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.

Septins are conserved GTP-binding proteins involved in membrane compartmentalization and remodeling. In budding yeast, five mitotic septins localize at the bud neck, where the plasma membrane is enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)). We previously established the subunit organization within purified yeast septin complexes and how these hetero-octamers polymerize into filaments in solution and on PtdIns4,5P(2)-containing lipid monolayers. How septin ultrastructure in vitro relates to the septin-containing filaments observed at the neck in fixed cells by thin-section electron microscopy was unclear. A morphological description of these filaments in the crowded space of the cell is challenging, given their small cross section. To examine septin organization in situ, sections of dividing yeast cells were analyzed by electron tomography of freeze-substituted cells, as well as by cryo-electron tomography. We found networks of filaments both perpendicular and parallel to the mother-bud axis that resemble septin arrays on lipid monolayers, displaying a repeat pattern that mirrors the molecular dimensions of the corresponding septin preparations in vitro. Thus these in situ structures most likely represent septin filaments. In viable mutants lacking a single septin, in situ filaments are still present, although more disordered, consistent with other evidence that the in vivo function of septins requires filament formation.

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.

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 β-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.

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE.

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.

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.

Microvilli are found on the surface of many cell types, including the mammalian oocyte, where they are thought to act in initial contact of sperm and oocyte plasma membranes. CD9 is currently the only oocyte protein known to be required for sperm-oocyte fusion. We found CD9 is localized to the oocyte microvillar membrane using transmission electron microscopy (TEM). Scanning electron microscopy (SEM) showed that CD9 null oocytes, which are unable to fuse with sperm, have an altered length, thickness and density of their microvilli. One aspect of this change in morphology was quantified using TEM by measuring the radius of curvature at the microvillar tips. A small radius of curvature is thought to promote fusibility and the radius of curvature of microvillar tips on CD9 wild-type oocytes was found to be half that of the CD9 null oocytes. We found that oocyte CD9 co-immunoprecipitates with two Ig superfamily cis partners, EWI-2 and EWI-F, which could have a role in linking CD9 to the oocyte microvillar actin core. We also examined latrunculin B-treated oocytes, which are known to have reduced fusion ability, and found altered microvillar morphology by SEM and TEM. Our data suggest that microvilli may participate in sperm-oocyte fusion. Microvilli could act as a platform to concentrate adhesion/fusion proteins and/or provide a membrane protrusion with a low radius of curvature. They may also have a dynamic interaction with the sperm that serves to capture the sperm cell and bring it into close contact with the oocyte plasma membrane.

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.

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.

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.