Synthetic Par polarity induces cytoskeleton asymmetry in unpolarized mammalian cells.

Polarized cells rely on a polarized cytoskeleton to function. Yet, how cortical polarity cues induce cytoskeleton polarization remains elusive. Here, we capitalized on recently established designed 2D protein arrays to ectopically engineer cortical polarity of virtually any protein of interest during mitosis in various cell types. This enables direct manipulation of polarity signaling and the identification of the cortical cues sufficient for cytoskeleton polarization. Using this assay, we dissected the logic of the Par complex pathway, a key regulator of cytoskeleton polarity during asymmetric cell division. We show that cortical clustering of any Par complex subunit is sufficient to trigger complex assembly and that the primary kinetic barrier to complex assembly is the relief of Par6 autoinhibition. Further, we found that inducing cortical Par complex polarity induces two hallmarks of asymmetric cell division in unpolarized mammalian cells: spindle orientation, occurring via Par3, and central spindle asymmetry, depending on aPKC activity.

Elongator stabilizes microtubules to control central spindle asymmetry and polarized trafficking of cell fate determinants.

Asymmetric cell division gives rise to two daughter cells that inherit different determinants, thereby acquiring different fates. Polarized trafficking of endosomes containing fate determinants recently emerged as an evolutionarily conserved feature of asymmetric cell division to enhance the robustness of asymmetric cell fate determination in flies, fish and mammals. In particular, polarized sorting of signalling endosomes by an asymmetric central spindle contributes to asymmetric cell division in Drosophila melanogaster. However, how central spindle asymmetry arises remains elusive. Here we identify a moonlighting function of the Elongator complex-an established protein acetylase and tRNA methylase involved in the fidelity of protein translation-as a key factor for central spindle asymmetry. Elongator controls spindle asymmetry by stabilizing microtubules differentially on the anterior side of the central spindle. Accordingly, lowering the activity of Elongator on the anterior side using nanobodies mistargets endosomes to the wrong cell. Molecularly, Elongator regulates microtubule dynamics independently of its acetylation and methylation enzymatic activities. Instead, Elongator directly binds to microtubules and increases their polymerization speed while decreasing their catastrophe frequency. Our data establish a non-canonical role of Elongator at the core of cytoskeleton polarity and asymmetric signalling.

Design of biologically active binary protein 2D materials.

Ordered two-dimensional arrays such as S-layers1,2 and designed analogues3-5 have intrigued bioengineers6,7, but with the exception of a single lattice formed with flexible linkers8, they are constituted from just one protein component. Materials composed of two components have considerable potential advantages for modulating assembly dynamics and incorporating more complex functionality9-12. Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building blocks, and use it to design a p6m lattice. The designed array components are soluble at millimolar concentrations, but when combined at nanomolar concentrations, they rapidly assemble into nearly crystalline micrometre-scale arrays nearly identical to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces, which we demonstrate can drive extensive receptor clustering, downstream protein recruitment and signalling. Using atomic force microscopy on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and that our material can therefore impose order onto fundamentally disordered substrates such as cell membranes. In contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work provides a foundation for a synthetic cell biology in which multi-protein macroscale materials are designed to modulate cell responses and reshape synthetic and living systems.

Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication.

Regions of the genome with the potential to form secondary DNA structures pose a frequent and significant impediment to DNA replication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciated DNA-binding domain that exhibits specific binding to G-quadruplex (G4) DNA structures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacent PARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicase DDX11. Loss of both Timeless and DDX11 causes epigenetic instability at G4-forming sequences and DNA damage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures by DDX11 to maintain processive DNA synthesis.