Moving beyond disease to function: Physiological roles for polyglutamine-rich sequences in cell decisions.

Glutamine-rich tracts, also known as polyQ domains, have received a great deal of attention for their role in multiple neurodegenerative diseases, including Huntington's disease (HD), spinocerebellar ataxia (SCA), and others [22], [27]. Expansions in the normal polyQ tracts are thus commonly linked to disease, but polyQ domains themselves play multiple important functional roles in cells that are being increasingly appreciated. The biochemical nature of these domains allows them to adopt a number of different structures and form large assemblies that enable environmental responsiveness, localized signaling, and cellular memory. In many cases, these involve the formation of condensates that have varied material states. In this review, we highlight known and emerging functional roles for polyQ tracts in normal cell physiology.

HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

mRNA structure determines specificity of a polyQ-driven phase separation.

RNA promotes liquid-liquid phase separation (LLPS) to build membraneless compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here, we report that secondary structure allows messenger RNAs (mRNAs) to self-associate and determines whether an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.

Clustered nuclei maintain autonomy and nucleocytoplasmic ratio control in a syncytium.

Nuclei in syncytia found in fungi, muscles, and tumors can behave independently despite cytoplasmic translation and the homogenizing potential of diffusion. We use a dynactin mutant strain of the multinucleate fungus Ashbya gossypii with highly clustered nuclei to assess the relative contributions of nucleus and cytoplasm to nuclear autonomy. Remarkably, clustered nuclei maintain cell cycle and transcriptional autonomy; therefore some sources of nuclear independence function even with minimal cytosol insulating nuclei. In both nuclear clusters and among evenly spaced nuclei, a nucleus' transcriptional activity dictates local cytoplasmic contents, as assessed by the localization of several cyclin mRNAs. Thus nuclear activity is a central determinant of the local cytoplasm in syncytia. Of note, we found that the number of nuclei per unit cytoplasm was identical in the mutant to that in wild-type cells, despite clustered nuclei. This work demonstrates that nuclei maintain autonomy at a submicrometer scale and simultaneously maintain a normal nucleocytoplasmic ratio across a syncytium up to the centimeter scale.

RNA Controls PolyQ Protein Phase Transitions.

Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However, the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities, is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets, indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information but also the biophysical properties of phase-separated compartments.

Septin Form and Function at the Cell Cortex.

Septins are GTP-binding proteins that form filaments and higher-order structures on the cell cortex of eukaryotic cells and associate with actin and microtubule cytoskeletal networks. When assembled, septins coordinate cell division and contribute to cell polarity maintenance and membrane remodeling. These functions manifest themselves via scaffolding of cytosolic proteins and cytoskeletal networks to specific locations on membranes and by forming diffusional barriers that restrict lateral diffusion of proteins embedded in membranes. Notably, many neurodegenerative diseases and cancers have been characterized as having misregulated septins, suggesting that their functions are relevant to diverse diseases. Despite the importance of septins, little is known about what features of the plasma membrane influence septin recruitment and alternatively, how septins influence plasma membrane properties. Septins have been localized to the cell cortex at the base of cilia, the mother-bud neck of yeast, and branch points of filamentous fungi and dendritic spines, in cleavage furrows, and in retracting membrane protrusions in mammalian cells. These sites all possess some degree of curvature and are likely composed of distinct lipid pools. Depending on the context, septins may act alone or in concert with other cytoskeletal elements to influence and sense membrane properties. The degree to which septins react to and/or induce changes in shape and lipid composition are discussed here. As septins are an essential player in basic biology and disease, understanding the interplay between septins and the plasma membrane is critical and may yield new and unexpected functions.

PolyQ-dependent RNA-protein assemblies control symmetry breaking.

Dendritic growth in fungi and neurons requires that multiple axes of polarity are established and maintained within the same cytoplasm. We have discovered that transcripts encoding key polarity factors including a formin, Bni1, and a polarisome scaffold, Spa2, are nonrandomly clustered in the cytosol to initiate and maintain sites of polarized growth in the fungus Ashbya gossypii. This asymmetric distribution requires the mRNAs to interact with a polyQ-containing protein, Whi3, and a Pumilio protein with a low-complexity sequence, Puf2. Cells lacking Whi3 or Puf2 had severe defects in establishing new sites of polarity and failed to localize Bni1 protein. Interaction of mRNAs with Whi3 and Puf2 promotes enrichment of transcripts at established sites of polarized growth and clustering of polarity transcripts throughout the cell body. Thus, aggregation-prone proteins make functional assemblies to position polarity transcripts, and nonrandom positioning of transcripts is required for symmetry-breaking events. This reveals a physiological function for polyQ-driven assemblies in regulating cell polarity.

Ploidy variation in multinucleate cells changes under stress.

Ploidy variation is found in contexts as diverse as solid tumors, drug resistance in fungal infection, and normal development. Altering chromosome or genome copy number supports adaptation to fluctuating environments but is also associated with fitness defects attributed to protein imbalances. Both aneuploidy and polyploidy can arise from multinucleate states after failed cytokinesis or cell fusion. The consequences of ploidy variation in syncytia are difficult to predict because protein imbalances are theoretically buffered by a common cytoplasm. We examined ploidy in a naturally multinucleate fungus, Ashbya gossypii. Using integrated lac operator arrays, we found that chromosome number varies substantially among nuclei sharing a common cytoplasm. Populations of nuclei range from 1N to >4N, with different polyploidies in the same cell and low levels of aneuploidy. The degree of ploidy variation increases as cells age. In response to cellular stress, polyploid nuclei diminish and haploid nuclei predominate. These data suggest that mixed ploidy is tolerated in these syncytia; however, there may be costs associated with variation as stress homogenizes the genome content of nuclei. Furthermore, the results suggest that sharing of gene products is limited, and thus there is incomplete buffering of ploidy variation despite a common cytosol.

Nuclear anarchy: asynchronous mitosis in multinucleated fungal hyphae.

Multinucleated cells are found in diverse contexts and include filamentous fungi, developing insect embryos, skeletal muscle and metastasizing tumor cells. Some multinucleated cells such as those in muscles arise from cell fusion events, but many are formed through specialized cell cycles in which nuclear and cell division are uncoupled. Recent work in the fungus Ashbya gossypii illustrates how unique spatial and temporal regulation of conserved cell cycle regulators directs mitosis in multinucleated cells.

Cdc42p, GTP hydrolysis, and the cell's sense of direction.

The GTPase Cdc42p is essential for polarity establishment in animals and fungi.(1) Human Cdc42p can functionally replace yeast Cdc42p,(2) indicating a high degree of evolutionary conservation. Current models of Cdc42p action generally follow the signaling paradigm established for Ras, in which receptors responding to an initiating stimulus cause guanine nucleotide exchange factors (GEFs) to trigger GTP-loading of Ras, leading to engagement of downstream effectors and ensuing cell proliferation. Key support for the Ras paradigm came from the finding that oncogenic forms of Ras, unable to hydrolyze GTP and therefore constitutively GTP-bound, mimicked the effect of constitutive signaling by the upstream receptors even in the absence of stimuli. Attempts to assess whether or not this paradigm is valid for Cdc42p-induced polarization of yeast cells have yielded conflicting results.(3-6) Here, we discuss the available information on this issue and conclude that unlike Ras signaling, Cdc42p directed polarity establishment additionally requires cycling between GTP- and GDP-bound forms. We suggest that such cycling is critical for a little-studied "function" of Cdc42p: its ability to designate a unique portion of the cell cortex to become the polarization site, and to become concentrated at that site.