Friday, May 9, 2025
5/9/2025
The role of biomolecular condensates in regulating the cytoskeleton. - Project Summary. The dynamic restructuring and precise positioning the actin cytoskeleton is essential for complex cell morphologies, cell motility, and cell signaling among other processes. While there is a large inventory of actin regulatory proteins and their biochemical activities, the spatial regulation of these biochemical activities throughout the cell still represents a key gap in understanding intracellular organization. Biomolecular condensates have emerged as a central mechanism for controlling diverse areas of biochemistry. Several studies from evolutionarily divergent systems point to the possibility that actin assembly may be controlled by condensates. Specifically, some actin regulators have hallmark features of intrinsically disordered regions (IDRs), and some sites where F-actin forms have biophysical properties ascribed to condensates. In some cases, these assemblies likely form by phase separation, but in others the condensates appear to emerge by different mechanisms. What isn’t clear is how biomolecular condensates specifically contribute to the localized assembly of the actin cytoskeleton and how this mode of regulation controls cell morphogenesis. In this proposal, I will identify the mechanisms by which ribonucleoprotein (RNP) condensates, containing both RNA and protein, pattern the assembly of the actin cytoskeleton in time and space. I will use the mycelial branching seen in the syncytial fungus Ashbya gossypii (“Ashbya”) as a model system for deciphering the links between condensates and actin regulation. It is known that focused enrichment of actin- interacting proteins leads to a local polarized cytoskeletal network at hyphal tips, and incipient branch sites in Ashbya. studies in the Gladfelter lab have shown the RNA-binding protein, Whi3, is required to promote formation of new polarity sites in Ashbya. Notably, Whi3 condenses with mRNA transcripts for the formin Bni1 and polarity protein Spa2 at existing and incipient branch sites. Ashbya provides a powerful system to study the role of condensates in actin regulation because the essential and physiological role of condensates can be genetically dissected in live cells. My preliminary data show Whi3-coated beads are capable of nucleating polarized actin networks in Ashbya cell-free extracts, opening up the ability to combine the power of genetics with cell-free extracts, a workhorse of cytoskeletal discovery. With this new assay, I will distinguish between two models for how condensates may regulate actin assembly through either (i) the local translation of or (ii) by changing the activity of condensate-controlled actin regulators in Aim 1. I will then identify how multiple Whi3 condensates in a common cytoplasm contribute to the complex morphology of Ashbya in Aim 2. This work will reveal mechanisms for how biomolecular condensates control spatial organization of the actin cytoskeleton, and how these assemblies drive complex cell morphology, an essential feature of many cells.
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