Biology subjects

Moores, C.

Publications and source records attributed to Moores, C..

11 recordsLinked to original sources

bioRxiv2026

STRUCTURAL PLASTICITY ALLOWS DOUBLECORTIN AND DRUG-STABILIZED MICROTUBULES TO ACCOMMODATE EACH OTHER

Microtubule architecture and dynamics are modulated by small molecules, but how these compounds alter lattice properties and interactions with cellular regulators remains incompletely understood. We used the neuronal microtubule-associated protein doublecortin (DCX), previously reported to preferentially recognize 13-protofilament microtubules, to probe lattices stabilized by different taxanes, including the fluorescent taxane Fchitax-3. We show that DCX binds 15-protofilament Fchitax-3-stabilized microtubules, demonstrating that DCX is not a specific marker of 13-protofilament architecture. Cryo-electron microscopy revealed that the fluorescent moiety of Fchitax-3 inserts into inter-protofilament lateral contacts, explaining its effects on microtubule architecture and dynamics. DCX binds Fchitax-3 microtubules through its N-terminal DC domain, which accommodates the narrower inter-protofilament binding site in the 15-protofilament lattice. Remarkably, although Fchitax-3 induces the longitudinal lattice expansion characteristic of taxanes, DCX remodels its binding site towards its preferred compacted configuration. Thus, taxanes can modify microtubule structure in distinct ways while preserving lattice plasticity and the capacity to undergo conformational remodelling by microtubule-associated proteins. This contrasts with the comparatively rigid lattice of GMPCPP-stabilized microtubules. Consequently, interpretations of microtubule lattice states based on comparisons between taxane- and GMPCPP-stabilized microtubules may conflate structural differences with different degrees of lattice plasticity. Our findings establish the microtubule lattice as a malleable structural substrate whose drug-induced conformations can be further remodelled by cellular binding partners.

biophysics↗

bioRxiv2025

Apicortin defines the Plasmodium apical conoid body during transmission but is dispensable for the parasite life cycle

Apicomplexan parasites such as Plasmodium spp. and Toxoplasma gondii possess unique tubulin-based structures, including subpellicular microtubules and apical polar rings, which are essential for parasite motility, host cell invasion, and replication. How the stability of these structures is maintained is poorly understood, but it may involve Apicortin, a microtubule-associated protein, so-far found only in apicomplexans and the placozoan Trichoplax adhaerens. Apicortin contains a doublecortin (DC) domain and a partial tubulin polymerisation-promoting protein (TPPP) domain, both implicated in microtubule binding and stabilization. In this study, we investigated the location and function of Apicortin in Plasmodium berghei. Live cell imaging of a transgenic parasite line expressing GFP-tagged Apicortin showed that it was present at the apical end of invasive parasites only during development of transmission stages within the mosquito vector. High-resolution imaging using super-resolution and expansion microscopy, revealed that Apicortin forms a distinct ring-like structure in the apical complex region at the apical end of ookinetes and sporozoites. However, deletion of the Apicortin gene had no effect on parasite development and transmission through the mosquito, indicating that this protein is not essential. This suggests that there may be redundancy or compensatory functions in the mechanisms that stabilise the apical complex.

cell biology↗

bioRxiv2025

Arp2/3-mediated bidirectional actin assembly by SPIN90 dimers in metazoans

Branched actin networks nucleated by the Arp2/3 complex play critical roles in various cellular processes, from cell migration to intracellular transport. However, when activated by WISH/DIP/SPIN90 family proteins, Arp2/3 nucleates linear actin filaments. Unexpectedly, we found that human SPIN90 is a dimer that can nucleate bidirectional actin filaments. To understand the basis for this, we determined a 3 [A] resolution structure of human SPIN90-Arp2/3 complex nucleating actin filaments. Our structure shows that SPIN90 dimerises via a 3-helix bundle and interacts with two Arp2/3 complexes. Each SPIN90 molecule binds both Arp2/3 complexes to promote their activation. Our analysis demonstrates that single filament nucleation by Arp2/3 is mechanistically more like branch formation than previously appreciated. The dimerisation domain in SPIN90 orthologues is conserved in metazoans, suggesting that this mode of bidirectional nucleation is a common strategy to generate anti-parallel actin filaments.

biophysics↗

bioRxiv2024

Arp2/3 iso-complexes limit t-tubule overgrowth to ensure myofiber formation and synchronous contraction

Muscle cells form a network of plasma membrane invaginations called t-tubules that control calcium release from the endoplasmic reticulum at the triads during muscle contraction. Although the importance of t-tubules for muscle physiology is well established, and abnormalities are found in multiple disorders, the mechanisms that mediate t-tubule growth are unknown. We show that the actomyosin cortex beneath the plasma membrane, regulated by Arp2/3 complexes containing Arpc5, acts as a gatekeeper for the membrane availability required for t-tubule growth. Enlarged t-tubules are formed upon disruption of Arpc5, impairing the synchronisation between plasma membrane depolarization and calcium release. Additionally, ablation of Arpc5 postnatally in myofibers results in muscle fatigue and t-tubule abnormalities, as observed in muscle disorders. We propose that the actomyosin cortex impacts muscle function, offering a potential pathophysiological mechanism for muscle disorders.

cell biology↗

bioRxiv2024

Mechanistic basis of temperature-adaptation in microtubule dynamics across frog species

Cellular processes are remarkably effective across diverse temperature ranges, even with highly conserved proteins. In the context of the microtubule cytoskeleton, which is critically involved in a wide range of cellular activities, this is particularly striking as tubulin is one of the most conserved proteins while microtubule dynamic instability is highly temperature sensitive. We thus lack a mechanistic framework that links functional adaptability with environmental pressures. Here, we leverage the diversity of natural tubulin variants from three closely related frog species that live at different temperatures: we combine in vitro reconstitution assays, quantitative biochemistry, and cryogenic electron microscopy to show how a small number of primary sequence changes influences the energy landscape of tubulin interactions and thereby mediates cold-adaptation and microtubule stability. This study thus broadens our conceptual framework for understanding microtubule dynamics and provides insights into how conserved cellular processes are tailored to different ecological niches.

biochemistry↗

bioRxiv2023

Cortactin stabilizes actin branches by bridging activated Arp2/3 to its nucleated actin filament

Regulation of the assembly and turnover of branched actin filament networks nucleated by the Arp2/3 complex is essential during many cellular processes including cell migration and membrane trafficking. Cortactin plays a key role in stabilizing actin filament branches by interacting with the Arp2/3 complex and actin filaments via its N-terminal Acidic domain (NtA) and 6.5 central unstructured 37 amino acid repeats, respectively 1, but the mechanism of this is unclear. We determined the structure of cortactin-stabilized Arp2/3 actin branches using cryo-electron microscopy. We find that cortactin interacts with the new daughter filament nucleated by the Arp2/3 complex at the branch site rather than the initial mother actin filament. Cortactin preferentially binds activated Arp3 in contrast to other nucleation promoting factors (NPFs) 2,3. Cortactin also stabilizes the F-actin-like interface of activated Arp3 with the first actin subunit of the new filament, and its central repeats extend along successive daughter filament subunits. Cortactin binding to Arp3 is incompatible with NPF interaction and its preference for activated Arp3 explains why it is retained at the actin branch. Our data have uncovered why cortactin displaces NPFs, while at the same time promoting synergy to regulate branched actin network dynamics.

biophysics↗

bioRxiv2023

A structural and dynamic visualization of the interaction between the microtubule-associated protein 7 (MAP7) and microtubules

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex.

biophysics↗

bioRxiv2020

Plasmodium berghei kinesin-5 associates with the spindle apparatus during cell division but is dispensable for parasite proliferation

Kinesin-5 motors play essential roles in spindle apparatus assembly during cell division, by generating forces to establish and maintain the spindle bipolarity essential for proper chromosome segregation. Kinesin-5 is largely conserved structurally and functionally in model eukaryotes, but its role is unknown in the Plasmodium parasite, an evolutionarily divergent organism with several atypical features of both mitotic and meiotic cell division. We have investigated the function and subcellular location of kinesin-5 during cell division throughout the Plasmodium berghei life cycle. Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with -tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella. Treatment of gametocytes with microtubule-specific inhibitors confirmed kinesin-5 association with nuclear spindles and not cytoplasmic axonemal microtubules. Altogether, our results demonstrate that kinesin-5 is associated with the spindle apparatus, expressed in proliferating parasite stages, and important for efficient production of infectious sporozoites.

cell biology↗

bioRxiv2019

A microtubule RELION-based pipeline for cryo-EM image processing

Microtubules are polar filaments built from {beta}-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of - and {beta}-tubulin. The determination of the {beta}-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.\n\nAbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

molecular biology↗

bioRxiv2019

Structural determinants of microtubule minus end preference in CAMSAP CKK domains

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which has lost minus-end specificity. Near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit subtly different footprints on microtubules. Whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. NMR experiments show that HsCKK is remarkably rigid, supporting this remodelling ability. Thus, in contrast to many MAPs, CKK domains can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

biophysics↗

bioRxiv2018

Near-atomic cryo-EM structure of yeast kinesin 5-microtubule complex reveals a distinct binding footprint

Kinesin-5s are essential members of the superfamily of microtubule-dependent motors that undertake conserved roles in cell division. We investigated coevolution of the motor-microtubule interface using cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to fission yeast microtubules. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation, with a closed nucleotide binding pocket and docked neck linker that supports cover neck bundle formation. Compared to mammalian tubulin microtubules, Cut7s footprint on S. pombe microtubule surface is subtly different because of their different architecture. However, the core motor-microtubule interaction that stimulates motor ATPase is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. The S. pombe microtubules were bound by the drug epothilone, which is visible in the taxane binding pocket. Stabilization of S. pombe microtubules is mediated by drug binding at this conserved site despite their noncanonical architecture and mechanochemistry.\n\nHighlightsO_LIS. pombe Cut7 has a distinct binding footprint on S. pombe microtubules\nC_LIO_LIThe core interface driving microtubule activation of motor ATPase is conserved\nC_LIO_LIThe neck linker is docked in AMPPNP-bound Cut7 and the cover neck bundle is formed\nC_LIO_LIEpothilone binds at the taxane binding site to stabilize S. pombe microtubules\nC_LI\n\neTOC textTo investigate coevolution of the motor-microtubule interface, we used cryo-electron microscopy to determine the near-atomic structure of the motor domain of Cut7, the fission yeast kinesin-5, bound to microtubules polymerized from natively purified fission yeast tubulin and stabilised by the drug epothilone.

biophysics↗