information on the chromatin body, cytoplasmic organization, membrane apposition between adjacent cells, and structure and distribution of pili and vesicles in the biofilm matrix is presented.

Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network.

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.

Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans.

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.

Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis.

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response.

The protein folding capacity of the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR senses unfolded proteins in the ER lumen and transmits that information to the cell nucleus, where it drives a transcriptional program that is tailored to re-establish homeostasis. Using thin section electron microscopy, we found that yeast cells expand their ER volume at least 5-fold under UPR-inducing conditions. Surprisingly, we discovered that ER proliferation is accompanied by the formation of autophagosome-like structures that are densely and selectively packed with membrane stacks derived from the UPR-expanded ER. In analogy to pexophagy and mitophagy, which are autophagic processes that selectively sequester and degrade peroxisomes and mitochondria, the ER-specific autophagic process described utilizes several autophagy genes: they are induced by the UPR and are essential for the survival of cells subjected to severe ER stress. Intriguingly, cell survival does not require vacuolar proteases, indicating that ER sequestration into autophagosome-like structures, rather than their degradation, is the important step. Selective ER sequestration may help cells to maintain a new steady-state level of ER abundance even in the face of continuously accumulating unfolded proteins.

Cytological analysis of meiosis in Caenorhabditis elegans.

The nematode Caenorhabditis elegans has emerged as an informative experimental system for analysis of meiosis, in large part because of the advantageous physical organization of meiotic nuclei as a gradient of stages within the germline. Here we provide tools for detailed observational studies of cells within the worm gonad, including techniques for light and electron microscopy.

Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.

Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features.

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole.

The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores approximately 4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.

Recent advances in high-pressure freezing: equipment- and specimen-loading methods.

This chapter is an update of material first published by McDonald in the first volume of this book. Here, we discuss the improvements in the technology and the methodology of high-pressure freezing (HPF) since that article was published. First, we cover the latest innovation in HPF, the Leica EM PACT2. This machine differs significantly from the BAL-TEC HPM 010 high-pressure freezer, which was the main subject of the former chapter. The EM PACT2 is a smaller, portable machine and has an optional attachment, the Rapid Transfer System (RTS). This RTS permits easy and reproducible loading of the sample and allows one to do correlative light and electron microscopy with high time resolution. We also place more emphasis in this article on the details of specimen loading for HPF, which is considered the most critical phase of the whole process. Detailed procedures are described for how to high-pressure freeze cells in suspension, cells attached to substrates, tissue samples, or whole organisms smaller than 300 microm, and tissues or organisms greater than 300 microm in size. We finish the article with a brief discussion of freeze substitution and recommend some sample protocols for this procedure.

Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+.

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1-2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase-specific inhibitor of SPB maturation whose expression is under control of cdc10(+). This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.