Biology subjects

Thomason, P. A.

Publications and source records attributed to Thomason, P. A..

8 recordsLinked to original sources

bioRxiv2026

Coordinated Assembly of WAVE and WASH complexes

WAVE and WASH polymerize Arp2/3-mediated branched actin at the leading edge of migrating cells and on the surface of endosomes, respectively. These two proteins are regulated within similar multiprotein complexes, the assembly mechanism of which remains poorly understood. Here we found using mass spectrometry that the two smallest subunits, BRK1 of the WAVE complex and CCDC53 of the WASH complex, interact with each other and with the assembly factor HSBP1. HSBP1 promotes WAVE and WASH assemblies and their respective activities, migration persistence and endosomal branched actin. Reduced levels of WAVE and WASH complexes caused by HSBP1 depletion can be corrected by providing cells with an excess of BRK1 or CCDC53 to assemble their specific complex. Unexpectedly, endogenous levels of BRK1 cross-regulate WASH assembly, whereas those of CCDC53 cross-regulate WAVE assembly. We found that various oligomers comprising BRK1, CCDC53 and HSBP1, including a heterotrimer containing one molecule of each, are formed through their promiscuous coiled coils. These oligomers play a coordinating role in the assembly of WAVE and WASH complexes.

cell biology↗

bioRxiv2026

In situ molecular architecture of PML bodies reveals open-state columnar trinucleosome assemblies within a porous, chromatin-permissive interior

Genome function in the nucleus is organised through membrane-less compartments enriched with specific proteins and nucleic acids. Promyelocytic leukemia (PML) bodies regulate telomere maintenance and DNA damage responses, but their internal molecular organisation remains poorly understood. We visualised the molecular composition of PML bodies directly within the nucleus using an advanced cryogenic-correlative light and electron microscopy (cryo-CLEM) pipeline. Cryo-electron tomography revealed eYFP-PML-I bodies as compartments with a molecular makeup distinct from the surrounding nucleoplasm. Cryogenic super-resolution correlative light and electron microscopy showed these comprise a diffuse PML-protein shell enclosing an inner core. A visual proteomics comparison of the core and surrounding regions through template matching and sub-tomogram averaging mapped nucleosomes, TRiC chaperonin complexes in both closed and open conformations, and single-capped PA28 proteasomes. Membrane structures of unknown function were additionally detected within some eYFP-PML-I bodies. Remarkably, the nucleosomes included density consistent with columnar trinucleosome assemblies (1), revealing that PML body interiors harbour discrete chromatin domains. Moreover, vault complexes were detected in poised positions in the proximal nucleoplasm. Together, these first in situ higher-resolution structural insights of PML bodies identify their critical functional components and an interior porous architecture that suggests a mechanism for content selection based on physical partitioning akin to size-exclusion chromatography. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/729032v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@19ad697org.highwire.dtl.DTLVardef@7e8590org.highwire.dtl.DTLVardef@72f0caorg.highwire.dtl.DTLVardef@1e8a1d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Macrophage chemotaxis steered by complex self-generated gradients of complement C5a

Macrophages rely on efficient chemotaxis to locate sites of infection and tissue damage. One general strategy that enhances chemotactic accuracy is the use of self-generated gradients, where cells locally deplete attractants to create or sharpen guidance cues. Here we show that mouse bone marrow-derived macrophages (BMDMs) migrate toward the complement component C5a using this strategy. Cells actively deplete C5a from their surroundings, establishing local gradients as fresh attractant diffuses inward. We visualized this process in real time with fluorescent C5a and reproduced its dynamics using computational models. C5a depletion is mediated primarily by C5aR1-dependent endocytosis. This mechanism produces complex responses, with different C5a concentrations inducing temporally distinct waves of migration, and maximal chemotaxis occurring below 10 nM C5a. As expected, increasing C5a concentrations recruit more cells. In contrast, human macrophages inactivate C5a mainly through carboxypeptidase-mediated enzymatic degradation, yielding a higher optimal concentration ([~]30 nM) and distinct migratory dynamics. Both species also deplete externally imposed C5a gradients, sharpening them and enhancing guidance. These findings identify C5a degradation as a critical mechanism by which macrophages extract directional information from their environment. Self-generated gradient formation, despite different mechanisms across species, emerges as a conserved and versatile strategy for immune navigation.

immunology↗

bioRxiv2025

Live-cell 3D-SIM of Rift Valley fever virus NSs filaments reveals a polygonal web architecture

A defining feature of Rift Valley fever virus (RVFV) is the incorporation of the NSs protein into large filamentous assemblies inside infected nuclei 1, as judged from fixed specimens. To gain insight into the three-dimensional (3D) structure of NSs filaments within nuclei of live-cells we used genetic-code expansion (GCE) to introduce an unnatural amino acid (uAA) fluorophore into the protein, and coupled this with live-cell structured illumination microscopy (SIM). Our super-resolved images revealed a micron-scale polygonal web of NSs fibres with discrete domain characteristics. Parallel experiments on fixed RVFV-infected cells confirmed that virally-encoded native NSs filaments also display this morphology. Overall, our 3D-SIM analysis sheds new light on the complex large-scale architecture of NSs filaments and provides context for programmable filamentous E3 ligases that promote virus replication. SignificanceThis work demonstrates how advanced labeling and imaging techniques can reveal unexpected organizational principles in viral protein assemblies. We bio-orthogonally labeled the Rift Valley fever virus NSs protein with bright tetrazine dyes using genetic-code expansion (GCE), that enabled imaging of NSs filaments in live-cells using 3D-SIM. GCE offers key advantages for studying small, genetically fragile viral proteins like NSs. It eliminates the need for bulky fluorescent proteins that can disrupt protein-protein interfaces critical for complex assembly and avoids fixation artifacts associated with traditional immunofluorescence microscopy. This integrative GCE and 3D-SIM approach revealed the complete native architecture of NSs filaments in 3D, dramatically enhancing our ability to visualize intricate polygonal web structures beyond simple linear filaments. The discrete structural domains identified here raise intriguing questions about potential mechanisms for spatially controlling E3 ligase activity in a programmable manner.

biochemistry↗

bioRxiv2025

EPHA2 and scavenger receptor-directed trafficking enhances endosomal leakiness and antisense therapy delivery

The potential for using therapeutic antisense oligonucleotides (ASOs) has been hampered by lack of understanding of how they enter cells and subsequently access their targets. Endocytosis contributes to ASO uptake, but the machinery mediating subsequent ASO trafficking to permit suppression of their target mRNAs has not been described. Here, we show that ASO engagement with a scavenger receptor (CD44) activates the ERK-RSK axis to promote serine phosphorylation of a receptor tyrosine kinase (EPHA2). Serine phosphorylation of EPHA2 permits endocytosis, trafficking, and accumulation of ASOs in nuclear-adjacent endosomes. These endosomes then become leaky, allowing ASOs to escape and effectively suppress target mRNA expression. Inhibition of stress granule-mediated repair of leaky endosomes further enhances ASO effectiveness. These data identify an endocytic route to the nucleus which may be exploited to maximise effectiveness of ASO-mediated therapies.

cell biology↗

bioRxiv2022

The Scar/WAVE complex drives normal actin protrusions without the Arp2/3 complex, but proline-rich domains are required

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. The pentameric Scar/WAVE regulatory complex (WRC) is the main catalyst of pseudopod and lamellipodium formation. Its actin nucleation activity has been attributed to its ability to combine monomeric actin and Arp2/3 complex through the VCA domain of Scar/WAVE, while other regions of the complex are typically thought to mediate spatial and temporal regulation and have no direct role in actin polymerization. Here we show that the Scar/WAVE with its VCA domain deleted can still induce the formation of morphologically normal actin protrusions. Equivalent results are seen in B16-F1 mouse melanoma cells and Dictyostelium discoideum cells. This actin polymerization occurs independently of the Arp2/3 complex, whose recruitment to the leading edge is greatly reduced by the loss of the VCA domain. We also expressed Scar/WAVE with VCA and polyproline domains both deleted. In Dictyostelium cells, these were only active if WASP (which contains its own proline-rich domain) was available. Similarly, in B16-F1 cells both Abi and WAVE proline-rich domains needed to be deleted before the function of the WRC was lost. Thus we conclude that proline-rich domains play a central role in actin nucleation. Our data demonstrate a new actin nucleation mechanism of the WRC that is independent of its VCA domain and the Arp2/3 complex. We also show that proline-rich domains are more fundamental than has been thought. Together, these findings suggest a new mechanism for WRC action.

cell biology↗

bioRxiv2019

WASP restricts active Rac to maintain cells’ front-rear polarisation

Efficient motility requires polarised cells, with anterior pseudopods and a retracting rear. This polarisation requires that the pseudopod catalyst Rac is restricted to the front. Here we show that the Arp2/3 complex regulator WASP is important for maintaining front-rear polarity, using a mechanism that limits where active Rac localises. Dictyostelium cells lacking WASP inappropriately activate Rac and SCAR/WAVE at their rears, leading to reduced cell speed. WASP facilitates the internalisation of clathrin-coated pits, and its Rac-binding CRIB motif is considered essential for its localisation and activity. However, WASP mutants with deleted CRIB domains, or harbouring a new mutation that prevents Rac binding, localise normally, recruit Arp2/3 complex, and drive actin polymerisation. Similarly, Rac inhibitors do not block WASP localisation or activation. Despite this, WASP CRIB mutants cannot restore polarisation of active Rac. Thus, WASPs interaction with Rac regulates Rac activity and cell polarity, but is dispensable for activating actin polymerization.

cell biology↗

bioRxiv2017

CYRI (FAM49) proteins are local inhibitors of Scar/WAVE induced lamellipodia that bind directly to active Rac1

Actin-based protrusions driving cell migration are reinforced through positive feedback, but it is unclear how the cell restricts the eventual size of a protrusion or limits positive signals to cause splitting or retraction. We have identified an evolutionarily conserved regulator of the protrusion machinery, which we name CYRI (CYFIP-related Rac interacting) protein. CYRI shows sequence similarity to the Scar/WAVE complex member CYFIP in a Domain of Unknown Function, DUF1394. CYRI binds specifically to activated Rac1 via a common motif shared with CYFIP, establishing DUF1394 as a new Rac1 binding domain. CYRI-depleted cells have broad, Scar/WAVE-enriched lamellipodia and enhanced Rac1 signaling. Conversely, CYRI overexpression suppresses spreading and dramatically sharpens protrusions into unproductive needles. CYRI proteins use dynamic inhibition of Scar/WAVE induced actin to focus positive protrusion signals and regulate pseudopod complexity. CYRI behaves like a \"local inhibitor\" predicted and described in widely accepted mathematical models, but not previously identified in living cells.

cell biology↗