Biology subjects

Renner, L. D.

Publications and source records attributed to Renner, L. D..

5 recordsLinked to original sources

bioRxiv2026

Intrafilament nucleotide exchange in a prokaryotic actin homolog

Polymerization and disassembly govern the cellular functions of cytoskeletal proteins. Canonical nucleotide-dependent polymers, including actin and microtubules, renew their nucleotide state through subunit subunit dissociation, nucleotide exchange in solution and repolymerization. Yet the assembly dynamics of the membrane-bound bacterial actin MreB remain unresolved. Here, using total internal reflection fluorescence microscopy and high-speed atomic force microscopy, we visualise Bacillus subtilis MreB assembly on supported lipid bilayers in real time. ATP binding drives polymerization into symmetrically elongating filaments, whereas ATP hydrolysis within filaments promotes disassembly. Unexpectedly, nucleotides continuously exchange within membrane-bound filaments without detectable subunit turnover, coupling filament stability to the surrounding nucleotide pool: ATP exchange stabilizes filaments, whereas ADP exchange triggers rapid fragmentation and disassembly. Monte-Carlo modelling and single-cell in vivo imaging support this mechanism. Thus, intrafilament nucleotide exchange enables MreB filaments to renew their nucleotide state without polymer turnover, revealing nucleotide-state rejuvenation as a new mode of biological polymer regulation.

microbiology↗

bioRxiv2026

A genetic screen unveils the polymerization cycle of the bacterial actin-like MreB

MreB, a bacterial actin homolog and polymerizing ATPase, is central to cell-shape maintenance and cell-wall integrity. Its functions rely on its ability to assemble into dynamic, membrane-associated polymers. However, how nucleotide binding and hydrolysis, MreB-MreB contacts, and membrane association are coordinated to enable polymer assembly and disassembly remains unclear. Here, we combined genetics and live-cell microscopy with biochemical approaches to dissect these processes. Using a highly sensitive reporter of MreB activity, we identified, through a genetic screen, residues critical for MreB function in Bacillus subtilis. Subsequent extensive characterization of corresponding stable variants of the homologous Geobacillus stearothermophilus MreB revealed that ATP binding, but not ATP hydrolysis, is required for polymerization. Productive longitudinal intraprotofilament contacts are required for efficient ATP hydrolysis and enhance membrane association. Perturbations predicted to weaken lateral interprotofilament contacts altered membrane association and modulated ATPase activity. Together, these effects provide experimental evidence consistent with long-range functional coupling among the longitudinal and lateral protofilament interfaces, the distant nucleotide-binding site, and membrane association dynamics. Moreover, impaired ATP hydrolysis delays disassembly of lipid-associated polymers, indicating that hydrolysis promotes polymer turnover. These results establish key mechanistic steps coordinating ATP-driven MreB polymerization and turnover and provide a basis for a complete MreB assembly-disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.

microbiology↗

bioRxiv2022

Polymerization cycle of actin homolog MreB from a Gram-positive bacterium

In most rod-shaped bacteria, the actin homologue MreB is an essential component of the protein complex effecting cell wall elongation. The polymerization cycle and filament properties of eukaryotic actin have studied for decades and are well characterized. However, purification and in vitro work on MreB proteins have proven very difficult. Current knowledge of MreB biochemical and polymerization properties remains limited and is based on MreB proteins from Gram-negative species. In this study, we report the first observation of organized filaments and the first 3D-structure of MreB from a Gram-positive bacterium. We have purified MreB from the thermophilic Geobacillus stearothermophilus and shown that it forms straight pairs of protofilaments in vitro, and that polymerization depends on the presence of both lipids and nucleotide triphosphate. Two spatially close short hydrophobic sequences mediate membrane anchoring. Importantly, we demonstrate that unlike eukaryotic actin, nucleotide hydrolysis is a prerequisite for MreB interaction with the membrane, and that binding to lipids then triggers polymerization. Based on our results, we propose a molecular model for the mechanism of MreB polymerization.

biochemistry↗

bioRxiv2021

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

Trypanosoma brucei, the causative agent of human African trypanosomiasis, employs a flagellum for dissemination within the parasites mammalian and insect hosts. T. brucei cells are highly motile in culture and must be able to move in all three dimensions for reliable cell division. These characteristics have made long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of a variety of important cell-cycle events. To address this issue, we have devised an imaging approach that confines cells to small volumes that can be imaged continuously for up to 24 h. This system employs cast agarose microwells generated using a PDMS stamp that can be made with different dimensions to maximize cell viability and imaging quality. Using this approach, we have imaged individual T. brucei through multiple rounds of cell division with high spatial and temporal resolution. We have employed this method to study the differential rate of T. brucei daughter cell division and show that the approach is compatible with loss-of-function experiments such as small molecule inhibition and RNAi. We have also developed a strategy that employs in-well "sentinel" cells to monitor potential toxicity due to imaging. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible. ImportanceTrypanosoma brucei causes severe diseases that affect humans and livestock in Sub-Saharan Africa. Efficient strategies for manipulating the T. brucei genome have provided a wealth of information about protein localization and function in diverse cellular processes. However, employing live-cell imaging for phenotypic analysis in T. brucei remains a significant challenge because immobilization of this highly motile parasite rapidly leads to morphologic defects and cell death. While fixed-cell imaging can provide snapshots of cellular events, it cannot provide the direct causal link or precise timing of events that comes from watching a living cell change over time. Our strategy using agarose microwells now allows long-term live cell T. brucei imaging with a simple apparatus that can be adapted for a wide range of experimental conditions.

microbiology↗

bioRxiv2017

MreB Filaments Create Rod Shape By Aligning Along Principal Membrane Curvature.

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion and increased glycan crosslinking. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

microbiology↗