Justin E. Molloy

Malaria parasite invasion of red blood cells is powered by Myosin A (MyoA), the actomyosin motor which is a core component of the glideosome. Our previous phosphoproteomic study identified a cyclic GMP-dependent phosphorylation site on MyoA at Ser19, suggesting that cyclic nucleotide signalling may directly regulate motor activity during invasion. However, the functional significance of this phosphorylation site remains unclear. Here, we show that Plasmodium falciparum MyoA phosphorylation at Ser19 is dynamically regulated during the intraerythrocytic cycle, peaking in extracellular merozoites and rapidly declining following invasion. Using conditional depletion of MyoA, we show that MyoA is essential for red blood cell invasion but dispensable for gametocyte development. Conditional complementation with mutant versions of MyoA that either mimic (S19D) or ablate phosphorylation (S19A) reveal markedly impaired asexual parasite proliferation, highlighting the importance of precise regulation of MyoA phosphorylation at Ser19. In vitro motility assays using parasite-derived MyoA showed that ablating phosphorylation either pharmacologically or by introducing an S19A mutation, reduces motor speed. Conversely, introducing a phosphomimetic S19D mutation restores motility levels comparable to phosphorylated wild type MyoA. Collectively, these results establish MyoA Ser19 phosphorylation as a regulatory switch for actomyosin motor activity.

Kinesin-1 microtubule motors are ATP-fuelled, twin-headed cargo transporters that step processively along microtubules, with a load-dependent directional bias. Here we show using single molecule optical trapping that 1 mM ATPγS, a slowly-hydrolysed analogue, substantially defeats the biasing mechanism, whereas 1 µM ATPγS supports it. Our data argue that kinesin re-registers itself between steps into an Await-Isomerisation (AI) state that engenders both on-axis steps and off-axis missteps, and that missteps can be rescued. In the AI state, ATP or ATPγS is bound but neck-linker docking and nucleotide hydrolysis are inhibited. Load-dependent exit from the AI state establishes hydrolytic competence via catalytic site closure and coupled neck-linker docking, which guides the tethered head to its next on-axis site. By overpopulating the AI state, ATPγS reveals its pivotal role in the biasing mechanism, whose control logic maximises steered diffusion-to-capture of the leading kinesin-1 head under load, at the expense of futile nucleotide turnover.

To improve access to advanced optical microscopy in educational and resource-limited settings we have developed the eduWOSM (educational Warwick Open Source Microscope), an open hardware platform for transmitted-light and epifluorescence imaging in up to 4 colours, including single molecule imaging. EduWOSMs are robust, bright, compact, portable and ultra-stable. They are controlled entirely by open source hardware and software, with an option for remote control from a webpage. Here we describe the core eduWOSM technology and benchmark its performance using 3 example projects, single fluorophore tracking of tubulin heterodimers within gliding microtubules, 4D (deconvolution) imaging/tracking of chromosome motions in dividing human cells, and automated single particle tracking in vitro and in live cells with classification into subdiffusive, diffusive and superdiffusive motion.

To improve access to advanced optical microscopy in educational and resource-limited settings we have developed the eduWOSM ( edu cational W arwick O pen S ource Mic roscope), an open hardware platform for transmitted-light and epifluorescence imaging in up to 4 colours, including single molecule imaging. EduWOSMs are robust, bright, compact, portable and ultra-stable. They are controlled entirely by open source hardware and software, with an option for remote control from a webpage. Here we describe the core eduWOSM technology and benchmark its performance using 3 example projects, single fluorophore tracking of tubulin heterodimers within gliding microtubules, 4D (deconvolution) imaging/tracking of chromosome motions in dividing human cells, and automated single particle tracking in vitro and in live cells with classification into subdiffusive, diffusive and superdiffusive motion.

The calcium calmodulin protein kinase II (CaMKII) is a multi-subunit ring assembly with a central hub formed by the association domains. There is evidence for hub polymorphism between and within CaMKII isoforms, but the link between polymorphism and subunit exchange has not been resolved. Here, we present near-atomic resolution cryogenic electron microscopy (cryo-EM) structures revealing that hubs from the α and β isoforms, either standalone or within an β holoenzyme, coexist as 12 and 14 subunit assemblies. Single-molecule fluorescence microscopy of Venus-tagged holoenzymes detects intermediate assemblies and progressive dimer loss due to intrinsic holoenzyme lability, and holoenzyme disassembly into dimers upon mutagenesis of a conserved inter-domain contact. Molecular dynamics (MD) simulations show the flexibility of 4-subunit precursors, extracted in-silico from the β hub polymorphs, encompassing the curvature of both polymorphs. The MD explains how an open hub structure also obtained from the β holoenzyme sample could be created by dimer loss and analysis of its cryo-EM dataset reveals how the gap could open further. An assembly model, considering dimer concentration dependence and strain differences between polymorphs, proposes a mechanism for intrinsic hub lability to fine-tune the stoichiometry of αβ heterooligomers for their dynamic localization within synapses in neurons.

: Myosin 10 (Myo10) is an actin-based molecular motor that is essential for filopodia formation and likely senses tension through interactions with integrins in filopodial tips. It possesses a single α-helical (SAH) domain at the end of its canonical lever, which amplifies the movement of the motor. We have shown the SAH domain can contribute to lever function and possesses the properties of a constant force spring. Here we investigate whether the SAH domain plays a role in tension sensing and whether it becomes extended under load at the filopodial tip. Previously, we found that removing the entire SAH domain and short anti-parallel coiled coil (CC) region at the C-terminal end of the SAH does not prevent Myo10 from moving to filopodial tips in cells. Exploiting this, we generated recombinant forms of Myo10, in which a tension-sensing module (TSMod), comprising a FRET-pair YPet and mCherry separated by a linker sequence of amino acids was then inserted between the Myo10 motor and tail domains, so as to replace the SAH domain and CC region. The linker sequence comprised either a portion of the native SAH domain, or control sequences that were either short (x1: stiff) or long (x5: flexible) repeats of “GPGGA”. As additional controls we also placed the TSMod construct at the N-terminus, where it should not experience force. Our FRET measurements indicate that the SAH domain of Myo10 may become extended at when the protein is stalled at the filopodial tips, so the SAH domain may therefore act as a force sensor.

Poly(ADP-ribose) polymerases (PARPs) play key roles in DNA damage repair pathways in eukaryotic cells. Human PARPs 1 and 2 are catalytically activated by damage in the form of both double-strand and single-strand DNA breaks. Recent structural work indicates that PARP2 can also bridge two DNA double-strand breaks (DSBs), revealing a potential role in stabilizing broken DNA ends. In this paper, we have developed a magnetic tweezers–based assay in order to measure the mechanical stability and interaction kinetics of proteins bridging across the two ends of a DNA DSB. We find that PARP2 forms a remarkably stable mechanical link (rupture force ~85 pN) across blunt-end 5′-phosphorylated DSBs and restores torsional continuity allowing DNA supercoiling. We characterize the rupture force for different overhang types and show that PARP2 switches between bridging and end-binding modes depending on whether the break is blunt-ended or has a short 5′ or 3′ overhang. In contrast, PARP1 was not observed to form a bridging interaction across blunt or short overhang DSBs and competed away PARP2 bridge formation, indicating that it binds stably but without linking together the two broken DNA ends. Our work gives insights into the fundamental mechanisms of PARP1 and PARP2 interactions at double-strand DNA breaks and presents a unique experimental approach to studying DNA DSB repair pathways.

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel–Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMEC_D), or from nominally healthy donors (controls; HCMEC_C). Using fluorescence microscopy, WPBs in HCMEC_C (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMEC_D (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMEC_D revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMEC_D WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMEC_D were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function.

Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.