Biology subjects

Way, M.

Publications and source records attributed to Way, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Myofibers drive postnatal myonuclear accretion through Arp2/3-dependent membrane protrusions

Skeletal muscle postnatal growth depends on myoblast fusion with pre-existing myofibers, but whether myofibers actively regulate this process remains unclear. The Arp2/3 complex, which nucleates branched actin networks, is required for myoblast fusion during embryogenesis, but its role in postnatal myofibers is unknown. Here, we investigated the role of the Arp2/3 complex in myofibers using an inducible skeletal muscle-specific Arpc4 knockout model. We find that loss of Arp2/3 in myofibers impairs myonuclear accretion, resulting in smaller myofibers and reduced muscle strength. Satellite cells are still activated and able to differentiate but accumulate around Arpc4-deficient myofibers rather than fusing, pointing to a myofiber-intrinsic defect. In co-culture, myofibers form long-lived membrane protrusions enriched in Arp2/3 at myoblast contact sites, and optogenetic activation of protrusion in myofibers induced myoblast fusion. Genetic depletion of Arpc4 in myofibers markedly reduces membrane protrusions both in vitro and in vivo. These findings unveil a fundamental role for the Arp2/3 complex in postnatal muscle growth, and reveal a mechanism by which myofibers actively extend membrane protrusions to drive myoblast fusion. This repositions myofibers as active fusogenic partners and cell-autonomous drivers of their own nuclear accretion.

cell biology↗

bioRxiv2025

SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion.

When stimulated by nucleation-promoting factors such as WAVE, the Arp2/3 complex generates branched actin networks. In contrast, when activated by SPIN90, the Arp2/3 complex generates linear actin filaments. The Arp2/3 complex in mammals, consists of 8 iso-complexes with dieerent properties as there are two isoforms of Arp3, ArpC1 and ArpC5. Here, using recombinant Arp2/3 iso-complexes with defined compositions, we show that SPIN90 selectively activates ArpC5L- rather than ArpC5- containing complexes to generate linear actin filaments. Consistent with this, SPIN90 at the leading edge of migrating cells enhances the recruitment of ArpC5L-, but not ArpC5- containing, Arp2/3 complexes to lamellipodia. These SPIN90-Arp2/3-ArpC5L complexes generate linear actin filaments that integrate into lamellipodia and impact protrusion eeiciency. Moreover, using polarised light microscopy, we show that loss of SPIN90 leads to an enrichment in actin filaments oriented more perpendicular to the plasma membrane. Our results demonstrate that SPIN90 regulates the architecture and dynamics of lamellipodial actin in an Arp2/3 iso-complex dependent fashion. SummarySPIN90 selectively recruits and activates ArpC5L containing Arp2/3 to shape the architecture and dynamics of lamellipodial actin.

cell biology↗

bioRxiv2025

In situ cryo-electron tomography of vaccinia virus exit from infected cells

Poxvirus-infected cells release newly assembled virions via Golgi-mediated envelopment and subsequent exocytosis at the plasma membrane, prior to cell lysis. Here, we used cryo-electron tomography and structured illumination microscopy to study vaccinia egress. Our 3D analysis reveals that Golgi-mediated envelopment is a flexible process that involves remodelling of the enfolding membrane and a final step that seals a small pore. During subsequent exocytosis, the viral outer membrane fuses with the plasma membrane but retains a distinct identity, beneath which septins and clathrin are independently recruited. Clathrin enhances actin-dependent viral spread, while septins suppress virus release from the cell. We found that septin filaments run parallel to the inner surface of the plasma membrane beneath virions attached to the cell surface. In contrast, clathrin induces the formation of plasma membrane invaginations in distinct subdomains. We propose that actin assembly at these subdomains provides a template for subsequent virus-induced actin polymerization.

cell biology↗

bioRxiv2025

Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

Vaccinia virus is a large enveloped DNA virus, which, like all poxviruses, replicates in the cytoplasm of infected cells. Vaccinia was historically thought to encode all the proteins required for its replication. However, more recent findings have shown that nuclear host proteins are redirected to the cytoplasm to facilitate viral replication. Among these, topoisomerase 2 (TOP2A) and 2{beta} (TOP2B), which mediate nuclear transcription, DNA replication, and chromosome segregation are the most abundant host proteins associated with nascent viral genomes. Here, we investigate the mechanisms driving TOP2A and TOP2B cytoplasmic translocation and their role in viral replication. We found that early viral protein synthesis induces the cytosolic relocalization of both isoforms, which are subsequently recruited to viral factories by an interaction of their C-terminal domains with the viral ligase, A50. TOP2A promotes replication by interacting with the vaccinia DNA replication machinery. In contrast, TOP2B suppresses replication by enhancing the formation of double-stranded RNA and antiviral granules, containing components of the tRNA splicing ligase complex. Our analysis provides new insights into host-pathogen interactions during poxvirus infection and the role of topoisomerase 2 outside of the nucleus.

cell biology↗

bioRxiv2025

Enhancing the efficiency of oncolytic vaccinia virus for ovarian cancer treatment

Vaccinia virus, known for its clinical safety has a tropism for primary and metastatic tumours as well as ovarian tissue. Consequently, oncolytic approaches with recombinant vaccinia viruses have emerged as attractive agents against ovarian cancer. Unfortunately, oncolytic vaccinia monotherapies are yet to live up to their potential promise. Given this, there is a need to identify combination agents that improve the effectiveness of vaccinia in ovarian cancer treatment. We screened 9,000 compounds to identify drugs that enhance the ability of a recombinant vaccinia virus lacking VGF and F1 ({Delta}VF) to induce death of ID8 Trp53-/- murine ovarian cancer cells. We identified a class of tubulin polymerisation inhibitors including vinorelbine. The combination of vinorelbine and vaccinia induces ID8 Trp53-/- cell death via apoptosis. In a syngeneic mouse model of high grade serous ovarian carcinoma, {Delta}VF virus lacking the viral thymidine kinase (TK), armed with GM-CSF and expressing NeonGreen ({Delta}VFTK-NG-GM-CSF) is tumour specific. A combination of the {Delta}VFTK-NG-GM-CSF virus with vinorelbine prolongs mouse survival compared to the treatment of mice with either agent alone. Our study suggests vinorelbine is a promising agent to combine with oncolytic vaccinia virus approaches for the management of ovarian cancer.

cancer 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

Sonoran Desert ex situ conservation gap analysis: charting the path towards conservation

Plant biodiversity is under threat. With two out of five plants at risk of extinction, preserving taxa through ex situ conservation approaches must be considered a priority. The Sonoran Desert (SD), a highly biodiverse ecoregion shared between Mexico and the United States is home to over 4000 native plant taxa. A method to determine conservation priorities was developed in collaboration with institutions from both countries. Herbaria records and data from nine seed banks were analysed to identify gaps in previous conservation efforts. The SD native taxa were prioritised based on a Priority Score (PS) and their potential distribution was modelled to identify priority regions. 4029 native taxa were reported. 1441 have accessions preserved in seed banks, but only 412 have been collected inside the SD. The PS considers some potentially endemic (126) and threatened taxa (112) as the most urgent to preserve. It also includes information about the storage behaviour (3236 orthodox), useful plants (1406), and taxa without populations in protected areas (230) to categorise the species. Although most of the SD flora is not represented in seed banks, at least 80% is predicted to produce desiccation-tolerant seeds and thus can be cost-effectively stored. Spatial analysis shows that the central region of the Baja California Peninsula stands out for its species richness and endemism. Our study presents the first ex situ conservation gap analysis of the SD flora and provides a replicable methodology for identifying priority species and potential areas for ex situ collection.

ecology↗

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

Branched actin networks in innate immune cells mediate host microbiota homeostasis.

Assembly of branched actin networks, driven by the Arp2/3 complex are essential for the function and integrity of the immune system. Patients with loss-of-function mutations in the ARPC5 subunit of the Arp2/3 complex develop inflammation and immunodeficiency after birth, leading to early mortality. However, the mechanistic basis for these phenotypes remains obscure. Here we demonstrate that loss of Arpc5 in the murine hematopoietic system, but not the corresponding Arpc5l isoform causes early-onset intestinal inflammation after weaning. This condition is initiated by microbiota breaching the ileal mucosa, leading to local and systemic inflammation. Macrophage and neutrophils infiltrate into the ileum, but in the absence of Arpc5 fail to restrict microbial invasion. Loss of Arpc5 compromises the ability of macrophages to phagocytose and kill intra-cellular bacteria. Our results underscore the indispensable role of Arpc5, but not Arpc5l containing Arp2/3 complexes in mononuclear phagocytes function and host-microbiota homeostasis. One-Sentence SummaryArpc5 containing Arp2/3 complexes are essential for host-microbiota homeostasis

immunology↗

bioRxiv2023

CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

The inhibitors, CK-666 and CK-869, are widely used to probe the function of actin nucleation by the Arp2/3 complex in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 is also only able to inhibit Arp3-but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

biochemistry↗

bioRxiv2023

Regeneration of actin filament branches from the same Arp2/3 complex

Branched actin filaments are found in many key cellular structures. Branches are nucleated by the Arp2/3 complex activated by nucleation-promoting factor (NPF) proteins and bound to the side of pre-existing mother filaments. Over time, branches dissociate from their mother filament, leading to network reorganization and turnover, but this mechanism is less understood. Here, using microfluidics and purified proteins, we examined the dissociation of individual branches under controlled biochemical and mechanical conditions. We observe that Arp2/3 remains bound to the mother filament after most debranching events, even when accelerated by force. Unexpectedly, this mother-remaining Arp2/3 readily nucleates a new actin filament branch, without being activated anew by an NPF: it simply needs to exchange its nucleotide and bind an actin monomer. The protein GMF, which accelerates debranching, prevents branch re-nucleation. Our results suggest that actin filament re-nucleation can provide a self-repair mechanism, helping branched networks to sustain mechanical stress in cells over extended periods of time.

biophysics↗

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

The level of Nck rather than N-WASP determines the rate of actin-based motility of Vaccinia

Vaccinia virus exiting from host cells activates Src/Abl kinases to phosphorylate A36, an integral membrane viral protein. Phosphorylated A36 binds the adaptors Nck and Grb2 which recruit N-WASP to activate Arp2/3-driven actin polymerisation to promote viral spread. A36 also recruits intersectin, which enhances actin polymerization via AP-2/clathrin and Cdc42. How many viral and host molecules does vaccinia hijack to induce actin polymerization? To advance our quantitative understanding of this process, we now determined absolute numbers of the essential molecules in the vaccinia signalling network using fluorescent molecule counting approaches in live cells. There are 1156{+/-}120 A36 molecules on virus particles inducing actin polymerization in HeLa cells. This number, however, is over 2000 in mouse embryonic fibroblasts (MEFs), suggesting that A36 levels on the virion are not fixed. In MEFs, viruses recruit 1032{+/-}200 Nck and 434{+/-}10 N-WASP molecules, suggesting a ratio of 4:2:1 for the A36:Nck:N-WASP signalling network. Loss of A36 binding to either secondary adaptors Grb2 or intersectin results in a 1.3- and 2.5-fold reduction in Nck respectively. Curiously, despite recruiting comparable numbers of the Arp2/3 activator, N-WASP (245{+/-}26 and 276{+/-}66), these mutant viruses move at different speeds that inversely correlate with the number of Nck molecules. Our analysis has uncovered two unexpected new aspects of Vaccinia virus egress, numbers of the viral protein A36 can vary in the virion membrane and the velocity of virus movement depends on the levels of the adaptor protein Nck.

cell biology↗

bioRxiv2023

ARPC5 deficiency leads to severe early onset systemic inflammation and early mortality

The seven subunit Arp2/3 complex drives the formation of branched actin networks that are essential for many cellular processes including cell migration. In humans, the ARPC5 subunit of the Arp2/3 complex is encoded by two paralogous genes (ARPC5 and ARPC5L), resulting in proteins with 67% identity. Through whole-exome sequencing, we identified a biallelic ARPC5 frameshift variant in a female child who presented with recurrent infections, multiple congenital anomalies, diarrhea, and thrombocytopenia, and suffered early demise from sepsis. Her consanguineous parents also had a previous child who died with similar clinical features. Using CRISPR/Cas9-mediated approaches, we demonstrate that loss of ARPC5 affects actin cytoskeleton organization and function, as well as chemokine-dependent cell migration in vitro. Homozygous Arpc5-/- mice do not survive past embryonic day 9 due to severe developmental defects, including loss of the second pharyngeal arch which contributes to craniofacial and heart development. Our results indicate that ARPC5 is important for both prenatal development and postnatal immune signaling, in a non-redundant manner with ARPC5L. Moreover, our observations add the ARPC5 locus to the list of genes that should be considered when patients present with syndromic early-onset immunodeficiency, particularly if recessive inheritance is suspected.

immunology↗

bioRxiv2023

Biomimetic actin cortices shape cell-sized lipid vesicles

Animal cells are shaped by a thin layer of actin filaments underneath the plasma membrane known as the actin cortex. This cortex stiffens the cell surface and thus opposes cellular deformation, yet also actively generates membrane protrusions by exerting polymerization forces. It is unclear how the interplay between these two opposing mechanical functions plays out to shape the cell surface. To answer this question, we reconstitute biomimetic actin cortices nucleated by the Arp2/3 complex inside cell-sized lipid vesicles. We show that thin Arp2/3-nucleated actin cortices strongly deform and rigidify the shapes of giant unilamellar vesicles and impart a shape memory on time scales that exceeds the time of actin turnover. In addition, actin cortices can produce finger-like membrane protrusions, showing that Arp2/3-mediated actin polymerization forces alone are sufficient to initiate protrusions in the absence of actin bundling or membrane curving proteins. Combining mathematical modeling and our experimental results reveals that the concentration of actin nucleating proteins, rather than actin polymerization speed, is crucial for protrusion formation. This is because locally concentrated actin polymerization forces can drive a positive feedback loop between recruitment of actin and its nucleators to drive membrane deformation. Our work paints a picture where the actin cortex can either drive or inhibit deformations depending on the local distribution of nucleators. Significance StatementThe cells in our body must actively change shape in order to migrate, grow and divide, but they also need to maintain their shape to withstand external forces during tissue development. Cellular shape control results from an interplay between the plasma membrane and its underlying cortex, a shell composed of crosslinked actin filaments. Using cell-free reconstitution and mathematical modelling, we show that minimal biomimetic actin cortices can mechanically rigidify lipid vesicles while at the same time driving membrane protrusion formation. Our observations suggest that the spatial distribution of actin nucleation determines whether the actin cortex drives or inhibits membrane deformations.

biophysics↗

bioRxiv2022

Cryo-ET of infected cells reveals that a succession of two lattices drives vaccinia virus assembly

During its cytoplasmic replication, vaccinia virus assembles non-infectious spherical immature virions (IV) coated by a viral D13 lattice. Subsequently, IV mature into infectious brick-shaped intracellular mature virus (IMV) that lack D13. Here, we performed cryo-electron tomography of frozen-hydrated vaccinia-infected cells to structurally characterise the maturation process in situ. During IMV formation a new viral core forms inside IV with a wall consisting of trimeric pillars arranged in a new pseudohexagonal lattice. This lattice appears as a palisade in cross-section. During maturation, which involves a 50% reduction in virion volume, the viral membrane becomes corrugated as it adapts to the newly formed viral core in a process that does not appear to require membrane removal. Our study suggests that the length of this core is determined by the D13 lattice and that the consecutive D13 and palisade lattices control virion shape and dimensions during vaccinia assembly and maturation.

microbiology↗

bioRxiv2022

Nucleation and stability of branched versus linear Arp2/3-generated actin filaments

Activation of the Arp2/3 complex by VCA-domain-bearing NPFs results in the formation of daughter actin filaments branching off the sides of pre-existing mother filaments. Alternatively, when stimulated by SPIN90, Arp2/3 directly nucleates linear actin filaments. Uncovering the similarities and differences of these two activation mechanisms is fundamental to understanding the regulation and function of Arp2/3. Analysis of individual filaments reveals that the catalytic VCA domain of WASP, N-WASP and WASH, accelerate the Arp2/3-mediated nucleation of linear filaments by SPIN90, in addition to their known branch-promoting activity. Unexpectedly, these VCA domains also destabilize existing branches, as well as SPIN90-Arp2/3 at filament pointed ends. Furthermore, cortactin and GMF, which respectively stabilize and destabilize Arp2/3 at branch junctions, have a similar impact on SPIN90-activated Arp2/3. However, unlike branch junctions, SPIN90-Arp2/3 at the pointed end of linear filaments is not destabilized by piconewton forces, and does not become less stable with time. It thus appears that linear and branched Arp2/3-generated filaments respond similarly to regulatory proteins, albeit with quantitative differences, and that they differ greatly in their responses to aging and to mechanical stress. These results indicate that SPIN90- and VCA-activated Arp2/3 complexes adopt similar yet non-identical conformations, and that their turnover in cells may be regulated differently.

biochemistry↗

bioRxiv2022

Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

Intracellular mature virions (IMV) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMV can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMV, like intracellular enveloped virions (IEV), the second form of vaccinia, undergo anterograde transport and recruit kinesin-1. In vitro reconstitution of virion transport reveals that IMV and IEV move toward microtubule plus-ends with respective velocities of 0.66 and 0.56 m/s. Quantitative imaging establishes IMV and IEV recruit an average of 65 and 115 kinesin-1 motor complexes respectively. In the absence of kinesin-1 there is a near-complete loss of in vitro motility and defects in the cellular spread of both virions. Our observations demonstrate kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to investigate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.

cell biology↗