Cell differentiation controls iron assimilation in a choanoflagellate.

Marine microeukaryotes have evolved diverse cellular features that link their life histories to surrounding environments. How those dynamic life histories intersect with the ecological functions of microeukaryotes remains a frontier to understand their roles in essential biogeochemical cycles. Choanoflagellates, phagotrophs that cycle nutrients through filter feeding, provide models to explore this intersection, for many choanoflagellate species transition between life history stages by differentiating into distinct cell types.

Chromatin profiling identifies putative dual roles for H3K27me3 in regulating transposons and cell type-specific genes in choanoflagellates.

Gene expression is tightly controlled during animal development to allow the formation of specialized cell types. Our understanding of how animals evolved this exquisite regulatory control remains elusive, but evidence suggests that changes in chromatin-based mechanisms may have contributed. To investigate this possibility, here we examine chromatin-based gene regulatory features in the closest relatives of animals, choanoflagellates.

A red algal polysaccharide influences the multicellular development of the choanoflagellate .

We uncovered an interaction between a choanoflagellate and alga, in which porphyran, a polysaccharide produced by the red alga , induces multicellular development in the choanoflagellate . We first noticed this possible interaction when we tested the growth of in media that was steeped with as a nutritional source. Under those conditions, formed multicellular rosette colonies even in the absence of any bacterial species that can induce rosette development.

An RFX transcription factor regulates ciliogenesis in the closest living relatives of animals.

Cilia allowed our protistan ancestors to sense and explore their environment, avoid predation, and capture bacterial prey. Regulated ciliogenesis was likely critical for early animal evolution, and in modern animals, deploying cilia in the right cells at the right time is crucial for development and physiology. Two transcription factors, RFX and FoxJ1, coordinate ciliogenesis in animals but are absent from the genomes of many other ciliated eukaryotes, raising the question of how the regulation of ciliogenesis in animals evolved.

Cell polarity in the protist-to-animal transition.

A signature feature of the animal kingdom is the presence of epithelia: sheets of polarized cells that both insulate the organism from its environment and mediate interactions with it. Epithelial cells display a marked apico-basal polarity, which is highly conserved across the animal kingdom, both in terms of morphology and of molecular regulators. How did this architecture first evolve? Although the last eukaryotic common ancestor almost certainly possessed a simple form of apico-basal polarity (marked by the presence of one or several flagella at a single cellular pole), comparative genomics and evolutionary cell biology reveal that the polarity regulators of animal epithelial cells have a surprisingly complex and stepwise evolutionary history.

The history of Salpingoeca rosetta as a model for reconstructing animal origins.

Choanoflagellates, the closest living relatives of animals, have the potential to reveal the genetic and cell biological foundations of complex multicellular development in animals. Here we describe the history of research on the choanoflagellate Salpingoeca rosetta. From its original isolation in 2000 to the establishment of CRISPR-mediated genome editing in 2020, S.

Genome editing enables reverse genetics of multicellular development in the choanoflagellate .

In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals.

Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa.

Predicted glycosyltransferases promote development and prevent spurious cell clumping in the choanoflagellate .

In a previous study we established forward genetics in the choanoflagellate and found that a C-type lectin gene is required for rosette development (Levin , 2014). Here we report on critical improvements to genetic screens in while also investigating the genetic basis for rosette defect mutants in which single cells fail to develop into orderly rosettes and instead aggregate promiscuously into amorphous clumps of cells. Two of the mutants, Jumble and Couscous, mapped to lesions in genes encoding two different predicted glycosyltransferases and displayed aberrant glycosylation patterns in the basal extracellular matrix (ECM).

Transfection of choanoflagellates illuminates their cell biology and the ancestry of animal septins.

As the closest living relatives of animals, choanoflagellates offer unique insights into animal origins and core mechanisms underlying animal cell biology. However, unlike traditional model organisms, such as yeast, flies, and worms, choanoflagellates have been refractory to DNA delivery methods for expressing foreign genes. Here we report a robust method for expressing transgenes in the choanoflagellate , overcoming barriers that have previously hampered DNA delivery and expression.

A structurally plastic ribonucleoprotein complex mediates post-transcriptional gene regulation in HIV-1.

HIV replication requires the nuclear export of essential, intron-containing viral RNAs. To facilitate export, HIV encodes the viral accessory protein Rev which binds unspliced and partially spliced viral RNAs and creates a ribonucleoprotein complex that recruits the cellular Chromosome maintenance factor 1 export machinery. Exporting RNAs in this manner bypasses the necessity for complete splicing as a prerequisite for mRNA export, and allows intron-containing RNAs to reach the cytoplasm intact for translation and virus packaging.

The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy.

Transition step during assembly of HIV Tat:P-TEFb transcription complexes and transfer to TAR RNA.

Transcription factors regulate eukaryotic RNA polymerase II (Pol II) activity by assembling and remodeling complexes at multiple steps in the transcription cycle. In HIV, we previously proposed a two-step model where the viral Tat protein first preassembles at the promoter with an inactive P-TEFb:7SK snRNP complex and later transfers P-TEFb to TAR on the nascent transcript, displacing the inhibitory snRNP and resulting in Pol II phosphorylation and stimulation of elongation. It is unknown how the Tat:P-TEFb complex transitions to TAR to activate the P-TEFb kinase.

Fabs enable single particle cryoEM studies of small proteins.

In spite of its recent achievements, the technique of single particle electron cryomicroscopy (cryoEM) has not been widely used to study proteins smaller than 100 kDa, although it is a highly desirable application of this technique. One fundamental limitation is that images of small proteins embedded in vitreous ice do not contain adequate features for accurate image alignment. We describe a general strategy to overcome this limitation by selecting a fragment antigen binding (Fab) to form a stable and rigid complex with a target protein, thus providing a defined feature for accurate image alignment.

Visualizing proteins and macromolecular complexes by negative stain EM: from grid preparation to image acquisition.

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast.

HIV Rev response element (RRE) directs assembly of the Rev homooligomer into discrete asymmetric complexes.

RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligomerization of Rev on the RRE scaffold and utilizes both protein-protein and protein-RNA interactions to organize complexes with high specificity.

Signal sequences activate the catalytic switch of SRP RNA.

The signal recognition particle (SRP) recognizes polypeptide chains bearing a signal sequence as they emerge from the ribosome, and then binds its membrane-associated receptor (SR), thereby delivering the ribosome-nascent chain complex to the endoplasmic reticulum in eukaryotic cells and the plasma membrane in prokaryotic cells. SRP RNA catalytically accelerates the interaction of SRP and SR, which stimulates their guanosine triphosphatase (GTPase) activities, leading to dissociation of the complex. We found that although the catalytic activity of SRP RNA appeared to be constitutive, SRP RNA accelerated complex formation only when SRP was bound to a signal sequence.