Modeling the Mechanisms by Which Coexisting Biomolecular RNA-Protein Condensates Form.

Liquid-liquid phase separation is an emerging mechanism for intracellular organization. This work presents a mathematical model to examine molecular mechanisms that yield phase-separated droplets composed of different RNA-protein complexes. Using a Cahn-Hilliard diffuse interface model with a Flory-Huggins free energy scheme, we explore how multiple (here two, for simplicity) protein-RNA complexes (species) can establish a heterogeneous droplet field where droplets with single or multiple species phase separate and evolve during coarsening. We show that the complex-complex de-mixing energy tunes whether the complexes co-exist or form distinct droplets, while the transient binding kinetics dictate both the timescale of droplet formation and whether distinct species phase separate into droplets simultaneously or sequentially. For specific energetics and kinetics, a field of droplets driven by the formation of only one protein-RNA complex will emerge. Slowly, the other droplet species will accumulate inside the preformed droplets of the other species, allowing them to occupy the same droplet space. Alternatively, unfavorable species mixing creates a parasitic relationship: the slow-to-form protein-RNA complex will accumulate at the surface of a competing droplet species, siphoning off the free protein as it is released. Once this competing protein-RNA complex has sufficiently accumulated on the droplet surface, it can form a new droplet that is capable of sharing an interface with the first complex droplet but is not capable of mixing. These results give insights into a wide range of phase-separation scenarios and heterogeneous droplets that coexist but do not mix within the nucleus and the cytoplasm of cells.

In vitro reconstitution of septin assemblies on supported lipid bilayers.

Septins are polymerizing eukaryotic proteins that play conserved roles in cell cortex organization and are essential in many cell types. How septin dynamics and protein-protein interactions determine their function at the plasma membrane remains a mystery. Here, we present a method for recapitulating septin polymerization and lipid interaction utilizing supported lipid bilayers to mimic the eukaryotic plasma membrane. Septins on supported lipid bilayers can be visualized with single-molecule sensitivity using total internal reflective fluorescence microscopy. Microscopy-based in vitro assays have revolutionized our understanding of actin, microtubules, and bacterial cytoskeletal systems, and will likely immediately advance our understanding of the septin proteins. As such, we hope that this technique will be adopted and widely utilized by those interested in uncovering septin properties and functions of septin interacting proteins.

The septin cortex at the yeast mother-bud neck.

A specialized cortical domain is organized by the septins at the necks of budding yeast cells. Recent findings suggest that this domain serves as a diffusion barrier and also as a local cell-shape sensor. We review these findings along with what is known about the organization of the septin cortex and its regulation during the cell cycle.

Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Isolation and characterization of effector-loop mutants of CDC42 in yeast.

The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.

CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.