Biology subjects

Harden, J. L.

Publications and source records attributed to Harden, J. L..

4 recordsLinked to original sources

bioRxiv2026

Self-centering steady-state flows emerge in confined actomyosin networks

The actin cytoskeleton drives shape changes and transport in cells and supports mechanical signal transmission. However, how cells control and use active cytoskeletal flows at the mesoscale is not well understood. We reconstituted an active cytoskeleton in water-in-oil emulsion droplets of Xenopus laevis egg extract and observed, above a critical droplet size, the emergence of a 3D radially convergent steady-state flow of polymeric actin, maintained by continuous actin turnover. The flow condensed lipid-rich cellular debris into a centered inclusion. Steady-state F-actin density and flow velocity profiles roughly collapse onto scale-invariant master curves. This behavior can be explained by a physical model representing the network as an isotropic active viscous fluid with a percolation threshold. The contracting network behaves as an active swimmer with complex internal dynamics that centers itself and the central inclusion inside the droplets without physical boundary attachment. Active contraction, crosslinking and polymerization dynamics in an actin network can thus generate cell-scale flow patterns that sense the confining geometry and external signals and exert forces that are likely sufficient to move and localize organelles in cells.

biophysics↗

bioRxiv2024

The formin FMNL2 plays a role in the response of melanoma cells to substrate stiffness

BackgroundCells are constantly sensing and responding to changes in their local environment to adapt their behaviour and morphology. These external stimuli include chemical and mechanical signals, and much recent work has revealed the complexity of the cellular response to changes in substrate stiffness. We investigated the effects of substrate stiffness on the morphology and motility of A2058 human melanoma cells. FMNL2, a formin protein associated with actin cytoskeleton dynamics, regulates cell morphology and motility but its role in stiffness sensing remains unclear. This study examines how A2058 cells respond to substrates of varying stiffness and evaluates the impact of FMNL2 depletion on these responses. ResultsWe found that with increasing substrate stiffness the cells transitioned from a rounded cell morphology to progressively more elongated morphologies with a concomitant increase in actin stress fiber alignment. Depletion of FMNL2 expression amplified these morphological changes, with knockdown cells showing consistently greater elongation and more pronounced stress fiber alignment compared to controls. Notably, the orientational order parameter (S) revealed higher alignment of actin filaments along the cells long axis in knockdown cells. Substrate stiffness also affected cell motility, indicated by an apparent optimal stiffness that maximized motility followed by a notable decrease in distance travelled during cell migration on progressively stiffer substrates. This decrease was largely attributable to a decrease in the time the cells spent in motion as the substrate stiffness increased. FMNL2 depletion significantly exacerbated this effect, with knockdown cells traveling shorter net distances and spending less time moving across all substrates. ConclusionsThis study demonstrates that substrate stiffness profoundly influences A2058 melanoma cell morphology and motility, with FMNL2 playing a pivotal regulatory role. Our observations suggest that FMNL2 is critical for maintaining motility and morphological adaptability under increased stiffness. Loss of FMNL2 enhanced stress fiber alignment and cell elongation while impairing motility, particularly on stiff substrates, revealing FMNL2 as a mechanosensitive effector. However, further biochemical work should be performed to determine the exact mechanisms by which FMNL2 participates in regulation of melanoma cell response to substrate stiffness.

cell biology↗

bioRxiv2023

The variable domain from the mitochondrial fission mechanoenzyme Drp1 promotes liquid-liquid phase separation

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" (VD) important for regulation. For the mitochondrial fission dynamin, Drp1, a regulatory role for the VD is demonstrated by mutations that can elongate, or fragment, mitochondria. How the VD encodes inhibitory and stimulatory activity is unclear. Here, isolated VD is shown to be intrinsically disordered (ID) yet undergoes a cooperative transition in the stabilizing osmolyte TMAO. However, the TMAO stabilized state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, raising the possibility that phase separation may enable rapid tuning of Drp1 assembly necessary for fission.

biophysics↗

bioRxiv2020

Engineered Tissue Interfaces for in vitro and in vivo Bioengineering

In regenerative medicine, the healing of the interfacial zone between tissues is a major challenge, yet approaches for studying the complex microenvironment of this interface remain lacking. Here, we study these complex living interfaces by manufacturing modular "blocks" of naturally porous decellularized plant-derived scaffolds with a computer numerical controlled mill. We demonstrate how each scaffold can be seeded with different cell types and easily assembled in a manner akin to LEGO bricks to create an engineered tissue interface (ETI). Cells migrate across the interface formed between an empty scaffold and a scaffold pre-seeded with cells. However, when both scaffolds contain cells, only a shallow cross-over zone of cell infiltration forms at the interface. As a proof-of-concept study, we use ETIs to investigate the interaction between lab grown bone and connective tissues. Consistent with the above, a cross-over zone of the two distinct cell types forms at the interface between scaffolds, otherwise the populations remain distinct. Finally, we demonstrate how ETIs are biocompatible in vivo, becoming vascularized and integrated into surrounding tissue after implantation. This work creates new tissue design avenues for understanding biological processes or the development of synthetic artificial tissues.

biophysics↗