Global Release of Translational Repression Across Plasmodium's Host-to-Vector Transmission Event.

Malaria parasites must be able to respond quickly to changes in their environment, including during their transmission between mammalian hosts and mosquito vectors. Therefore, before transmission, female gametocytes proactively produce and translationally repress mRNAs that encode essential proteins that the zygote requires to establish a new infection. This essential regulatory control requires the orthologues of DDX6 (DOZI), LSM14a (CITH), and ALBA proteins to form a translationally repressive complex in female gametocytes that associates with many of the affected mRNAs. However, while the release of translational repression of individual mRNAs has been documented, the details of the global release of translational repression have not. Moreover, the changes in spatial arrangement and composition of the DOZI/CITH/ALBA complex that contribute to translational control are also not known. Therefore, we have conducted the first quantitative, comparative transcriptomics and DIA-MS proteomics of Plasmodium parasites across the host-to-vector transmission event to document the global release of translational repression. Using female gametocytes and zygotes of P. yoelii, we found that nearly 200 transcripts are released for translation soon after fertilization, including those with essential functions for the zygote. However, we also observed that some transcripts remain repressed beyond this point. In addition, we have used TurboID-based proximity proteomics to interrogate the spatial and compositional changes in the DOZI/CITH/ALBA complex across this transmission event. Consistent with recent models of translational control, proteins that associate with either the 5' or 3' end of mRNAs are in close proximity to one another during translational repression in female gametocytes and then dissociate upon release of repression in zygotes. This observation is cross-validated for several protein colocalizations in female gametocytes via ultrastructure expansion microscopy and structured illumination microscopy. Moreover, DOZI exchanges its interaction from NOT1-G in female gametocytes to the canonical NOT1 in zygotes, providing a model for a trigger for the release of mRNAs from DOZI. Finally, unenriched phosphoproteomics revealed the modification of key translational control proteins in the zygote. Together, these data provide a model for the essential translational control mechanisms used by malaria parasites to promote their efficient transmission from their mammalian host to their mosquito vector.

Mesoscale structure-function relationships in mitochondrial transcriptional condensates.

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized in vitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates in vitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed in vitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates.

Function moves biomolecular condensates in phase space.

Phase separation underlies the formation of biomolecular condensates. We hypothesize the cellular processes that occur within condensates shape their structural features. We use the example of transcription to discuss structure-function relationships in condensates. Various types of transcriptional condensates have been reported across the evolutionary spectrum in the cell nucleus as well as in mitochondrial and bacterial nucleoids. In vitro and in vivo observations suggest that transcriptional activity of condensates influences their supramolecular structure, which in turn affects their function. Condensate organization thus becomes driven by differences in miscibility among the DNA and proteins of the transcription machinery and the RNA transcripts they generate. These considerations are in line with the notion that cellular processes shape the structural properties of condensates, leading to a dynamic, mutual interplay between structure and function in the cell.

Droplets take DNA by force.

Single-molecule experiments can now quantify the surface forces that compete to package tethered DNA into a protein-rich condensate - providing much-needed mechanistic insight into the phase behaviour of the entangled genome in the nucleus.

Phase separation in genome organization across evolution.

Phase separation is emerging as a paradigm to explain the self-assembly and organization of membraneless bodies in the cell. Recent advances show that this principle also extends to nucleoprotein complexes, including DNA-based structures. We discuss here recent observations on the role of phase separation in genome organization across the evolutionary spectrum from bacteria to mammals. These findings suggest that molecular interactions amongst DNA-binding proteins evolved to form a variety of biomolecular condensates with distinct material properties that affect genome organization and function. We suggest that phase separation contributes to genome organization across evolution and that the resulting phase behavior of genomes may underlie regulatory mechanisms and disease.

Self-assembly of multi-component mitochondrial nucleoids via phase separation.

Mitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size control of the mitochondrial nucleoid (mt-nucleoid). The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into droplets with slow internal dynamics. TFAM and mtDNA form heterogenous, viscoelastic structures in vitro, which recapitulate the dynamics and behavior of mt-nucleoids in vivo. Mt-nucleoids coalesce into larger droplets in response to various forms of cellular stress, as evidenced by the enlarged and transcriptionally active nucleoids in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS). Our results point to phase separation as an evolutionarily conserved mechanism of genome organization.

How Hierarchical Interactions Make Membraneless Organelles Tick Like Clockwork.

Biomolecular condensates appear throughout the cell, serving many different biochemical functions. We argue that condensate functionality is optimized when the interactions driving condensation vary widely in affinity. Strong interactions provide structural specificity needed to encode functional properties but carry the risk of kinetic arrest, while weak interactions allow the system to remain dynamic but do not restrict the conformational ensemble enough to sustain specific functional features. To support our opinion, we describe illustrative examples of the interplay of strong and weak interactions that are found in the nucleolus, SPOP/DAXX condensates, polySUMO/polySIM condensates, chromatin, and stress granules. The common feature of these systems is a hierarchical assembly motif in which weak, transient interactions condense structurally defined functional units.

Controlling the material properties and rRNA processing function of the nucleolus using light.

The nucleolus is a prominent nuclear condensate that plays a central role in ribosome biogenesis by facilitating the transcription and processing of nascent ribosomal RNA (rRNA). A number of studies have highlighted the active viscoelastic nature of the nucleolus, whose material properties and phase behavior are a consequence of underlying molecular interactions. However, the ways in which the material properties of the nucleolus impact its function in rRNA biogenesis are not understood. Here we utilize the Cry2olig optogenetic system to modulate the viscoelastic properties of the nucleolus. We show that above a threshold concentration of Cry2olig protein, the nucleolus can be gelled into a tightly linked, low mobility meshwork. Gelled nucleoli no longer coalesce and relax into spheres but nonetheless permit continued internal molecular mobility of small proteins. These changes in nucleolar material properties manifest in specific alterations in rRNA processing steps, including a buildup of larger rRNA precursors and a depletion of smaller rRNA precursors. We propose that the flux of processed rRNA may be actively tuned by the cell through modulating nucleolar material properties, which suggests the potential of materials-based approaches for therapeutic intervention in ribosomopathies.

Blank spots on the map: some current questions on nuclear organization and genome architecture.

The past decades have provided remarkable insights into how the eukaryotic cell nucleus and the genome within it are organized. The combined use of imaging, biochemistry and molecular biology approaches has revealed several basic principles of nuclear architecture and function, including the existence of chromatin domains of various sizes, the presence of a large number of non-membranous intranuclear bodies, non-random positioning of genes and chromosomes in 3D space, and a prominent role of the nuclear lamina in organizing genomes. Despite this tremendous progress in elucidating the biological properties of the cell nucleus, many questions remain. Here, we highlight some of the key open areas of investigation in the field of nuclear organization and genome architecture with a particular focus on the mechanisms and principles of higher-order genome organization, the emerging role of liquid phase separation in cellular organization, and the functional role of the nuclear lamina in physiological processes.

Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles.

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including 'high concentration' binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼3-8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

Coexisting Liquid Phases Underlie Nucleolar Subcompartments.

The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.

Soft viscoelastic properties of nuclear actin age oocytes due to gravitational creep.

The actin cytoskeleton helps maintain structural organization within living cells. In large X. laevis oocytes, gravity becomes a dominant force and is countered by a nuclear actin network that prevents liquid-like nuclear bodies from immediate sedimentation and coalescence. However, nuclear actin's mechanical properties, and how they facilitate the stabilization of nuclear bodies, remain unknown. Using active microrheology, we find that nuclear actin forms a weak viscoelastic network, with a modulus of roughly 0.1 Pa. Embedded probe particles subjected to a constant force exhibit continuous displacement, due to viscoelastic creep. Gravitational forces also cause creep displacement of nuclear bodies, resulting in their asymmetric nuclear distribution. Thus, nuclear actin does not indefinitely support the emulsion of nuclear bodies, but only kinetically stabilizes them by slowing down gravitational creep to ~2 months. This is similar to the viability time of large oocytes, suggesting gravitational creep ages oocytes, with fatal consequences on long timescales.

Remodeling nuclear architecture allows efficient transport of herpesvirus capsids by diffusion.

The nuclear chromatin structure confines the movement of large macromolecular complexes to interchromatin corrals. Herpesvirus capsids of approximately 125 nm assemble in the nucleoplasm and must reach the nuclear membranes for egress. Previous studies concluded that nuclear herpesvirus capsid motility is active, directed, and based on nuclear filamentous actin, suggesting that large nuclear complexes need metabolic energy to escape nuclear entrapment. However, this hypothesis has recently been challenged. Commonly used microscopy techniques do not allow the imaging of rapid nuclear particle motility with sufficient spatiotemporal resolution. Here, we use a rotating, oblique light sheet, which we dubbed a ring-sheet, to image and track viral capsids with high temporal and spatial resolution. We do not find any evidence for directed transport. Instead, infection with different herpesviruses induced an enlargement of interchromatin domains and allowed particles to diffuse unrestricted over longer distances, thereby facilitating nuclear egress for a larger fraction of capsids.

A nuclear F-actin scaffold stabilizes ribonucleoprotein droplets against gravity in large cells.

The size of a typical eukaryotic cell is of the order of ∼10 µm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog Xenopus laevis grow to a diameter ≥1 mm. They have a correspondingly large nucleus (germinal vesicle) of ∼450 µm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that germinal vesicles contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that on actin disruption, ribonucleoprotein droplets, including nucleoli and histone locus bodies, undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ∼10 µm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large Xenopus oocytes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control.

Large-scale phosphoproteomic analysis of membrane proteins in renal proximal and distal tubule.

Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.