Jannette Rusch

Colony Organization in the Green Alga Botryococcus braunii (Race B) Is Specified by a Complex Extracellular Matrix

ABSTRACT 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 β-1, 4- and/or β-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.

The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

ABSTRACT 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 cells 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.

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

Conditional depletion of the chloroplast protease ClpP in the alga Chlamydomonas affects plastid protein homeostasis and leads to an autophagocytic and plastid unfolded protein-like response. It involves vacuolization of the cytoplasm and increased accumulation of small heat shock proteins, specific chaperones, proteases, and proteins implicated in thylakoid membrane maintenance and biogenesis. 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.

Genome-wide identification of Drosophila Hb9 targets reveals a pivotal role in directing the transcriptome within eight neuronal lineages, including activation of nitric oxide synthase and Fd59a/Fox-D.

Hb9 is a homeodomain-containing transcription factor that acts in combination with Nkx6, Lim3, and Tail-up (Islet) to guide the stereotyped differentiation, connectivity, and function of a subset of neurons in Drosophila. The role of Hb9 in directing neuronal differentiation is well documented, but the lineage of Hb9(+) neurons is only partly characterized, its regulation is poorly understood, and most of the downstream genes through which it acts remain at large. Here, we complete the lineage tracing of all embryonic Hb9(+) neurons (to eight neuronal lineages) and provide evidence that hb9, lim3, and tail-up are coordinately regulated by a common set of upstream factors. Through the parallel use of micro-array gene expression profiling and the Dam-ID method, we searched for Hb9-regulated genes, uncovering transcription factors as the most over-represented class of genes regulated by Hb9 (and Nkx6) in the CNS. By a nearly ten-to-one ratio, Hb9 represses rather than activates transcription factors, highlighting transcriptional repression of other transcription factors as a core mechanism by which Hb9 governs neuronal determination. From the small set of genes activated by Hb9, we characterized the expression and function of two - fd59a/foxd, which encodes a transcription factor, and Nitric oxide synthase. Under standard lab conditions, both genes are dispensable for Drosophila development, but Nos appears to inhibit hyper-active behavior and fd59a appears to act in octopaminergic neurons to control egg-laying behavior. Together our data clarify the mechanisms through which Hb9 governs neuronal specification and differentiation and provide an initial characterization of the expression and function of Nos and fd59a in the Drosophila CNS.

The complement system supports normal postnatal development and gonadal function in both sexes

Male and female infertility are clinically managed and classified as distinct diseases, and relatively little is known about mechanisms of gonadal function common to both sexes. We used genome-wide genetic analysis on 74,896 women and men to find rare genetic variants that modulate gonadal function in both sexes. This uncovered an association with variants disrupting CSMD1, a complement regulatory protein located on 8p23, in a genomic region with an exceptional evolution. We found that Csmd1 knockout mice display a diverse array of gonadal defects in both sexes, and in females, impaired mammary gland development that leads to increased offspring mortality. The complement pathway is significantly disrupted in Csmd1 mice, and further disruption of the complement pathway from joint inactivation of C3 leads to more extreme reproductive defects. Our results can explain a novel human genetic association with infertility and implicate the complement system in the normal development of postnatal tissues.

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

Multispecies Purification of Testicular Germ Cells

ABSTRACT Advanced methods of cellular purification are required to apply genome technology to the study of spermatogenesis. One approach, based on flow cytometry of murine testicular cells stained with Hoechst-33342 (Ho-FACS), has been extensively optimized and currently allows the isolation of nine germ cell types. This staining technique is straightforward to implement, is highly effective at purifying specific germ cell types, and yields sufficient cell numbers for high-throughput studies. Ho-FACS is a technique that does not require species-specific markers, but whose applicability to other species is largely unexplored. We hypothesized that, because of the similar cell physiology of spermatogenesis across mammals, Ho-FACS could be used to produce highly purified subpopulations of germ cells in mammals other than mouse. To test this hypothesis, we applied Ho-FACS to four mammalian species that are widely used in testis research: Rattus norvegicus, Cavia porcellus, Canis familiaris, and Sus scrofa domesticus. We successfully isolated four germ cell populations from these species with average purity of 79% for spermatocytes, 90% for spermatids, and 66% for spermatogonia. Additionally, we compare the performance of mechanical and chemical dissociation for each species, and propose an optimized gating strategy to better discriminate round and elongating spermatids in the mouse, which can potentially be applied to other species. Our work indicates that spermatogenesis may be uniquely accessible among mammalian developmental systems, as a single set of reagents may be sufficient to isolate germ cell populations from many different mammalian species, opening new avenues in the fields of development and male reproductive biology.