Eukaryotic Sexual Reproduction Evoked "with a Little Help from My Friends".

Bacteria and eukaryotes interact in many ways-from the microbiome that educates the mammalian immune system and enhances nutrition to relationships that are commensal, symbiotic, or parasitic. Now in an unexpected twist, King and colleagues have expanded the repertoire of prokaryotic influence over eukaryotic physiology to include mating.

Early sexual origins of homeoprotein heterodimerization and evolution of the plant KNOX/BELL family.

Developmental mechanisms that yield multicellular diversity are proving to be well conserved within lineages, generating interest in their origins in unicellular ancestors. We report that molecular regulation of the haploid-diploid transition in Chlamydomonas, a unicellular green soil alga, shares common ancestry with differentiation pathways in land plants. Two homeoproteins, Gsp1 and Gsm1, contributed by gametes of plus and minus mating types respectively, physically interact and translocate from the cytosol to the nucleus upon gametic fusion, initiating zygote development. Their ectopic expression activates zygote development in vegetative cells and, in a diploid background, the resulting zygotes undergo a normal meiosis. Gsm1/Gsp1 dyads share sequence homology with and are functionally related to KNOX/BELL dyads regulating stem-cell (meristem) specification in land plants. We propose that combinatorial homeoprotein-based transcriptional control, a core feature of the fungal/animal radiation, may have originated in a sexual context and enabled the evolution of land-plant body plans.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom.

The ecological prominence of diatoms in the ocean environment largely results from their superior competitive ability for dissolved nitrate (NO3-). To investigate the cellular and genetic basis of diatom NO3- assimilation, we generated a knockout in the nitrate reductase gene (NR-KO) of the model pennate diatom Phaeodactylum tricornutum In NR-KO cells, N-assimilation was abolished although NO3- transport remained intact. Unassimilated NO3- accumulated in NR-KO cells, resulting in swelling and associated changes in biochemical composition and physiology. Elevated expression of genes encoding putative vacuolar NO3- chloride channel transporters plus electron micrographs indicating enlarged vacuoles suggested vacuolar storage of NO3- Triacylglycerol concentrations in the NR-KO cells increased immediately following the addition of NO3-, and these increases coincided with elevated gene expression of key triacylglycerol biosynthesis components. Simultaneously, induction of transcripts encoding proteins involved in thylakoid membrane lipid recycling suggested more abrupt repartitioning of carbon resources in NR-KO cells compared with the wild type. Conversely, ribosomal structure and photosystem genes were immediately deactivated in NR-KO cells following NO3- addition, followed within hours by deactivation of genes encoding enzymes for chlorophyll biosynthesis and carbon fixation and metabolism. N-assimilation pathway genes respond uniquely, apparently induced simultaneously by both NO3- replete and deplete conditions.

LHCSR1 induces a fast and reversible pH-dependent fluorescence quenching in LHCII in Chlamydomonas reinhardtii cells.

To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

Characterization of a newly isolated freshwater Eustigmatophyte alga capable of utilizing far-red light as its sole light source.

Oxygenic phototrophs typically utilize visible light (400-700 nm) to drive photosynthesis. However, a large fraction of the energy in sunlight is contained in the far-red region, which encompasses light beyond 700 nm. In nature, certain niche environments contain high levels of this far-red light due to filtering by other phototrophs, and in these environments, organisms with photosynthetic antenna systems adapted to absorbing far-red light are able to thrive. We used selective far-red light conditions to isolate such organisms in environmental samples. One cultured organism, the Eustigmatophyte alga Forest Park Isolate 5 (FP5), is able to absorb far-red light using a chlorophyll (Chl) a-containing antenna complex, and is able to grow under solely far-red light. Here we characterize the antenna system from this organism, which is able to shift the absorption of Chl a to >705 nm.

Quantitative biogeography of picoprasinophytes establishes ecotype distributions and significant contributions to marine phytoplankton.

Bathycoccus and Ostreococcus are broadly distributed marine picoprasinophyte algae. We enumerated small phytoplankton using flow cytometry and qPCR assays for phylogenetically distinct Bathycoccus clades BI and BII and Ostreococcus clades OI and OII. Among 259 photic-zone samples from transects and time-series, Ostreococcus maxima occurred in the North Pacific coastal upwelling for OI (36 713 ± 1485 copies ml-1 ) and the Kuroshio Front for OII (50 189 ± 561 copies ml-1 ) and the two overlapped only in frontal regions. The Bathycoccus overlapped more often with maxima along Line-P for BI (10 667 ± 1299 copies ml-1 ) and the tropical Atlantic for BII (4125 ± 339 copies ml-1 ). Only BII and OII were detected at warm oligotrophic sites, accounting for 34 ± 13% of 1589 ± 448 eukaryotic phytoplankton cells ml-1 (annual average) at Station ALOHA's deep chlorophyll maximum. Significant distributional and molecular differences lead us to propose that Bathycoccus clade BII represents a separate species which tolerates higher temperature oceanic conditions than Bathycoccus prasinos (BI). Morphological differences were not evident, but quick-freeze deep-etch electron microscopy provided insight into Bathycoccus scale formation. Our results highlight the importance of quantitative seasonal abundance data for inferring ecological distributions and demonstrate significant, differential picoprasinophyte contributions in mesotrophic and open-ocean waters.

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control.

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

Historical perspective on Chlamydomonas as a model for basic research: 1950-1970.

During the period 1950-1970, groundbreaking research on the genetic mapping of Chlamydomonas reinhardtii and the use of mutant strains to analyze photosynthesis was conducted in the laboratory of R. Paul Levine at Harvard University. An account of this era, based in part on interviews with Levine, is presented.

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens.

Eisosomes are among the few remaining eukaryotic cellular differentations that lack a defined function(s). These trough-shaped invaginations of the plasma membrane have largely been studied in Saccharomyces cerevisiae, in which their associated proteins, including two BAR domain proteins, have been identified, and homologues have been found throughout the fungal radiation. Using quick-freeze deep-etch electron microscopy to generate high-resolution replicas of membrane fracture faces without the use of chemical fixation, we report that eisosomes are also present in a subset of red and green microalgae as well as in the cysts of the ciliate Euplotes. Eisosome assembly is closely correlated with both the presence and the nature of cell walls. Microalgal eisosomes vary extensively in topology and internal organization. Unlike fungi, their convex fracture faces can carry lineage-specific arrays of intramembranous particles, and their concave fracture faces usually display fine striations, also seen in fungi, that are pitched at lineage-specific angles and, in some cases, adopt a broad-banded patterning. The conserved genes that encode fungal eisosome-associated proteins are not found in sequenced algal genomes, but we identified genes encoding two algal lineage-specific families of predicted BAR domain proteins, called Green-BAR and Red-BAR, that are candidate eisosome organizers. We propose a model for eisosome formation wherein (i) positively charged recognition patches first establish contact with target membrane regions and (ii) a (partial) unwinding of the coiled-coil conformation of the BAR domains then allows interactions between the hydrophobic faces of their amphipathic helices and the lipid phase of the inner membrane leaflet, generating the striated patterns.

Ultrastructure and composition of the Nannochloropsis gaditana cell wall.

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (~75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix.

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous ß-1, 4- and/or ß-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Structural correlates of cytoplasmic and chloroplast lipid body synthesis in Chlamydomonas reinhardtii and stimulation of lipid body production with acetate boost.

Light microscopy and deep-etch electron microscopy were used to visualize triacylglyceride (TAG)-filled lipid bodies (LBs) of the green eukaryotic soil alga Chlamydomonas reinhardtii, a model organism for biodiesel production. Cells growing in nitrogen-replete media contain small cytoplasmic lipid bodies (α-cyto-LBs) and small chloroplast plastoglobules. When starved for N, ß-cyto-LB formation is massively stimulated. ß-Cyto-LBs are intimately associated with both the endoplasmic reticulum membrane and the outer membrane of the chloroplast envelope, suggesting a model for the active participation of both organelles in ß-cyto-LB biosynthesis and packaging. When sta6 mutant cells, blocked in starch biosynthesis, are N starved, they produce ß-cyto-LBs and also chloroplast LBs (cpst-LBs) that are at least 10 times larger than plastoglobules and eventually engorge the chloroplast stroma. Production of ß-cyto-LBs and cpst-LBs under the conditions we used is dependent on exogenous 20 mM acetate. We propose that the greater TAG yields reported for N-starved sta6 cells can be attributed to the strain's ability to produce cpst-LBs, a capacity that is lost when the mutant is complemented by a STA6 transgene. Provision of a 20 mM acetate "boost" during N starvation generates sta6 cells that become so engorged with LBs-at the expense of cytoplasm and most organelles-that they float on water even when centrifuged. This property could be a desirable feature for algal harvesting during biodiesel production.

Sex-linked deubiquitinase establishes uniparental transmission of chloroplast DNA.

Most sexual organisms inherit organelles from one parent, commonly by excluding organelles from the smaller gametes. However, post-mating elimination of organelles derived from one gamete ensures uniparental inheritance, where the underlying mechanisms to distinguish organelles by their origin remain obscure. Mating in Chlamydomonas reinhardtii combines isomorphic plus and minus gametes, but chloroplast DNA from minus gametes is selectively degraded in zygotes. Here, we identify OTU2p (otubain protein 2), encoded in the plus mating-type locus MT+, as the protector of plus chloroplast. Otu2p is an otubain-like deubiquitinase, which prevents proteasome-mediated degradation of the preprotein translocase of the outer chloroplast membrane (TOC) during gametogenesis. Using OTU2p-knockouts and proteasome inhibitor treatment, we successfully redirect selective DNA degradation in chloroplasts with reduced TOC levels regardless of mating type, demonstrating that plus-specific Otu2p establishes uniparental chloroplast DNA inheritance. Our work documents that a sex-linked organelle quality control mechanism drives the uniparental organelle inheritance without dimorphic gametes.

50 Years of Women in Cell Biology: Where have we been? Where are we going?

It's been 50 years since Women in Cell Biology (WICB) was founded by junior women cell biologists who found themselves neither represented at the American Society for Cell Biology (ASCB) presentations nor receiving the information, mentoring, and sponsorship they needed to advance their careers. Since then, gender parity at ASCB has made significant strides: WICB has become a standing ASCB committee, women are regularly elected president of the ASCB, and half the symposia speakers are women. Many of WICB's pioneering initiatives for professional development, including career panels, workshops, awards for accomplishments in science and mentoring, and career mentoring roundtables, have been incorporated and adapted into broader "professional development" that benefits all members of ASCB. The time has passed when we can assume that all women benefit equally from progress. By strategically, thoughtfully, and honestly recognizing the challenges to women of the past and today, we may anticipate those new challenges that will arise in the next 50 years. WICB, in collaboration with the ASCB, can lead in data collection and access and can promote diversity, equity, and inclusion. This work will be a fitting homage to the women who, half a century ago, posted bathroom stall invitations to the first Women in Cell Biology meetup.

Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii.

When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid "popped-cell" microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are approximately 50% saturated (C(16) and C(18)) fatty acids and approximately 50% unsaturated fatty acids, half of which are in the form of oleic acid (C(18:1)). The FFA are approximately 50% C(16) and approximately 50% C(18). The LB-derived TAG yield from a liter of sta6 cells at 10(7) cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.

Acidocalcisomes: Ultrastructure, Biogenesis, and Distribution in Microbial Eukaryotes.

Acidocalcisomes are membrane-enclosed organelles with acidic lumens that accumulate polyphosphate, often in granular form, and sequester calcium and metals. They carry a transmembrane polyphosphate polymerase and two classes of proton pumps: H+-pyrophosphatases (H+-PPases) and V-type ATPases. This report describes acidocalcisomes that were snap-frozen in living cells, primarily the green alga Chlamydomonas reinhardtii, and then fractured and etched (QFDEEM). Polyphosphate granules prove to be uncommon in log-phase C. reinhardtii cells and abundant in stressed cells, where they are also found within autophagy-related vacuoles. Their E (ectoplasmic) fracture face adopts a unique rugose morphology with etching, and displays ∼14nm globular domains in broken cell preparations. Using etched membrane morphology as a guide, acidocalcisomes were identified during assembly in the trans-Golgi and were recognized in QFDEEM replicas of 18 additional algae and protists. Phylogenetic analysis documents that the eukaryotic gene encoding the signature acidocalcisomal H+-PPase pump has homologues in three widespread eukaryotic clades and has been lost in opisthokonts and Amoebozoa. The eukaryotic clades are related to three functionally diverged prokaryotic PPase pumps, one of which transports Na+. Our data indicate that the Last Eukaryotic Common Ancestor (LECA) encoded two bacteria-derived pumps and one Asgard-archaea-derived pump.