Biology subjects

Blake, T. C. A.

Publications and source records attributed to Blake, T. C. A..

4 recordsLinked to original sources

bioRxiv2025

Phosphoinositide lipids have bidirectional and spatially distinct roles in filopodial dynamics

Neural circuits are assembled by filopodia, dynamic actin-rich protrusions that connect environment sensing with structural changes. Using rapid timelapse imaging and acute pharmacological perturbation in Xenopus laevis retinal ganglion cells, we reveal that phosphoinositide lipid conversions impose spatially distinct control over filopodial dynamics. The class I phosphoinositide 3-kinase (PI3K) inhibitor alpelisib and Oculocerebrorenal syndrome of Lowe protein/Inositol polyphosphate 5-phosphatase inhibitor YU142670 markedly reduced filopodial tip motility. Class II PI3K inhibitor PITCOIN4 and YU142670 curtailed base dynamics, indicating location-specific lipid requirements. Despite no detectable change in PI(4,5)P2, both alpelisib and YU142670 depleted PI(3,4)P2 at filopodial tips. Their co-application was non-additive, with partial alleviation of YU142670-induced stalling by alpelisib, consistent with networked control of lipid conversion and actin remodelling. Live imaging with a TAPP1-3xcPH probe combined with quantitative cross-correlation and Granger causality analysis showed that PI(3,4)P2 at filopodial tips both generates tip extension and responds to forward filopodial movement. Disrupting actin polymerisation rapidly erased tip PI(3,4)P2 prior to filopodial stalling. These findings define a vulnerable node with implications for neurodevelopmental miswiring.

cell biology↗

bioRxiv2023

Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions

Filopodia are narrow actin-rich protrusions with important roles in neuronal development. The neuronally-enriched TOCA-1/CIP4 family of F-BAR and SH3 domain adaptor proteins have emerged as upstream regulators that link membrane interactions to actin binding proteins in lamellipodia and filopodia, including WAVE and N-WASP nucleation promoting factors and formins. Here, we demonstrate a direct interaction between TOCA-1 and Ena/VASP actin filament elongators that is mediated by clustered SH3 domain interactions. Using Xenopus retinal ganglion cell axonal growth cones, where Ena/VASP proteins have a native role in filopodia extension, we show that TOCA-1 localises to filopodia and lamellipodia, with a retrograde flow of puncta, and correlates with filopodial protrusion. Two-colour single molecule localization microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters coalesce at advancing lamellipodia and filopodia and operate synergistically with Ena to promote filopodial protrusion dependent on a functional SH3 domain. In analogous yet distinct ways to lamellipodin and IRSp53, we propose that transient TOCA-1 clusters recruit and promote Ena activity to orchestrate filopodial protrusion.

cell biology↗

bioRxiv2020

Structure of Full Length Plasmodium Myosin A and its light chain PfELC, dual targets against malaria parasite pathogenesis

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity and ultimately pathogenesis of this parasite relies on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future antimalarials targeting both the glideosome motor and its regulatory elements.HighlightsThe first structures of the full length PfMyoA motor in two states of its motor cycle.A unique priming of the PfMyoA lever arm results from specific lever arm/motor domain interactions, which allows for a larger powerstroke to enhance speed.Sequence adaptations within the motor domain and degenerate IQ motifs in the lever arm dictate PfMyoA motor properties.PfELC is essential for blood cell invasion and is a weak link in the assembly of a fully functional motor, providing a second novel target for antimalarial drug design.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

All symptoms of malaria disease are associated with the asexual blood stages of development, involving cycles of red blood cell (RBC) invasion and egress by the Plasmodium spp. merozoite. Merozoite invasion is rapid and is actively powered by a parasite actomyosin motor. The current accepted model for actomyosin force generation envisages arrays of parasite myosins, pushing against short actin filaments connected to the external milieu that drive the merozoite forwards into the RBC. In Plasmodium falciparum, the most virulent human malaria species, Myosin A (PfMyoA) is critical for parasite replication. However, the precise function of PfMyoA in invasion, its regulation, the role of other myosins and overall energetics of invasion remain unclear. Here, we developed a conditional mutagenesis strategy combined with live video microscopy to probe PfMyoA function and that of the auxiliary motor PfMyoB in invasion. By imaging conditional mutants with increasing defects in force production, based on disruption to a key PfMyoA phospho-regulation site, the absence of the PfMyoA essential light chain, or complete motor absence, we define three distinct stages of incomplete RBC invasion. These three defects reveal three energetic barriers to successful entry: RBC deformation (pre-entry), mid-invasion initiation, and completion of internalisation, each requiring an active parasite motor. In defining distinct energetic barriers to invasion, these data illuminate the mechanical challenges faced in this remarkable process of protozoan parasitism, highlighting distinct myosin functions and identifying potential targets for preventing malaria pathogenesis.

microbiology↗