Postma, H. W. C. | Search bioRxiv

bioRxiv2026

Energetic gradients emerge in developing motor-microtubule structures

Living matter produces a variety of beautiful spatiotemporal structures and patterns that are not enduringly present in their nonliving counterparts. These ordered, non-equilibrium steady states are often sustained through the consumption of energy. Here, we investigate the energetic cost of assembling an ordered aster from an initially disordered, uniform mixture of cytoskeletal microtubules and kinesin motors. Using a calibrated fluorescent ATP reporter, we measure reproducible radial ATP gradients on scales of tens of microns that establish within, and persist over, tens of minutes, alongside coupled spatial gradients in motor density. These appreciable gradients are predicted by a reaction-diffusion model that acknowledges the localization of ATP consumption to regions where both molecular motors and microtubules are sufficiently abundant to encourage consumption, as confirmed by finite element modeling. With our results, we compare the power per volume required by our cytoskeletal networks with the known power per volume expenditure in cells. Comparison of our measured results with estimates of the dissipative processes available to motor-microtubule mixtures leads to the hypothesis that maintaining spatial motor gradients dominates the energetic demand in this system. Our direct quantification of energetic fluxes across space unlocks future explorations of what steady states are accessible to cells, and how the cytoskeleton drives broad spatial organization. SignificanceHow much energy do organisms pay to form and maintain their organizing biochemical patterns? Existing measurements of cellular metabolism and energy expenditures largely resolve net or supply-side biochemical fluxes, without spatial information, impeding the study of this basic question. Here, we develop an experimental approach to directly measure the distributions of biochemical energy that respond to power expenditures of cytoskeletal motor-microtubule networks as they form aster structures, reminiscent of those found in the mitotic spindle. As these structures self-assemble, calibrated readouts in real molecular units register large, reproducible, and long-lived gradients of ATP. We interpret these measurements by developing theory to account for the functional destinies of energy expenditure. These advances clarify outstanding questions of energy in living matter.

biophysics↗

bioRxiv2020

In Situ Unzipping of Long, High Molecular Weight DNA in Solid State Nanopores

Nanopores are an established paradigm in genome sequencing technology, with remarkable advances still being made today. All efforts continually address the challenges associated with rapid, accurate, high-throughput, and low cost detection, particularly with long-read length DNA. We report on the in situ melting and unzipping of long, high molecular weight DNA. At varying salt concentration, we directly compare the translocation conductance and speeds between SiN and graphene nanopores at sub-10 nm pore diameters. We observe the force-induced unzipping of dsDNA at higher salt concentrations than previously reported in literature. We observe free running translocation without secondary structures of ssDNA that is an order of magnitude longer than reported before. We hypothesize that the frayed single strands at the molecules end get captured with a higher likelihood than both ends together. In understanding this phenomena for long-read lengths, we continue to address the challenges revolving around future generations of sequencing technology. Statement of SignificanceGenome sequencing is an advancing field with applications in clinical diagnostics. However, the challenges of providing accurate identification of longer DNA molecules at low cost are still developing. While detection of long DNA molecules is established, the identification of its individual nucleotides presents its own set of challenges. By separating the hydrogen bonds between the two strands, individual nucleotides are made directly measurable. However, identification is hindered from the formation of secondary structures, where the single-stranded DNA sticks to itself. Previous studies only included short DNA molecules. We report in situ force-induced unzipping and translocation of long DNA without secondary structures almost an order of magnitude longer than reported before. Our findings present new experimental conditions and insights that progress the field towards high accuracy sequencing of individual long molecules.

biophysics↗