Abstract Enzyme cascades enable complex biochemical transformations, but their optimization is resource-intensive, requiring navigation through high-dimensional parameter spaces encompassing reaction conditions, enzyme ratios, and buffer composition. Here we introduce CascadeMAP, an autonomous microfluidic platform for closed-loop optimization of enzyme cascades, integrating high-throughput microfluidics with Bayesian optimization and multi-agent AI system. We demonstrate the platform across two cascades: (i) a glycerol detection pathway monitored by fluorescence and (ii) a 1,2,3-trichloropropane degradation pathway monitored by label-free Raman spectroscopy providing orthogonal detection modalities. Bayesian optimization identified optimal conditions three times faster than Design of Experiments. Multi-agent AI system automated hypothesis generation, processing 11 GB of experimental data, pattern recognition, and insight synthesis. Operating without human intervention for 7 days, CascadeMAP processed ∼220,000 reactions across ∼7,400 different conditions. This capability establishes a generalizable framework for the autonomous optimization of enzyme cascades and metabolic pathways and accelerates the development of biocatalytic and synthetic biological systems.

The α/β-hydrolase (ABH) superfamily is a widespread and functionally versatile protein fold recognized for its ability to adapt to diverse molecular functions across all three domains of life. One such spectacular example of evolutionary adaptation at the ABH fold is an acquisition of oxygenolytic luciferase reaction that occurred within the hydrolytic haloalkane dehalogenase family. The molecular details of this evolution remain puzzling. In this work, we determine crystal structures and explore dynamical behaviour of a bifunctional ancestral ABH-fold enzyme, highlighting molecular features associated with the transition from hydrolytic to oxygenolytic catalysis at this fold. Structures showed a canonical αβα-sandwich shielded with a helical cap domain. The catalytic pocket is voluminous enough to accommodate a bulky substrate. Molecular dynamics simulations demonstrated that coelenterazine entry does not present a major energetic barrier and identified a preferred binding orientation important for oxygenolytic catalysis. Comparisons between ancestral and extant enzymes highlighted specific amino acids and sequence motifs characteristic for oxygenolytic luciferases. Collectively, our results provide an expanded view of the evolutionary transition in which ABH-fold enzymes, originally using water to cleave chemical bonds, adapted to utilize dioxygen for bioluminescence.

Cellular actin cytoskeleton networks compete for limited actin monomers. New in vitro reconstitutions with purified proteins reveal that without network turnover, the strongest networks monopolize all the resources. However, with turnover, weaker networks survive and coexist with stronger networks.

There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.

Spatial and temporal actin assembly and disassembly is precisely regulated by the coordinated action of diverse sets of actin binding proteins (ABPs) with complementary properties. How the C. elegans actin sequestering protein tetrathymosinβ (TTH-1) functions alone and in combination with other ABPs to help maintain a pool of unassembled actin for rapid F-actin network assembly is poorly understood. TTH-1 consists of 4 human thymosinβ4-like repeats, and thymosinβ4 is known to sequester G-actin, preventing nucleation and elongation.

In vitro studies of actin filament networks crosslinked with dynamic actin binding proteins provide critical insights into cytoskeletal mechanics as well as inspiration for new adaptive materials design. However, discontinuous variance in the physiochemical properties of actin binding proteins impedes holistic relationships between crosslinker molecular parameters, network structure, and mechanics. Bio-synthetic constructs composed of synthetic polymer backbones and actin binding motifs would enable crosslinkers with engineered physiochemical properties to directly target the desired structure-property relationships. As a proof of concept, bio-synthetic crosslinkers composed of highly flexible polyethylene glycol (PEG) polymers functionalized with the actin binding peptide LifeAct, are explored as actin crosslinkers. Using bulk rheology and fluorescence microscopy, these constructs are shown to modulate actin filament network structure and mechanics in a contour length dependent manner, while maintaining the stress-stiffening behavior inherent to actin filament networks. These results encourage the design of more diverse and complex peptide-polymer crosslinkers to interrogate and control semi-flexible polymer networks. Bio-synthetic telechelics consisting of polyethylene glycol chains end-capped with the actin-binding peptide, LifeAct, are effective F-actin crosslinkers with contour length dependent control over network mechanics and structure.

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

While it is well-established that F-actin networks with specific organizations and dynamics are tightly regulated by distinct sets of associated actin-binding proteins (ABPs), how ABPs self-sort to particular F-actin networks remains largely unclear. We report that actin assembly factors Arp2/3 complex and formin Cdc12 tune the association of ABPs fimbrin Fim1 and tropomyosin Cdc8 to different F-actin networks in fission yeast. Genetic and pharmacological disruption of F-actin networks revealed that Fim1 is preferentially directed to Arp2/3-complex mediated actin patches, whereas Cdc8 is preferentially targeted to formin Cdc12-mediated filaments in the contractile ring. To investigate the role of Arp2/3 complex- and formin Cdc12-mediated actin assembly, we used four-color TIRF microscopy to observe the in vitro reconstitution of ABP sorting with purified proteins. Fim1 or Cdc8 alone bind similarly well to filaments assembled by either assembly factor. However, in ‘competition’ reactions containing both actin assembly factors and both ABPs, ∼2.0-fold more Fim1 and ∼3.5-fold more Cdc8 accumulates on Arp2/3 complex branch points and formin Cdc12-assembled actin filaments, respectively. These findings indicate that F-actin assembly factors Arp2/3 complex and formin Cdc12 help facilitate the recruitment of specific ABPs, thereby tuning ABP sorting and subsequently establishing the identity of F-actin networks in fission yeast.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

Vasodilator-stimulated phosphoprotein (VASP) family proteins play a crucial role in mediating the actin network architecture in the cytoskeleton. The Ena/VASP homology 2 (EVH2) domain in each of the four identical arms of the tetrameric VASP consists of a loading poly-Pro region, a G-actin-binding domain (GAB), and an F-actin-binding domain (FAB). Together, the poly-Pro, GAB, and FAB domains allow VASP to bind to sides of actin filaments in a bundle, and recruit profilin-G-actin to processively elongate the filaments. The atomic resolution structure of the ternary complex, consisting of the loading poly-Pro region and GAB domain of VASP with profilin-actin, has been solved over a decade ago; however, a detailed structure of the FAB-F-actin complex has not been resolved to date. Experimental insights, based on homology of the FAB domain with the C region of WASP, have been used to hypothesize that the FAB domain binds to the cleft between subdomains 1 and 3 of F-actin. Here, in order to develop our understanding of the VASP-actin complex, we first augment known structural information about the GAB domain binding to actin with the missing FAB domain-actin structure, which we predict using homology modeling and docking simulations. In earlier work, we used mutagenesis and kinetic modeling to study the role of domain-level binding-unbinding kinetics of Ena/VASP on actin filaments in a bundle, specifically on the side of actin filaments. We further look at the nature of the side-binding of the FAB domain of VASP at the atomistic level using our predicted structure, and tabulate effective mutation sites on the FAB domain that would disrupt the VASP-actin complex. We test the binding affinity of Ena with mutated FAB domain using total internal reflection fluorescence microscopy experiments. The binding affinity of VASP is affected significantly for the mutant, providing additional support for our predicted structure.

Actin filament networks are exposed to mechanical stimuli, but the effect of strain on actin filament structure has not been well established in molecular detail. This is a critical gap in understanding because the activity of a variety of actin-binding proteins has recently been determined to be altered by actin filament strain. We therefore used all-atom molecular dynamics simulations to apply tensile strains to actin filaments and find that changes in actin subunit organization are minimal in mechanically strained, but intact, actin filaments. However, a conformational change disrupts the critical D-loop to W-loop connection between longitudinal neighboring subunits, which leads to a metastable cracked conformation of the actin filament whereby one protofilament is broken prior to filament severing. We propose that the metastable crack presents a force-activated binding site for actin regulatory factors that specifically associate with strained actin filaments. Through protein-protein docking simulations, we find that 43 evolutionarily diverse members of the dual zinc-finger-containing LIM-domain family, which localize to mechanically strained actin filaments, recognize two binding sites exposed at the cracked interface. Furthermore, through its interactions with the crack, LIM domains increase the length of time damaged filaments remain stable. Our findings propose a new molecular model for mechanosensitive binding to actin filaments.

It was proposed from cellular studies that S. pombe tropomyosin Cdc8 (Tpm) segregates into two populations due to the presence or absence of an amino-terminal acetylation that specifies which formin-mediated F-actin networks it binds, but with no supporting biochemistry. To address this mechanism in vitro , we developed methods for S. pombe actin expression in Sf9 cells. We then employed 3-color TIRF microscopy using all recombinant S. pombe proteins to probe in vitro multicomponent mechanisms involving actin, acetylated and unacetylated Tpm, formins, and myosins. Acetyl-Tpm exhibits tight binding to actin in contrast to weaker binding by unacetylated Tpm. In disagreement with the differential recruitment model, Tpm showed no preferential binding to filaments assembled by the FH1-FH2-domains of two S. pombe formins, nor did Tpm binding have any bias towards the growing formin-bound actin filament barbed end. Although our in vitro findings do not support a direct formin-tropomyosin interaction, it is possible that formins bias differential tropomyosin isoform recruitment through undiscovered mechanisms. Importantly, despite a 12% sequence divergence between skeletal and S. pombe actin, S. pombe myosins Myo2 and Myo51 exhibited similar motile behavior with these two actins, validating key prior findings with these myosins that used skeletal actin.

Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development.

Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.