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Multiple biomolecular condensates coexist at the pre- and post- synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns. Synapsin-1 is essential for condensation of SVs, and its C-terminal IDR has been shown to be a key driver of condensation. Focusing on this IDR, we dissected the contributions of two conserved features namely the segregation of polar and proline residues along the linear sequence, and the compositional preference for arginine over lysine. Scrambling the blocks of polar and proline residues weakens the driving forces for forming micron-scale condensates. However, the extent of clustering in subsaturated solutions remains equivalent to that of the wild-type synapsin-1. In contrast, substituting arginine with lysine significantly weakens both the driving forces for condensation and the extent of clustering in subsaturated solutions. Co-expression of the scrambled variant of synapsin-1 with synaptophysin results in a gain-of-function phenotype in cells, whereas arginine to lysine substitutions eliminate condensation in cells. We report an emergent consequence of synapsin-1 condensation, which is the generation of interphase pH gradients that is realized via differential partitioning of protons between coexisting phases. This pH gradient is likely to be directly relevant for vesicular ATPase functions and the loading of neurotransmitters. Our studies highlight how conserved IDR grammars serve as drivers of synapsin-1 condensation. 

Abstract Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area. 

Abstract Intrinsically disordered protein regions (IDRs) are highly dynamic sequences that rapidly sample a collection of conformations over time. In the past several decades, IDRs have emerged as a major component of many proteomes, comprising ~30% of all eukaryotic protein sequences. Proteins with IDRs function in a wide range of biological pathways and are notably enriched in signaling cascades that respond to environmental stresses. Here, we identify and characterize intrinsic disorder in the soluble cytoplasmic N‐terminal domains of MSL8, MSL9, and MSL10, three members of the MscS‐like (MSL) family of mechanosensitive ion channels. In plants, MSL channels are proposed to mediate cell and organelle osmotic homeostasis. Bioinformatic tools unanimously predicted that the cytosolic N‐termini of MSL channels are intrinsically disordered. We examined the N‐terminus of MSL10 (MSL10 N ) as an exemplar of these IDRs and circular dichroism spectroscopy confirms its disorder. MSL10 N adopted a predominately helical structure when exposed to the helix‐inducing compound trifluoroethanol (TFE). Furthermore, in the presence of molecular crowding agents, MSL10 N underwent structural changes and exhibited alterations to its homotypic interaction favorability. Lastly, interrogations of collective behavior via in vitro imaging of condensates indicated that MSL8 N , MSL9 N , and MSL10 N have sharply differing propensities for self‐assembly into condensates, both inherently and in response to salt, temperature, and molecular crowding. Taken together, these data establish the N‐termini of MSL channels as intrinsically disordered regions with distinct biophysical properties and the potential to respond uniquely to changes in their physiochemical environment. 

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.