Biology subjects

Lacey, S. E.

Publications and source records attributed to Lacey, S. E..

5 recordsLinked to original sources

bioRxiv2026

In situ structures of the ciliary tip show the multi-step mechanism of axoneme assembly

Motile cilia are highly complex, ordered organelles which extend from the cell surface to generate fluid flow. During motile cilia assembly, components rapidly self-organize at the ciliary tip. Here, over two hundred different components bind microtubules to form an intricate, repeating structure called the axoneme. Despite in-depth structural characterization of axonemal complexes, we have limited understanding of how these structures are generated. Here, we directly visualize the steps of axonemal assembly in situ by performing cryo-electron tomography of Chlamydomonas reinhardtii cilia tips. Using subtomogram averaging and classification, we identified conserved microtubule luminal proteins that establish periodicity and show that controlled microtubule lattice perforation can facilitate incorporation of late-binding components. By uncovering unexpected assembly intermediates, we explain how structural integrity and the emergence of motility are coordinated within the axoneme. Overall, we demonstrate that self-assembly proceeds through regulated, sequential construction steps and provide a molecular framework for understanding axoneme biogenesis.

molecular biology↗

bioRxiv2022

The Molecular Structure of Anterograde Intraflagellar Transport Trains

Anterograde intraflagellar transport trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFTA and IFTB proteins that link structural and signalling cargoes to microtubule motors for import into cilia. It remains unknown how the IFTA/B proteins are arranged into complexes and how these complexes polymerise into functional trains. Here, we use in situ cryo-electron tomography and Alphafold2 protein structure predictions to generate the first molecular model of the entire anterograde train. We show how the conformation of both IFTA and IFTB is dependent on lateral interactions with neighbouring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. The retrograde dynein motor binding site is a composite surface involving multiple IFTB repeats, ensuring that dynein can only form a strong interaction with IFTB upon train assembly. Finally, we reveal how IFTB extends two flexible tethers to maintain a connection with IFTA that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that are involved in cilia assembly.

molecular biology↗

bioRxiv2021

Microtubule Lattice Spacing Governs Cohesive Envelope Formation of Tau Family Proteins

Tau is an intrinsically-disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here, we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau-envelope disassembly. Investigating other members of the tau-MAP family, we find MAP2 similarly forms envelopes governed by lattice-spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct segments. We conclude that the interdependent allostery between lattice-spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

biophysics↗

bioRxiv2020

Cryo-EM reveals the complex architecture of dynactin's shoulder and pointed end

Dynactin is a 1.1 MDa complex that activates the molecular motor, dynein, for ultra-processive transport along microtubules. In order to do this it forms a tripartite complex with dynein and a coiled-coil adaptor. Dynactin consists of an actin-related filament whose length is defined by its flexible shoulder domain. Despite previous cryo-EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high-resolution information in these regions. Here we combine multiple cryo-EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder positions the four identical p50 subunits in different conformations to bind dynactins filament and securely houses the p150 subunit. The pointed end map allows us to build the first structure of p62, and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.

molecular biology↗

bioRxiv2019

Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule

Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of the axoneme in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a long coiled-coil stalk. Here we address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using the stand-alone Relion package. We show the majority of the MTBD is remarkably rigid upon binding, with the transition to the high affinity state controlled by the movement of a single helix at the MTBD interface. In addition DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that reaches over and contacts the adjacent protofilament. Unexpectedly we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.

molecular biology↗