Biology subjects

Basalla, J. L.

Publications and source records attributed to Basalla, J. L..

6 recordsLinked to original sources

bioRxiv2025

Viscoelasticity and interface properties of multi-component condensates govern protein sequestration and suppression of amyloid formation

Stress granules (SGs) are multi-component biomolecular condensates widely implicated as sites of protein aggregation by virtue of the high concentrations of amyloidogenic RNA-binding proteins they contain. This model, in which SGs are viewed as crucibles for amyloid formation, has not been rigorously tested. Here, we employed twelve multi-component protein-nucleic acid condensate systems as SG-mimics with diverse physicochemical features. Utilizing three fibril-forming proteins, hnRNPA1, Tau, and FUS, which are concentrated more than 50-fold in condensates, we report that multi-component biomolecular condensates robustly suppress, rather than promote, amyloid formation. Multiscale experimental analyses, including quantitative kinetic measurements, rheology, and microscopy, combined with computational modelling, reveal that condensates serve as sinks for soluble protein, and fibrils form in the dilute phase, although interfaces can promote nucleation. Three key physicochemical properties of condensates govern suppression of fibril formation: First, condensate-mediated sequestration lowers the concentration of fibril-forming proteins in the dilute phase. Second, condensate viscoelasticity constrains efflux-driven fibril growth in the dilute phase. And third, dilution of fibril-forming proteins at condensate interfaces mitigates fibril nucleation. The sink potential of SG-mimics is recapitulated in G3BP1-RNA condensates and SGs reconstituted in mammalian cell lysate, suggesting that SGs may have evolved to suppress stress-induced protein aggregation.

biophysics↗

bioRxiv2023

An invariant C-terminal tryptophan in McdB mediates its interaction and positioning function with carboxysomes

Bacterial microcompartments (BMCs) are widespread, protein-based organelles that regulate metabolism. The model for studying BMCs is the carboxysome, which facilitates carbon-fixation in several autotrophic bacteria. Carboxysomes can be distinguished as type or {beta}, which are structurally and phyletically distinct. We recently characterized the Maintenance of Carboxysome Distribution (Mcd) systems responsible for spatially regulating - and {beta}-carboxysomes, consisting of the proteins McdA and McdB. McdA is an ATPase that drives carboxysome positioning, and McdB is the adaptor protein that directly interacts with carboxysomes to provide cargo specificity. The molecular features of McdB proteins that specify their interactions with carboxysomes, and whether these are similar between - and {beta}-carboxysomes, remain unknown. Here, we identify C-terminal motifs containing an invariant tryptophan necessary for - and {beta}-McdBs to associate with - and {beta}-carboxysomes, respectively. Substituting this tryptophan with other aromatic residues reveals corresponding gradients of carboxysome colocalization and positioning by McdB in vivo. Intriguingly, these gradients also correlate with the ability of McdB to form condensates in vitro. The results reveal a shared mechanism underlying McdB adaptor protein binding to carboxysomes, and potentially other BMCs. Our findings also implicate condensate formation as playing a key role in this association. SIGNIFICANCE STATEMENTO_LIMaintenance of carboxysome distribution protein B (McdB) is necessary for positioning a widespread class of protein-based organelles in bacteria that regulate metabolism. Without McdB, these organelles aggregate and lose functionality. How McdB interacts with and positions these organelles is unknown. C_LIO_LIWe determine that an invariant tryptophan is necessary for McdB to interact with and position its organelle. A similar mechanism occurs in two diverse bacterial cell types, both relying on the invariant tryptophan. C_LIO_LIThis class of bacterial organelle includes compartments involved in bacterial pathogenesis and carbon fixation. Our results therefore advance our understanding and applications of these organelles. C_LI

microbiology↗

bioRxiv2022

Cell Surface-localized CsgF Condensate is a Gatekeeper in Bacterial Curli Subunit Secretion

Curli are functional amyloids present on the outer membrane of E. coli. Cell-surface aggregation of CsgA, the major subunit of curli, is a well-orchestrated process. CsgB, the minor subunit of curli, nucleates the aggregation of CsgA while CsgF, a curli accessory protein, ensures proper anchoring of CsgB to the cell surface. The molecular basis of the interactions between CsgF and curli subunits is unclear. Here, we show that CsgF undergoes phase separation in vitro and that the ability of CsgF variants to phase separate tightly correlated with CsgF function in cells during curli biogenesis. Substitution of phenylalanine residues in the CsgF N-terminus both reduced the propensity of CsgF to phase-separate and impaired curli biogenesis. Exogenous addition of purified CsgF complemented csgF - cells. This exogenous addition assay was used to assess the ability of CsgF variants to complement csgF - cells. The presence of CsgF on the cell surface modulated the secretion of CsgA to the cell surface. We also found that the CsgB nucleator protein is a CsgF client. CsgB can form SDS-insoluble aggregates within the dynamic CsgF condensate, and we propose that these multi-component CsgF-B condensates form a nucleation-competent complex that templates CsgA amyloid formation on the cell surface. Together, our study provides insight into the ability of CsgF to phase separate, regulate CsgA secretion, and promote CsgB aggregation in curli assembly.

biochemistry↗

bioRxiv2022

The disordered N-terminus of McdB modulates phase separation via pH-sensitive hydration and folds upon interaction with McdA

Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well-studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to fine-tune condensate solubility. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

biochemistry↗

bioRxiv2020

The McdAB system positions α-carboxysomes in proteobacteria

Carboxysomes are protein-based organelles essential for carbon fixation in cyanobacteria and proteobacteria. Previously, we showed that the cyanobacterial nucleoid is utilized as a surface for the equidistant-spacing of {beta}-carboxysomes across cell lengths by a two-component system (McdAB) in the model cyanobacterium Synechococcus elongatus PCC 7942. More recently, we found that McdAB systems are widespread among {beta}-cyanobacteria, which possess {beta}-carboxysomes, but are absent in -cyanobacteria, which possess structurally distinct -carboxysomes. Since cyanobacterial -carboxysomes are thought to have arisen in proteobacteria and were subsequently horizontally transferred into cyanobacteria, this raised the question whether -carboxysome containing proteobacteria possess a McdAB system for positioning -carboxysomes. Here, using the model chemoautotrophic proteobacterium H. neapolitanus, we show that a McdAB system distinct from that of {beta}-cyanobacteria operates to position -carboxysomes across cell lengths. We further show that this system is widespread among -carboxysome containing proteobacteria and that cyanobacteria likely inherited an -carboxysome operon from a proteobacterium lacking the mcdAB locus. These results demonstrate that McdAB is a cross-phylum two-component system necessary for positioning - and {beta}-carboxysomes. The findings have further implications for understanding the positioning of other bacterial protein-based organelles involved in diverse metabolic processes.

microbiology↗

bioRxiv2019

The McdAB Carboxysome Positioning System is Widespread Among β-cyanobacteria

Carboxysomes are protein-based organelles that are essential for allowing cyanobacteria to fix CO2. Previously we identified a two-component system, McdAB, responsible for equidistantly positioning carboxysomes in the model cyanobacterium Synechococcus elongatus PCC 7942. McdA, a ParA-type ATPase, non-specifically binds the nucleoid in the presence of ATP. McdB, a novel factor that directly binds carboxysomes, displaces McdA from the nucleoid. Removal of McdA from the nucleoid in the vicinity of carboxysomes by McdB causes a global break in McdA symmetry, and carboxysome motion occurs via a Brownian-ratchet based mechanism towards the highest concentration of McdA. Despite the importance for cyanobacteria to properly position their carboxysomes, whether the McdAB system is widespread among cyanobacteria remains an open question. Here, we used neighborhood analysis to show that the McdAB system is widespread among {beta}-cyanobacteria and often clusters near carboxysome-related components. Moreover, we show that two distinct McdAB systems exist in {beta}-cyanobacteria, with Type 2 systems being the most abundant (>98% of {beta}-cyanobacteria) and Type 1 systems, like that of S. elongatus, possibly being acquired more recently. Surprisingly, our analysis suggests that the McdAB system is completely absent in -cyanobacteria. Lastly, all McdB proteins we identified share the sequence signatures of a protein capable of undergoing Liquid-Liquid Phase Separation (LLPS). Indeed, we find that S. elongatus McdB undergoes LLPS in vitro, the first example of a ParA-type ATPase partner protein exhibiting this behavior. This is an intriguing finding given the recent demonstration of LLPS activity by {beta}-carboxysome core components. Our results have broader implications for understanding carboxysome biogenesis and positioning across all {beta}-cyanobacteria.\n\nIn BriefWe found that the McdAB carboxysome positioning system is widespread among {beta}-cyanobacteria, absent in -cyanobacteria, exists in two distinct forms, and that S. elongatus McdB undergoes liquid-liquid phase separation.

microbiology↗