Patricia Occhipinti

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 assemblies form by diffusion-driven annealing on membranes

Significance The mechanisms and location of polymerization and disassembly direct the function of cytoskeletal proteins. Septins are far less understood than other cytoskeletal elements such as actin and microtubules, yet they have a conserved function acting as scaffolds at cell membranes and are implicated in cancers, neurodegenerative diseases, and microbial pathogenesis. We have defined a key role of the membrane in directing septin filament formation in live cells and reconstituted dynamic septin polymerization, using purified components. We find that septins grow into filaments and form higher-order structures by diffusing, colliding, and annealing on the plasma membrane. This work is important because it defines previously unidentified basic steps of polymerization and construction of higher-order assemblies of septin proteins. Septins assemble into filaments and higher-order structures that act as scaffolds for diverse cell functions including cytokinesis, cell polarity, and membrane remodeling. Despite their conserved role in cell organization, little is known about how septin filaments elongate and are knitted together into higher-order assemblies. Using fluorescence correlation spectroscopy, we determined that cytosolic septins are in small complexes, suggesting that septin filaments are not formed in the cytosol. When the plasma membrane of live cells is monitored by total internal reflection fluorescence microscopy, we see that septin complexes of variable size diffuse in two dimensions. Diffusing septin complexes collide and make end-on associations to form elongated filaments and higher-order structures, an assembly process we call annealing. Septin assembly by annealing can be reconstituted in vitro on supported lipid bilayers with purified septin complexes. Using the reconstitution assay, we show that septin filaments are highly flexible, grow only from free filament ends, and do not exchange subunits in the middle of filaments. This work shows that annealing is a previously unidentified intrinsic property of septins in the presence of membranes and demonstrates that cells exploit this mechanism to build large septin assemblies.

Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

Polarized fluorescence microscopy reveals that septins across diverse species assemble into similar higher-order structures consisting of dynamic, paired filaments.

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

Fungal and human septins can distinguish between different degrees of micron-scale curvature in cells, suggesting that this property of the septin cytoskeleton provides a cell with a mechanism to sense its local shape.

Regulation of Distinct Septin Rings in a Single Cell by Elm1p and Gin4p Kinases

Septins are conserved, GTP-binding proteins that assemble into higher order structures, including filaments and rings with varied cellular functions. Using four-dimensional quantitative fluorescence microscopy of Ashbya gossypii fungal cells, we show that septins can assemble into morphologically distinct classes of rings that vary in dimensions, intensities, and positions within a single cell. Notably, these different classes coexist and persist for extended times, similar in appearance and behavior to septins in mammalian neurons and cultured cells. We demonstrate that new septin proteins can add through time to assembled rings, indicating that septins may continue to polymerize during ring maturation. Different classes of rings do not arise from the presence or absence of specific septin subunits and ring maintenance does not require the actin and microtubule cytoskeletons. Instead, morphological and behavioral differences in the rings require the Elm1p and Gin4p kinases. This work demonstrates that distinct higher order septin structures form within one cell because of the action of specific kinases.

Protein Aggregation Behavior Regulates Cyclin Transcript Localization and Cell-Cycle Control

Little is known about the active positioning of transcripts outside of embryogenesis or highly polarized cells. We show here that a specific G1 cyclin transcript is highly clustered in the cytoplasm of large multinucleate cells. This heterogeneous cyclin transcript localization results from aggregation of an RNA-binding protein, and deletion of a polyglutamine stretch in this protein results in random transcript localization. These multinucleate cells are remarkable in that nuclei cycle asynchronously despite sharing a common cytoplasm. Notably, randomization of cyclin transcript localization significantly diminishes nucleus-to-nucleus differences in the number of mRNAs and synchronizes cell-cycle timing. Thus, nonrandom cyclin transcript localization is important for cell-cycle timing control and arises due to polyQ-dependent behavior of an RNA-binding protein. There is a widespread association between polyQ expansions and RNA-binding motifs, suggesting that this is a broadly exploited mechanism to produce spatially variable transcripts and heterogeneous cell behaviors. PAPERCLIP:

PolyQ-dependent RNA–protein assemblies control symmetry breaking

Transcripts encoding polarity factors such as Bni1 and Spa2 are nonrandomly clustered in the cytosol to initiate and maintain sites of polarized growth in the fungus Ashbya gossypii.

Cellular requirements for the small molecule forchlorfenuron to stabilize the septin cytoskeleton.

The septins are filament‐forming, GTP‐binding proteins that are conserved from yeast to humans. Septins assemble into higher‐order structures such as rings, bars, and gauzes with diverse functions including serving as membrane diffusion barriers and scaffolds for cell signaling. The basis for septin filament polymerization and the rules governing septin polymer dynamics are presently not well understood. Pharmacological agents are essential tools in studying such properties of the actin and microtubule cytoskeletons however there are only limited reports of a drug specific to the septin cytoskeleton. Forchlorfenuron (FCF) is a synthetic plant cytokinin used in agriculture which has been shown to alter septin organization in yeast and mammalian tissue culture cells. Here we assess cellular requirements and properties of septin‐based structures induced by FCF. Treatment of the filamentous fungus Ashbya gossypii with FCF leads to assembly of extensive septin fibers throughout hyphae which is rapidly reversed upon removal of the drug. These fibers do not exchange or add septin subunits after assembly, indicating that FCF suppresses normal septin dynamics and stabilizes the polymers. While FCF‐induced septin fibers do not co‐localize to actin or microtubules, a polarized F‐actin cytoskeleton is likely required for the assembly of drug‐induced septin fibers. Thus, FCF is a potent inducer of septin polymerization and acts as a reversible stabilizer of extended septin polymers. This drug will be a powerful tool for studying mechanisms of septin polymerization and function, particularly in cell types where molecular analyses are complicated by the presence of multiple isoforms and limited genetics.

Septin Phosphorylation and Coiled-Coil Domains Function in Cell and Septin Ring Morphology in the Filamentous Fungus Ashbya gossypii

ABSTRACT Septins are a class of GTP-binding proteins conserved throughout many eukaryotes. Individual septin subunits associate with one another and assemble into heteromeric complexes that form filaments and higher-order structures in vivo. The mechanisms underlying the assembly and maintenance of higher-order structures in cells remain poorly understood. Septins in several organisms have been shown to be phosphorylated, although precisely how septin phosphorylation may be contributing to the formation of high-order septin structures is unknown. Four of the five septins expressed in the filamentous fungus, Ashbya gossypii, are phosphorylated, and we demonstrate here the diverse roles of these phosphorylation sites in septin ring formation and septin dynamics, as well as cell morphology and viability. Intriguingly, the alteration of specific sites in Cdc3p and Cdc11p leads to a complete loss of higher-order septin structures, implicating septin phosphorylation as a regulator of septin structure formation. Introducing phosphomimetic point mutations to specific sites in Cdc12p and Shs1p causes cell lethality, highlighting the importance of normal septin modification in overall cell function and health. In addition to discovering roles for phosphorylation, we also present diverse functions for conserved septin domains in the formation of septin higher-order structure. We previously showed the requirement for the Shs1p coiled-coil domain in limiting septin ring size and reveal here that, in contrast to Shs1p, the coiled-coil domains of Cdc11p and Cdc12p are required for septin ring formation. Our results as a whole reveal novel roles for septin phosphorylation and coiled-coil domains in regulating septin structure and function.

A conserved G1 regulatory circuit promotes asynchronous behavior of nuclei sharing a common cytoplasm

Synthesis and accumulation of conserved cell cycle regulators such as cyclins are thought to promote G1/S and G2/M transitions in most eukaryotes. 1 When cells at different stages of the cell cycle are fused to form heterokaryons, the shared complement of regulators in the cytoplasm induces the nuclei to become synchronized.2 However, multinucleate fungi often display asynchronous nuclear division cycles, even though the nuclei inhabit a shared cytoplasm. 3 Similarly, checkpoints can induce nuclear asynchrony in multinucleate cells by arresting only the nucleus that receives damage. 4-6 The cell biological basis for nuclear autonomy in a common cytoplasm is not known. Here we show that in the filamentous fungus Ashbya gossypii, sister nuclei born from one mitosis immediately lose synchrony in the subsequent G1 interval. A conserved G1 transcriptional regulatory circuit involving the Rb-analogue Whi5p promotes the asynchronous behavior yetWhi5 protein is uniformly distributed among nuclei throughout the cell cycle. The homologous Whi5p circuit in S. cerevisiae employs positive feedback to promote robust and coherent entry into the cell cycle. We propose that positive feedback in this same circuit generates timing variability in a multinucleate cell. These unexpected findings indicate that a regulatory program whose products (mRNA transcripts) are translated in a common cytoplasm can nevertheless promote variability in the individual behavior of sister nuclei.