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

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 and isolated The mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching.

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.

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 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.

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 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).

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.

The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture.

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.

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.

Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material.

This article presents the best current practices for preparation of biological samples for examination as thin sections in an electron microscope. The historical development of fixation, dehydration, and embedding procedures for biological materials are reviewed for both conventional and low temperature methods. Conventional procedures for processing cells and tissues are usually done over days and often produce distortions, extractions, and other artifacts that are not acceptable for today's structural biology standards.

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.

Transmission electron microscopy of thin sections of Drosophila: preparation of embryos using n-heptane and glutaraldehyde.

This protocol describes the simultaneous permeabilization of Drosophila embryos with n-heptane and initial fixation with glutaraldehyde. Even though the vitelline membrane around the embryo is chemically permeabilized, it must be manually removed to achieve infiltration with embedding resins. Once the embryo is embedded, it can be sectioned for transmission electron microscopy (TEM).

Preparation of Drosophila specimens for examination by transmission electron microscopy.

There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue.

Postembedding immunolabeling of thin sections of Drosophila tissues for transmission electron microscopy.

Postembedding immunolabeling using resin sections is the recommended method for beginners carrying out electron microscopy (EM) immunolabeling. Postembedding labeling refers to labeling on sections, which is a method of gaining access to the interior of the cell without the harshness of detergent or ionic extraction as is performed with preembed labeling. Investigators already familiar with routine EM-sectioning techniques find EM immunolabeling using resin sections easiest to do, as procedures are similar to those used when performing light microscopy (LM) immunolabeling, but using a different resin.

Immunolabeling of thin sections of Drosophila tissues for transmission electron microscopy.

The main advantage of electron microscopy (EM) for immunolabeling is resolution, but there is also another aspect that is often overlooked. For many investigators, the definitive image of an organelle is the one generated by EM. This is especially true for membranous organelles, with the possible exception of the nucleus and plant vacuoles.

Transmission electron microscopy of thin sections of Drosophila: conventional chemical fixation of embryos using trialdehyde.

High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well.

Transmission electron microscopy of thin sections of Drosophila: high-pressure freezing and freeze-substitution.

The state of the art in fine-structure preservation for thin sectioning can be achieved by using fast-freezing technology followed by freeze substitution and embedding in resin. Samples prepared by high-pressure freezing are estimated to be "fixed" in 20-50 msec. Fast freezing also freezes every cell component regardless of its chemistry.

Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae.

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.

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.

RAB-5- and RAB-11-dependent vesicle-trafficking pathways are required for plasma membrane repair after attack by bacterial pore-forming toxin.

Pore-forming toxins (PFTs) secreted by pathogenic bacteria are the most common bacterial protein toxins and are important virulence factors for infection. PFTs punch holes in host cell plasma membranes, and although cells can counteract the resulting membrane damage, the underlying mechanisms at play remain unclear. Using Caenorhabditis elegans as a model, we demonstrate in vivo and in an intact epithelium that intestinal cells respond to PFTs by increasing levels of endocytosis, dependent upon RAB-5 and RAB-11, which are master regulators of endocytic and exocytic events.

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.

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.

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.

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.

Oocyte CD9 is enriched on the microvillar membrane and required for normal microvillar shape and distribution.

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).

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.