Biology subjects

Heermann, T.

Publications and source records attributed to Heermann, T..

4 recordsLinked to original sources

bioRxiv2022

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanisms ancient origin.

cell biology↗

bioRxiv2022

One-pot synthesis of prenylated proteins utilizing E. coli cell-free expression

Bottom-up synthetic biology is a powerful tool for uncovering the mechanisms underlying vital biological processes, such as signaling and cell polarization. The core principle of reconstituting cellular functions in their minimal forms can be achieved through modular protein design. However, assembling multiple purified proteins into a functional and synchronized system remains a technical challenge. The fact that many regulatory proteins show direct or indirect membrane interactions further exacerbates the complications. Here, we introduce the Cell-Free prenylated Protein Synthesis (CFpPS) system which enables the production of prenylated proteins in a single reaction mix, through reconstituted prenylation machinery. Not only does the CFpPS system offer a fast and reliable method for producing solubilized prenylated proteins, but it can also produce the protein of interest directly in the vicinity of biomimetic membranes, thus enabling microscopy-based functional assessment. As proof of principle, we demonstrate synthesis and solubilization of various important signaling proteins from the Ras superfamily, as well as membrane binding and extraction of the key polarity regulator Cdc42. Furthermore, our method can be used to confer membrane affinity to any protein, simply by adding a 4-peptide motif to the C-terminus of the protein. In sum, the CFpPS system offers a versatile and effective platform for designing peripheral membrane proteins for synthetic biology applications.

synthetic biology↗

bioRxiv2022

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take place, are inherently difficult to assess with standard biophysical methods. The introduction of mass-sensitive particle tracking (MSPT) constitutes an important step towards a thorough quantitative characterization of such processes. Technically, this was made possible through the advent of interferometric scattering microscopy (iSCAT)-based mass photometry (MP). When the background removal strategy is optimized to reveal the two-dimensional motion of membrane-associated particles, this technique allows the real-time analysis of both diffusion and molecular mass of unlabeled macromolecules on biological membranes. Here, a detailed protocol to perform and analyze mass-sensitive particle tracking of membrane-associated systems is described. Measurements performed on a commercial mass photometer achieve time resolution in the millisecond regime and, depending on the MP system, a mass detection limit down to 50 kDa. To showcase the potential of MSPT for the in-depth analysis of membrane-catalyzed macromolecule dynamics in general, results obtained for exemplary protein systems such as the native membrane interactor annexin V are presented. SUMMARYThis protocol describes an iSCAT-based image processing and single-particle tracking approach that enables the simultaneous investigation of the molecular mass and the diffusive behavior of macromolecules interacting with lipid membranes. Step-by-step instructions for sample preparation, mass-to-contrast conversion, movie acquisition, and post-processing are provided along-side directions to prevent potential pitfalls.

biophysics↗

bioRxiv2021

Mass-sensitive particle tracking (MSPT) to elucidate the membrane-associated MinDE reactioncycle

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. So far, it has been consensus that their observation with sufficient sensitivity and time resolution requires the introduction of - predominantly fluorescent-labels to the system. However, the recent advent of mass photometry to quantify mass distributions of unlabelled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, we introduce mass-sensitive particle tracking (MSPT), enabling simultaneous label-free tracking and monitoring of molecular masses of single biomolecules diffusing on lipid membranes. We applied this approach to the highly non-linear reaction cycles underlying MinDE protein self-organisation. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. We found that MinD assembles into complexes larger than the commonly postulated dimer, through lateral interactions of membrane-bound complexes and subunit recruitment from solution. Furthermore, the ATPase-activating protein MinE interconnects MinD into high-molecular-weight heteromeric complexes and affects their subunit turnover and concerted membrane release. This study demonstrates the potential of MSPT to enhance our quantitative understanding of both prokaryotic and eukaryotic membrane-associated biological systems.

biophysics↗