Biology subjects

Franzmann, T. M.

Publications and source records attributed to Franzmann, T. M..

8 recordsLinked to original sources

bioRxiv2024

Biomolecular condensates sustain pH gradients at equilibrium driven by charge neutralisation

Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here, we show in contrast that condensed biomolecular systems sustain significant pH gradients without any external energy input. By studying individual condensates on the micron scale using a microdroplet platform, we reveal dense phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that by doing so protein condensates can drive substantial alkaline and acidic gradients which are compositionally tuneable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterisation of human proteomic condensate networks, we further highlight potential wide ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a unique regulatory mechanism to modulate biochemical activity in living systems.

biophysics↗

bioRxiv2024

Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

cell biology↗

bioRxiv2023

Single-molecule digital sizing of proteins in solution

Proteins constitute the molecular machinery of life and exert their biological function by interacting with other proteins, as well as by assembling into biomolecular complexes and higher order structures. Characterizing the sizes, interactions, and assembly states of proteins is thus key for understanding the normal functional behavior of proteins and for elucidating aberrant processes and interactions that can lead to dysfunction and disease. However, the physical characterization of proteins has remained a challenging problem due to the inherent compositional heterogeneity of protein mixtures as well as the polydisperse nature of protein complexes. Here, we address this challenge by demonstrating measurements of molecular diffusivity of single proteins and protein assemblies in microchannels using single-molecule fluorescence detection. The approach, termed single-molecule microfluidic diffusional sizing (smMDS), allows individual molecules to be counted directly, that is, in a digital manner, to enable calibration-free single-molecule diffusional-sizing-based monitoring of protein hydrodynamic radii even within heterogenous multicomponent mixtures. Applying smMDS to a variety of protein systems, we show that the high sensitivity provided by smMDS enables ultrasensitive sizing of proteins down to the femtomolar concentration range. We further demonstrate the applicability of the approach towards affinity profiling of protein interactions at the single-molecule level and illustrate the potential of smMDS in resolving different assembly states of high- and low-molecular weight protein oligomers. Furthermore, we highlight the digital nature of the detection process by sizing multiple protein species within complex aggregation mixtures. Finally, we apply the approach to characterize nanoscale clusters of a phase separating protein system. Taken together, smMDS constitutes a versatile approach for digital, in-solution characterization of the sizes, interactions, and assembly states of proteins. We anticipate that smMDS will facilitate the discovery of new biomolecular mechanisms of proteins and will find broad applicability in the analysis of protein complexes in the biological, biophysical, and biomedical sciences, and beyond.

biophysics↗

bioRxiv2023

Condensate partitioning governs the mechanism of action of FUS phase separation modulators

Biomolecular phase separation plays a pivotal role in governing critical biological functions and arises from the collective interactions of large numbers of molecules. Characterising the underlying collective interactions of phase separation, however, has proven to be challenging with currently available tools. Here, we propose a general and easily accessible strategy to quantify collective interactions in biomolecular phase separation with respect to composition and energetics. By measuring the dilute phase concentration of one species only, we determine tie line gradients and free energy dominance as dedicated descriptors of collective interactions. We apply this strategy to dissect the role of salts and small molecules on phase separation of the protein fused in sarcoma (FUS). We discover that monovalent salts can display both exclusion from or preferential partitioning into condensates to either counteract charge screening or enhance non-ionic interactions. Moreover, we show that the common hydrophobic interaction disruptor 1,6-hexanediol inhibits FUS phase separation by acting as a solvation agent capable of expanding the protein polypeptide chain. Taken together, our work presents a widely applicable strategy that enables quantification of collective interactions and provides unique insights into the underlying mechanisms of condensate formation and modulation.

biophysics↗

bioRxiv2022

A conserved and tunable mechanism for the temperature-controlled condensation of the translation factor Ded1p

Heat shock promotes the assembly of translation factors into condensates to facilitate the production of stress-protective proteins. How translation factors detect heat and assemble into condensates is not well understood. Here, we investigate heat-induced condensate assembly by the translation factor Ded1p from five different fungi, including Ded1p from Saccharomyces cerevisiae. Using targeted mutagenesis and in vitro reconstitution biochemistry, we find that heat-induced Ded1p assembly is driven by a conformational rearrangement of the folded helicase domain. This rearrangement determines the assembly temperature and the assembly of Ded1p into nanometer-sized particles, while the flanking intrinsically disordered regions engage in intermolecular interactions to promote assembly into micron-sized condensates. Using protein engineering, we identify six amino acid substitutions that determine most of the thermostability of a thermophilic Ded1p ortholog, thereby providing a molecular understanding underlying the adaptation of the Ded1p assembly temperature to the specific growth temperature of the species. We conclude that heat-induced assembly of Ded1p into translation factor condensates is regulated by a complex interplay of the structured domain and intrinsically disordered regions which is subject to evolutionary tuning.

biochemistry↗

bioRxiv2022

Phase separating RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation, especially at low endogenous concentrations found in cells, are thought to follow the tenets of classical nucleation theory describing a sharp transition between a dense phase and a dilute phase characterized by dispersed monomers. Here, we used in vitro biophysical studies to study subsaturated solutions of phase separating RNA binding proteins with intrinsically disordered prion like domains (PLDs) and RNA binding domains (RBDs). Surprisingly, we find that subsaturated solutions are characterized by heterogeneous distributions of clusters comprising tens to hundreds of molecules. These clusters also include low abundance mesoscale species that are several hundreds of nanometers in diameter. Our results show that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. Interestingly, however, cluster formation and phase separation can be decoupled from one another using solutes that impact the solubilities of phase separating proteins. They can also be decoupled by specific types of mutations. Overall, our findings implicate the presence of distinct, sequence-specific energy scales that contribute to the overall phase behaviors of RNA binding proteins. We discuss our findings in the context of theories of associative polymers. Significance StatementMembraneless biomolecular condensates are molecular communities with distinct compositional preferences and functions. Considerable attention has focused on phase separation as the process that gives rise to condensates. Here, we show that subsaturated solutions of RNA binding proteins form heterogeneous distributions of clusters in subsaturated solutions. The formation of clusters in subsaturated solutions and condensates in supersaturated solution are coupled through sequence-specific interactions. Given the low endogenous concentrations of phase separating proteins, our findings suggest that clusters in subsaturated conditions might be of functional relevance in cells.

biophysics↗

bioRxiv2018

Filament formation by the translation factor eIF2B regulates protein synthesis in starved cells

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. The mechanism by which starved cells regulate the process of protein synthesis is largely unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants of eIF2B suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through protein assembly is a widespread mechanism that allows cells to adapt to fluctuating environments.

cell biology↗

bioRxiv2018

Intracellular mass density increase is accompanying but not sufficient for stiffening and growth arrest of yeast cells

Many organisms, including yeast cells, bacteria, nematodes and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography and atomic force microscopy. We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

biophysics↗