The endoplasmic reticulum (ER) is the major compartment for the processing and quality control of newly synthesized proteins. Green fluorescent protein (GFP) was used as a noninvasive probe to determine the viscous properties of the aqueous lumen of the ER. GFP was targeted to the ER lumen of CHO cells by transient transfection with cDNA encoding GFP (S65T/F64L mutant) with a C-terminus KDEL retention sequence and upstream prolactin secretory sequence. Repeated laser illumination of a fixed 2-micrometers diameter spot resulted in complete bleaching of ER-associated GFP throughout the cell, indicating a continuous ER lumen. A residual amount (<1%) of GFP-KDEL was perinuclear and noncontiguous with the ER, presumably within a pre- or cis-Golgi compartment involved in KDEL-substrate retention. Quantitative spot photobleaching with a single brief bleach pulse indicated that GFP was fully mobile with a t1/2 for fluorescence recovery of 88 +/- 5 ms (SE; 60x lens) and 143 +/- 8 ms (40x). Fluorescence recovery was abolished by paraformaldehyde except for a small component of reversible photobleaching with t1/2 of 3 ms. For comparison, the t1/2 for photobleaching of GFP in cytoplasm was 14 +/- 2 ms (60x) and 24 +/- 1 ms (40x). Utilizing a mathematical model that accounted for ER reticular geometry, a GFP diffusion coefficient of 0.5-1 x 10(-7) cm2/s was computed, 9-18-fold less than that in water and 3-6-fold less than that in cytoplasm. By frequency-domain microfluorimetry, the GFP rotational correlation time was measured to be 39 +/- 8 ns, approximately 2-fold greater than that in water but comparable to that in the cytoplasm. Fluorescence recovery after photobleaching using a 40x lens was measured (at 23 degrees C unless otherwise indicated) for several potential effectors of ER structure and/or lumen environment: t1/2 values (in ms) were 143 +/- 8 (control), 100 +/- 13 (37 degrees C), 53 +/- 13 (brefeldin A), and 139 +/- 6 (dithiothreitol). These results indicate moderately slowed GFP diffusion in a continuous ER lumen.

A comprehensive genome-scale metabolic network of Chlamydomonas reinhardtii, including a detailed account of light-driven metabolism, is reconstructed and validated. The model provides a new resource for research of C. reinhardtii metabolism and in algal biotechnology.

Terrestrial fungi play critical roles in nutrient cycling and food webs and can shape macroorganism communities as parasites and mutualists. Although estimates for the number of fungal species on the planet range from 1.5 to over 5 million, likely fewer than 10% of fungi have been identified so far. To date, a relatively small percentage of described species are associated with marine environments, with ∼1,100 species retrieved exclusively from the marine environment. Nevertheless, fungi have been found in nearly every marine habitat explored, from the surface of the ocean to kilometers below ocean sediments. Fungi are hypothesized to contribute to phytoplankton population cycles and the biological carbon pump and are active in the chemistry of marine sediments. Many fungi have been identified as commensals or pathogens of marine animals (e.g., corals and sponges), plants, and algae. Despite their varied roles, remarkably little is known about the diversity of this major branch of eukaryotic life in marine ecosystems or their ecological functions. This perspective emerges from a Marine Fungi Workshop held in May 2018 at the Marine Biological Laboratory in Woods Hole, MA. We present the state of knowledge as well as the multitude of open questions regarding the diversity and function of fungi in the marine biosphere and geochemical cycles.

Mutualistic symbioses shape the evolution of species and ecosystems and catalyze the emergence of biological complexity, yet how such symbioses first form is unclear. We show that an obligate mutualism between the yeast Saccharomyces cerevisiae and the alga Chlamydomonas reinhardtii—two model eukaryotes with very different life histories—can arise spontaneously in an environment requiring reciprocal carbon and nitrogen exchange. This capacity for mutualism is phylogenetically broad, extending to other Chlamydomonas and fungal species. Furthermore, we witnessed the spontaneous association of Chlamydomonas algal cells physically interacting with filamentous fungi. These observations demonstrate that under specific conditions, environmental change induces free-living species to become obligate mutualists and establishes a set of experimentally tractable, phylogenetically related, synthetic systems for studying the evolution of symbiosis.

The green alga Chlamydomonas reinhardtii is a popular unicellular organism for studying photosynthesis, cilia biogenesis and micronutrient homeostasis. Ten years since its genome project was initiated, an iterative process of improvements to the genome and gene predictions has propelled this organism to the forefront of the “omics” era. Housed at Phytozome, the Joint Genome Institute’s (JGI) plant genomics portal, the most up-to-date genomic data include a genome arranged on chromosomes and high-quality gene models with alternative splice forms supported by an abundance of RNA-Seq data. Here, we present the past, present and future of Chlamydomonas genomics. Specifically, we detail progress on genome assembly and gene model refinement, discuss resources for gene annotations, functional predictions and locus ID mapping between versions and, importantly, outline a standardized framework for naming genes.

We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ;3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.

This paper presents results from a high spatial resolution survey of 33 main-belt asteroids with diameters >40 km using the Keck II Adaptive Optics (AO) facility. Five of these (45 Eugenia, 87 Sylvia, 107 Camilla, 121 Hermione, 130 Elektra) were confirmed to have satellite. Assuming the same albedo as the primary, these moonlets are relatively small (∼5% of the primary size) suggesting that they are fragments captured after a disruptive collision of a parent body or captured ejecta due to an impact. For each asteroid, we have estimated the minimum size of a moonlet that can positively detected within the Hill sphere of the system by estimating and modeling a 2-σ detection profile: in average on the data set, a moonlet located at 2/100 × R Hill (1/4 × R Hill ) with a diameter larger than 6 km (4 km) would have been unambiguously seen. The apparent size and shape of each asteroid was estimated after deconvolution using a new algorithm called AIDA. The mean diameter for the majority of asteroids is in good agreement with IRAS radiometric measurements, though for asteroids with a D < 200 km, it is underestimated on average by 6-8%. Most asteroids had a size ratio that was very close to those determined by lightcurve measurements. One observation of 104 Klymene suggests it has a bifurcated shape. The bi-lobed shape of 121 Hermione described in Marchis et al. [Marchis, F., Hestroffer, D., Descamps, P., Berthier, J., Laver, C., de Pater, I., 2005c. Icarus 178, 450-464] was confirmed after deconvolution. The ratio of contact binaries in our survey, which is limited to asteroids larger than 40 km, is surprisingly high (∼6%), suggesting that a non-single configuration is common in the main-belt. Several asteroids have been analyzed with lightcurve inversions. We compared lightcurve inversion models for plane-of-sky predictions with the observed images (9 Metis, 52 Europa, 87 Sylvia, 130 Elektra, 192 Nausikaa, and 423 Diotima, 511 Davida). The AO images allowed us to determine a unique photometric mirror pole solution, which is normally ambiguous for asteroids moving close to the plane of the ecliptic (e.g., 192 Nausikaa and 52 Europa). The photometric inversion models agree well with the AO images, thus confirming the validity of both the lightcurve inversion method and the AO image reduction technique.

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