Biology subjects

Zakharov, A.

Publications and source records attributed to Zakharov, A..

3 recordsLinked to original sources

bioRxiv2023

Actin polymerization drives lumen formation in a human epiblast model

Lumens or fluid-filled cavities are a ubiquitous feature of mammals and are often evolutionarily linked to the origin of body-plan complexity. Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. While osmotic pressure gradients drive lumen formation in many developmental contexts, mechanisms of human epiblast lumenogenesis are unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based model of the epiblast using engineered hydrogels that model the confinement faced by the epiblast in the blastocyst. Actin polymerization into a dense mesh-like network at the apical surface generates forces to drive early lumen expansion, as leaky junctions prevent osmotic pressure gradients. Theoretical modeling reveals that apical actin polymerization into a stiff network drives lumen opening, but predicts that a switch to pressure driven lumen growth at larger lumen sizes is required to avoid buckling of the cell layer. Consistent with this prediction, once the lumen reaches a radius of around 12 m, tight junctions mature, and osmotic pressure gradients develop to drive further lumen growth. Human epiblasts show a transcriptional signature of actin polymerization during early lumenogenesis. Thus, actin polymerization drives lumen opening in the human epiblast, and may serve as a general mechanism of lumenogenesis.

developmental biology↗

bioRxiv2023

Clots reveal anomalous elastic behavior of fiber networks

The mechanical properties of many soft natural and synthetic biological materials are relevant to their function. The emergence of these properties from the collective response of the structural components of the material to external stress as well as to intrinsic cell traction, remains poorly understood. Here, we examine the nonlinear elastic behavior of blood clots by combining microscopy and rheological measurements with an elastic network model that accounts for the stretching, bending, and buckling of constituent fibrin fibers. We show that the inhibition of fibrin crosslinking reduces fiber bending stiffness and introduces an atypical fiber buckling-induced softening regime at intermediate shear, before the well-characterized stiffening regime. We also show that crosslinking and platelet contraction significantly alter force propagation in the network in a strain-dependent manner. Our mechanics-based model, supported by experiments, provides a framework to understand the origins of characteristic and anomalous regimes of non-linear elastic response not only in blood clots, but also more generally in active biopolymer networks.

biophysics↗

bioRxiv2022

A comprehensive phenotypic screening strategy to identify modulators of cargo translocation by the bacterial Type IVB secretion system

Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage co-culture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines. ImportanceMulti-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogens Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.

microbiology↗