Septins are a conserved family of cytoskeletal proteins known for sensing micron-scale membrane curvature via amphipathic helix (AH) domains. While cooperative interactions in septin assembly have been suggested, the molecular mechanisms governing membrane binding and assembly remain unclear. Building on prior findings, we use all-atom molecular dynamics simulations to examine how single and paired extended AH domains, derived from Cdc12, interact with lipid bilayers. We find that a single membrane-bound AH adopts a bent conformation upon membrane association. In solution, a second AH peptide preferentially interacts with the bound peptide through conserved salt bridges, favoring an antiparallel arrangement. Simulations of covalently linked AH tandems confirm the stability of this configuration. When two AH domains are membrane-bound, they induce localized lipid packing defects, reduce tail order, and exhibit slight peptide displacement on planar bilayers. These observations suggest a cooperative AH binding mechanism and are consistent with models in which lipid packing defects facilitate multivalent AH engagement in curved membrane environments. Our findings advance the mechanistic understanding of septin-membrane interactions and highlight the role of cooperative AH domain binding in stabilizing higher-order structures.

Cells employ cytoskeletal polymers to move, divide, and pass information inside and outside of the cell. Previous work on eukaryotic cytoskeletal elements such as actin, microtubules, and intermediate filaments investigating the mechanisms of polymerization have been critical to understand how cells control the assembly of the cytoskeleton. Most biophysical analyses have considered cooperative versus isodesmic modes of polymerization; this framework is useful for specifying functions of regulatory proteins that control nucleation and understanding how cells regulate elongation in time and space. The septins are considered a fourth component of the eukaryotic cytoskeleton, but they are poorly understood in many ways despite their conserved roles in membrane dynamics, cytokinesis, and cell shape, and in their links to a myriad of human diseases. Because septin function is intimately linked to their assembled state, we set out to investigate the mechanisms by which septin polymers elongate under different conditions. We used simulations, in vitro reconstitution of purified septin complexes, and quantitative microscopy to directly interrogate septin polymerization behaviors in solution and on synthetic lipid bilayers of different geometries. We first used reactive Brownian dynamics simulations to determine if the presence of a membrane induces cooperativity to septin polymerization. We then used fluorescence correlation spectroscopy to assess septins' ability to form filaments in solution at different salt conditions. Finally, we investigated septin membrane adsorption and polymerization on planar and curved supported lipid bilayers. Septins clearly show signs of salt-dependent cooperative assembly in solution, but cooperativity is limited by binding a membrane. Thus, septin assembly is dramatically influenced by extrinsic conditions and substrate properties and can show properties of both isodesmic and cooperative polymers. This versatility in assembly modes may explain the extensive array of assembly types, functions, and subcellular locations in which septins act.

mRNA-protein assemblies play a fundamental role in forming membraneless compartments within cells, whose functions may include activating, inhibiting, and localizing reactions. Recruitment of proteins into droplets can diminish cell to cell variability in protein abundance. However, the extent to which mRNA-protein assemblies may also buffer noise arising from transcription is not understood. Complicating study of this question is that models of kinetics typically treat this as a phase separation process, when mRNA-protein assemblies can contain as few as 2 mRNA transcripts, far below the thermodynamic thresholds for phase separation. Here, through stochastic simulations and asymptotic analysis, we quantify noise suppression by mRNA-protein assemblies as a function of gene expression kinetic parameters, and show that assemblies formed from just a handful of mRNAs effectively regulate transcript abundances and suppress fluctuations. We place particular emphasis on how this mechanism can facilitate regulated transcription by reducing noise even in the context of infrequent bursts of transcription. We investigate two biologically relevant models in which mRNA assembly acts to either "buffer" noise by storing mRNA in inert droplets, or "filter" assembled mRNAs by accelerating their decay, and quantify expression noise as a function of kinetic parameters. In either case, the most controlled expression occurs when bursts produce mRNAs close to the assembly threshold, which we find to be broadly consistent with observations of an RNP-droplet forming cyclin in multinucleate \textit{Ashbya gossypii} cells.

Morphogenesis in fungi and animals is directed by the polarization of the small GTPases Cdc42 and Rac. In the budding yeast Saccharomyces cerevisiae, competition between polarity patches results in one polarized patch and the growth of a single bud. Here, we describe cell polarity in the yeast Aureobasidium pullulans, which establishes multiple coexisting polarity sites yielding multiple buds during a single cell-division cycle. Polarity machinery components oscillate in their abundance in these coexisting sites but do so independently of one another, pointing to a lack of global coupling between sites. Previous theoretical work has demonstrated that negative feedback in a polarity circuit could promote the coexistence of multiple polarity sites, and time-delayed negative feedback is known to cause oscillations. We show that both these features of negative feedback depend on a protein we identified as Pak1 and that Pak1 requires Rac1 but not Cdc42 for its localization. This work shows how conserved signaling networks can be modulated for distinct morphogenic programs even within the constraints of fungal budding.

The syncytiotrophoblast (STB) is a multinucleated cell layer that forms the outer surface of human chorionic villi. Its unusual structure, with billions of nuclei in a single cell, makes it difficult to resolve using conventional single-cell methods. To better understand STB differentiation, we performed single-nucleus and single-cell RNA sequencing on placental tissue and trophoblast organoids (TOs). Single-nucleus RNA-seq was essential for capturing STB populations, revealing three nuclear subtypes: a juvenile subtype co-expressing CTB and STB markers, one enriched in oxygen sensing genes, and another in transport and GTPase signaling. Organoids grown in suspension culture (STBout) showed higher expression of STB markers, hormones, and a greater proportion of the transport-associated nuclear subtype while TOs grown with an inverted polarity (STBin) exhibited a higher proportion of the oxygen sensing nuclear subtype. Gene regulatory analysis identified conserved STB markers, including the chromatin remodeler RYBP. Although RYBP knockout did not impair fusion, it downregulated CSH1 and upregulated oxygen-sensing genes. Comparing STB expression in first trimester, term, and TOs revealed shared features but context-dependent variability. These findings establish TOs as a robust platform to model STB differentiation and nuclear heterogeneity, providing insight into the regulatory networks that shape placental development and function.

Heteromeric membrane proteins play crucial physiological roles, yet how they are formed remains poorly understood. Heteromeric hERG1a/1b ion channels, essential for maintaining normal cardiac rhythm, assemble via cotranslational association of their encoding mRNAs. We hypothesized that direct hERG1a and 1b mRNA interactions facilitate this process. Using fluorescence colocalization and free energy of binding predictions, we found that hERG1a and 1b mRNAs form specific heterotypic condensates in vitro, suggesting direct interactions. When hERG1a mRNA was altered by synonymous mutations predicted to reduce its structural diversity and ability to interact with other mRNAs, overlap with hERG1b was dramatically diminished both in vitro and in cells, indicating weakened interactions. Reducing hERG1a structural diversity also influenced its translational complexes, defined by overlapping between fluorescently labeled mRNA and encoded protein (centroids within 400 nm). Whereas most of the wild-type hERG1a mRNA translates within heterotypic complexes, likely reflecting the biogenesis of hERG1a/1b heteromeric assemblies, reducing hERG1a structural diversity yielded more homotypic translational condensates and fewer hERG1a/1b heterotypic ones. This result suggests that the strength of mRNA interactions impact ion channel biogenesis. Further analysis of the heterotypic translational complexes revealed two distinct classes: a) simultaneous translation of both subunits and b) sequential association of fully translated hERG1b with translating hERG1a. Notably, reducing hERG1a structural diversity and interactions with 1b shifted translation toward the sequential mode. These findings identify a new role of mRNA sequence, structure, and interactions in orchestrating the cotranslational association of important heteromeric membrane proteins.

Budding yeasts present an especially challenging geometry for segregation of chromosomes, which must be delivered across the narrow mother-bud neck into the bud. Studies in the model yeast Saccharomyces cerevisiae have revealed an elaborate set of mechanisms that selectively orient one mitotic spindle pole toward the bud and then drive spindle elongation along the mother-bud axis, ensuring nuclear segregation between mother and bud. It is unclear how these pathways might be adapted to yield similar precision in more complex cell geometries. Here, we provide the first description of the dynamics of mitosis in a multinucleate, multibudding yeast, Aureobasidium pullulans, and identify many unexpected differences from uninucleate yeasts. Mitotic spindles do not orient along the mother-bud axis prior to anaphase, and accurate nuclear segregation often occurs after spindle disassembly. Cortical Num1-dynein forces pull highly mobile nuclei into buds, and once a nucleus enters a bud, it discourages others from entering, ensuring that most daughters inherit only one nucleus.

Cells must limit RNA-RNA interactions to avoid irreversible RNA entanglement. Cells may prevent deleterious RNA-RNA interactions by genome organization to avoid complementarity however, RNA viruses generate long, perfectly complementary antisense RNA during replication. How do viral RNAs avoid irreversible entanglement? One possibility is RNA sequestration into biomolecular condensates. To test this, we reconstituted critical SARS-CoV-2 RNA-RNA interactions in Nucleocapsid condensates. We observed that RNAs with low propensity RNA-RNA interactions resulted in more round, liquid-like condensates while those with high sequence complementarity resulted in more heterogeneous networked morphology independent of RNA structure stability. Residue-resolution molecular simulations and direct sequencing-based detection of RNA-RNA interactions support that these properties arise from degree of trans RNA contacts. We propose that extensive RNA-RNA interactions in cell and viral replication are controlled via a combination of genome organization, timing, RNA sequence content, RNA production ratios, and emergent biomolecular condensate material properties.

Eukaryotes are distinguished from prokaryotes by the presence of a nucleus. However, at some or all stages in their life cycle many fungal cells constitutively contain at least two nuclei in the same cytoplasm, resulting in a multinucleate 'coenocyte' (from the Greek for 'common' and 'cell') or a 'syncytium' (from the Greek for 'together' and 'cell'). Such organization is ancient and has evolved repeatedly. Crucially, multinucleation presents challenges and opportunities for gene expression programs, cytoplasm patterning, environmental sensing and evolution. Here, we focus on multinucleation in fungi where it is a ubiquitous strategy for cell organization (Figure 1). In this primer, we first give an overview of the evolutionary origins, prevalence and diversity of fungal multinucleation. We next describe the two distinct mechanisms that give rise to multinucleation in fungi. The first involves a specialized cell division called endomitosis, in which repeated nuclear divisions occur without cytokinesis or laying down septal cross walls. The other mechanism is cell-cell fusion, whereby two distinct fungal cells recognize and grow towards each other, fuse and allow their respective nuclei to intermingle within the same cytoplasm. Finally, we touch on some aspects of how fungal multinucleation impacts cytoplasmic organization, self/non-self recognition, genetic individuality, and perhaps even cross-species interactions in these organisms.

A new study provides a robust global estimate of pelagic fungal biomass. Their findings reveal fungi as ecologically significant components of the ocean carbon cycle, marking a turning point for integrating fungi into marine microbial ecology.

Septins assemble into scaffolds that direct cell growth and morphology that are often localized to the plasma membrane. While septins preferentially bind convex membranes via amphipathic helices, their assembly on varied geometries in cells suggests additional localization cues. We tested the hypothesis that lipid composition directs septin assembly through lipid packing properties. Lipid mixtures varying in lipid packing were designed by molecular dynamics simulations and incorporated onto supported lipid bilayers to measure septin adsorption in vitro. Septins strongly favor loosely-packed, disordered lipid bilayers but additional geometry cues act in conjunction with this membrane property. Introducing tighter lipid packing in cells disrupted septin structures in a curvature dependent manner, specifically limiting septin assembly and retention along flat regions of the plasma membrane. This work demonstrates that packing defects and geometry jointly regulate septin localization and highlights how multiple membrane properties are integrated to control organization of the septin cytoskeleton.

The realization that the cell is abundantly compartmentalized into biomolecular condensates has opened new opportunities for understanding the physics and chemistry underlying many cellular processes1, fundamentally changing the study of biology2. The term biomolecular condensate refers to non-stoichiometric assemblies that are composed of multiple types of macromolecules in cells, occur through phase transitions, and can be investigated by using concepts from soft matter physics3. As such, they are intimately related to aqueous two-phase systems4 and water-in-water emulsions5. Condensates possess tunable emergent properties such as interfaces, interfacial tension, viscoelasticity, network structure, dielectric permittivity, and sometimes interphase pH gradients and electric potentials6–14. They can form spontaneously in response to specific cellular conditions or to active processes, and cells appear to have mechanisms to control their size and location15–17. Importantly, in contrast to membrane-enclosed organelles such as mitochondria or peroxisomes, condensates do not require the presence of a surrounding membrane.

Amphipathic helices (AHs) are secondary structures that can facilitate binding of proteins to the membrane by folding into a helix with hydrophobic and hydrophilic faces that interact with the same surfaces in the lipid membrane. Septins are cytoskeletal proteins that preferentially bind to domains of micron-scale curvature on the cell membrane. Studies have shown that AH domains in septin are essential for curvature sensing. We present the first computational study of septin AH interactions with lipid bilayers. Using all-atom simulations and metadynamics-enhanced sampling, we study the effect of charge distribution at the flanking ends of septin AH on the energy for helical folding and its consequences on the binding configuration and affinity to the membrane. This is relevant to septins, since the net positive charge on the flanking C-terminal amino acids is a conserved property across several organisms. Simulations revealed that the energy barrier for folding in the neutral-capped AH is much larger than the charge-capped AH, leading to a small fraction of AH folding and integration to the membrane compared to a significantly folded configuration in the bound charge-capped AH. These observations are consistent with the binding measurements of synthetic AH constructs with variable helicity to lipid vesicles. Additionally, we examined an extended AH sequence including eight amino acids upstream and downstream of the AH to mimic the native protein. Again, simulations and experiments show that the extended peptide, with a net positive charge at C-terminus, adopts a strong helical configuration in solution, giving rise to a higher membrane affinity. Altogether, these results identify the energy cost for folding of AHs as a regulator of AH binding configuration and affinity and provide a basic template for parameterizing AH-membrane interactions as a starting point for the future multiscale simulations for septin-membrane interactions.

Condensates are found at synapses where they support the clustering of synaptic vesicles (SVs) and neurotransmitter release. The possibility of RNA promoting the formation or function of these condensates is not known. Here, we set out to assess whether RNA affects molecular condensates in the synapse or could be regulated at these sites, focusing on synapsin-1, which is essential for synapse condensates. Using in vitro reconstitution systems, cell lines and neurons, we show that RNA drives synapsin-1 coacervation, with some bias toward structured RNAs being more effective at promoting demixing. The importance of RNA was confirmed in living synapses, where acute disruption of native RNA induces a dispersion of SVs and synapsin. Conversely, ectopically expressed SV-like condensates have the ability to recruit RNA species and the translational machinery, which accumulates within condensate-microdomains, resulting in increased translation efficacy. Our work indicates a novel role of RNAs in modulating SV condensates, as well as translation, at the synapse. ### Competing Interest Statement The authors have declared no competing interest.

The syncytiotrophoblast (STB) is a tissue-sized multinucleate cell in the human placenta that performs essential functions including molecular transport, metabolism, and hormone production-roles typically distributed across many cell types in multiple organs. To achieve these diverse tasks, the human STB is thought to regionally separate these functions by adapting its cytoplasmic structure, organelle distribution, and molecular composition across placental villous subtypes to meet local functional demands. How can a single, tissue-sized cytoplasm create localized cytoplasmic zones? Recent advances in single nucleus RNA sequencing (snRNAseq) in the human placenta and trophoblast organoids have revealed distinct nuclear subtypes within the STB, including a juvenile population recently incorporated into the syncytia, an oxygen-sensing nuclear subtype, and one that specializes in GTPase signaling and hormone production. Notably, the distribution of these subtypes changes throughout gestation in vivo and under varying culture conditions in vitro. These findings highlight a dynamic process of nuclear specialization that mirrors the functional diversity of the STB cytoplasm. In this review, we highlight research demonstrating both nuclear and cytoplasmic specialization in the STB and provide models for how this could be functionally established. Understanding the molecular mechanisms that enable the STB's ultrastructure to coordinate its diverse functions could illuminate novel therapeutic strategies for addressing pregnancy complications.

Aureobasidium pullulans is a polyextremotolerant black yeast that exhibits impressive morphological plasticity. Consequently, it shows promise as a model system for investigating mechanisms of cell adaptation to different environments and the regulation of cell shape. Here, we build upon the current toolkit for working with A. pullulans and design and test 25 vectors with seven different codon-optimized fluorophores and three selection cassettes. This includes vectors that allow for dual expression of green fluorescent protein and mCherry-tagged proteins at the URA3 locus and vectors that enable homology-based deletion or C-terminal tagging of endogenous genes without the need for cloning. This versatile vector series for working with A. pullulans will enable a broad range of experiments in this emerging model system.