Metal-binding propensity in the mitochondrial dynamin-related protein 1

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Unlike that seen with the native mitochondrial lipid cardiolipin, metal-bound chelator lipids recruit Drp1 to the membrane but is rendered functionally inactive in membrane fission. Metal-bound chelator beads also recruit Drp1 and represents a potential strategy to deplete or purify the protein from native tissue lysates.

Total In Vitro Biosynthesis of the Thioamitide Thioholgamide and Investigation of the Pathway

Thioholgamides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent activity against cancerous cell lines and an unprecedented structure. Despite being one of the most structurally and chemically complex RiPPs, very few biosynthetic steps have been elucidated. Here, we report the complete in vitro reconstitution of the biosynthetic pathway. We demonstrate that thioamidation is the first step and acts as a gatekeeper for downstream processing. Thr dehydration follows thioamidation, and our studies reveal that both these modifications require the formation of protein complexes – ThoH/I and ThoC/D. Harnessing the power of AlphaFold we deduce that ThoD acts as a lyase and also propose putative catalytic residues. ThoF catalyzes the oxidative decarboxylation of the terminal Cys and the subsequent macrocyclization is facilitated by ThoE. This is followed by Ser dehydration, which is also carried out by ThoC/D. ThoG is responsible for histidine bis-N-methylation, which is a prerequisite for His β-hydroxylation – a modification carried out by ThoJ. The last step of the pathway is the removal of the leader peptide by ThoK to afford mature thioholgamide.

Microtubule plus-end regulation by centriolar cap proteins

Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, grow very slowly and are very stable. The complex of centriolar proteins CP110 and CEP97 forms a cap that stabilizes the distal centriole end and prevents its over-elongation. Here, we used in vitro reconstitution assays to show that whereas CEP97 does not interact with microtubules directly, CP110 specifically binds microtubule plus ends, potently blocks their growth and induces microtubule pausing. Cryo-electron tomography indicated that CP110 binds to the luminal side of microtubule plus ends and reduces protofilament peeling. Furthermore, CP110 directly interacts with another centriole biogenesis factor, CPAP/SAS-4, which tracks growing microtubule plus ends, slows down their growth and prevents catastrophes. CP110 and CPAP synergize in inhibiting plus-end growth, and this synergy depends on their direct binding. Together, our data reveal a molecular mechanism controlling centriolar microtubule plus-end dynamics and centriole biogenesis.

Size-dependent secondary nucleation and amplification of α-synuclein amyloid fibrils

The size of the amyloid seeds is known to modulate their autocatalytic amplification and cellular toxicity. However, the seed size-dependent secondary nucleation mechanism, toxicity, and disease-associated biological processes mediated by α-synuclein (α-Syn) fibrils are largely unknown. Using the cellular model and in vitro reconstitution, we showed that the size of α-Syn fibril seeds not only dictates its cellular internalization and associated cell death; but also the distinct mechanisms of fibril amplification pathways involved in the pathological conformational change of α-Syn. Specifically, small-sized fibril seeds showed elongation possibly through monomer addition at the fibril termini; whereas longer fibrils template the fibril amplification by surface-mediated nucleation as demonstrated by super-resolution microscopy. The distinct mechanism of fibril amplification, and cellular uptake along with toxicity suggest that breakage of fibrils into different sizes of seeds determine the underlying pathological outcome of synucleinopathies.

A synthetic membrane shaper for controlled liposome deformation

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

EB3-informed dynamics of the microtubule stabilizing cap during stalled growth

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

Control of neurotransmitter release by two distinctmembrane-binding faces of the Munc13-1 C­1C2B region

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C­1C2B region with the plasma membrane: i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here we have tested this model and investigated the role of the C­1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.

Using quantitative reconstitution to investigate multi-component condensates

Many biomolecular condensates are thought to form via liquid-liquid phase separation (LLPS) of multivalent macromolecules. For those that form through this mechanism, our understanding has benefitted significantly from biochemical reconstitutions of key components and activities. Reconstitutions of RNA-based condensates to date have mostly been based on relatively simple collections of molecules. However, proteomics and sequencing data indicate that natural RNA-based condensates are enriched in hundreds to thousands of different components, and genetic data suggest multiple interactions can contribute to condensate formation to varying degrees. In this perspective we describe recent progress in understanding RNA-based condensates through different levels of biochemical reconstitutions, as a means to bridge the gap between simple in vitro reconstitution and cellular analyses. Complex reconstitutions provide insight into the formation, regulation, and functions of multi-component condensates. We focus on two RNA-protein condensate case studies: stress granules and RNA processing bodies (P bodies), and examine the evidence for cooperative interactions among multiple components promoting LLPS. An important concept emerging from these studies is that composition and stoichiometry regulate biochemical activities within condensates. Based on the lessons learned from stress granules and P bodies we discuss forward-looking approaches to understand the thermodynamic relationships between condensate components, with the goal of developing predictive models of composition and material properties, and their effects on biochemical activities. We anticipate that quantitative reconstitutions will facilitate understanding of the complex thermodynamics and functions of diverse RNA-protein condensates.