Biology subjects

Fritsch, A. W.

Publications and source records attributed to Fritsch, A. W..

8 recordsLinked to original sources

bioRxiv2024

inPhase - A simple, accurate and fast approach to determine phase diagrams of protein condensates

Protein phase separation has become a widely studied phenomena in biology with implications in cell metabolism and disease. The study of phase separating proteins often relies on the precise determination of their phase diagrams. These phase diagrams give information on the protein concentration required for condensate formation and the respective concentration inside the condensate at defined external conditions (temperature, salt, pH). However, it has so far often proven difficult to accurately measure phase diagrams. Here, we report a method that is based on mass and volume conservation and defined reaction volumes, which we call inPhase. We can use this method to determine accurate values for both dilute and condensed branch protein concentrations. With this information we can produce accurate phase diagrams. We compare our method to the widely used quantitative fluorescence approach and find that it underestimates the partition factor into condensates at least two-fold for FUS and PGL-3. The accessibility of our method opens the possibility for the thermodynamic assessment of entire protein families, generating sufficient quantitative data for testing theory, and for the screening of drugs in pharmaceutical research.

biochemistry↗

bioRxiv2024

Actin polymerization counteracts prewetting of N-WASP on supported lipid bilayers

Cortical condensates, transient punctate-like structures rich in actin and the actin nucleation pathway member N-WASP, form during activation of the actin cortex in the C. elegans oocyte. Their emergence and spontaneous dissolution is linked to a phase separation process driven by chemical kinetics. However, the physical process that drives the onset of cortical condensate formation near membranes remains unexplored. Here, using a reconstituted phase separation assay of cortical condensate proteins, we demonstrate that the key component, N-WASP, can collectively undergo surface condensation on supported lipid bilayers via a prewetting transition. Actin partitions into the condensates, where it polymerizes and counteracts the N-WASP prewetting transition. Taken together, the dynamics of condensate-assisted cortex formation appear to be controlled by a balance between surface-assisted condensate formation and polymer-driven condensate dissolution. This opens new perspectives for understanding how the formation of complex intracellular structures is affected and controlled by phase separation.

biophysics↗

bioRxiv2022

Predicting disordered regions driving phase separationof proteins under variable salt concentration

We investigate intrinsically disordered regions (IDRs) of phase separating proteins regarding their impact on liquid-liquid phase separation (LLPS) of the full protein. Our theoretical approach uses a mean-field theory that accounts for sequence-dependent electrostatic interactions via a random-phase approximation (RPA) and in addition allows for variable salt concentration for the condensed and dilute protein phases. The numerical solution of the complete phase diagrams together with the tie lines that we derive for this model system leaves two parameters to be determined by fitting experimental data on concentrations of all species involved in the system. For our comparisons, we focus on two proteins, PGL-3 and FUS, known to undergo LLPS. For PGL-3 we predict that its long IDR near the C-terminus promotes LLPS, which we validate through direct comparison with in vitro experimental results under the same physiological conditions. For the structurally more complex protein FUS the role of the low complexity (LC) domain in LLPS has been intensively studied. Apart from the LC domain we here investigate theoretically two IDRs, one near the N-terminus and another near the C-terminus. Our theoretical analysis of these domains predict that the IDR at the N-terminus (aa 1-285) is the main driver of LLPS of FUS by comparison to in vitro experiments of the full length protein under the same physiological temperature and salt conditions. SIGNIFICANCEIntrinsically disordered proteins are drivers of cellular liquid-liquid phase separation. However, it remains a challenge to directly predict the phase behaviour of a protein based on its primary sequence, and under physiological conditions. We present a random-phase approximation that allows for variable salt concentration and thus accounts for salt partitioning. We use this to link the sequence of the disordered regions with the behaviour of the complete protein through direct comparisons to in vitro phase-separation assays. In particular, for FUS we determine the exact region responsible for LLPS, weighting in a long-standing debate.

biophysics↗

bioRxiv2021

Local thermodynamics governs the formation and dissolution of protein condensates in living cells

Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm C. elegans which play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams which are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intra-cellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the non-equilibrium nature of the cytoplasm is manifested on larger scales. SIGNIFICANCE STATEMENTLiving cells rely on a continuous flux of energy to spatially organize biochemical processes. It remained unclear whether cells can achieve this spatial organization via thermodynamic principles. Here, we report the striking behavior of a cold-blooded organism that reacts to environmental temperature changes similar to a thermodynamic system at local equilibrium. Our key finding is that protein-rich droplets form and dissolve reversibly with temperature due to changes in the organism?s entropy. We show that the organism uses a specific molecule to extend droplet stability to the natural temperature range of the organisms habitat. Due to the relevance of such protein droplets for the organism?s fertility, our works shed light on how molecular components could facilitate biological functions via thermodynamic principles.

biophysics↗

bioRxiv2020

Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

biophysics↗

bioRxiv2020

A hydraulic instability drives the cell death decision in the nematode germline

Oocytes are large and resourceful. During oogenesis some germ cells grow, typically at the expense of others that undergo apoptosis. How germ cells are selected to live or die out of a homogeneous population remains unclear. Here we show that this cell fate decision in C. elegans is mechanical and related to tissue hydraulics. Germ cells become inflated when the pressure inside them is lower than in the common cytoplasmic pool. This condition triggers a hydraulic instability which amplifies volume differences and causes some germ cells to grow and others to shrink. Shrinking germ cells are extruded and die, as we demonstrate by reducing germ cell volumes via thermoviscous pumping. Together, this reveals a robust mechanism of mechanochemical cell fate decision making in the germline.

developmental biology↗

bioRxiv2019

Material aging causes centrosome weakening and disassembly during mitotic exit

Centrosomes must resist microtubule-mediated forces for mitotic chromosome segregation. During mitotic exit, however, centrosomes are deformed and fractured by those same forces, which is a key step in centrosome disassembly. How the functional material properties of centrosomes change throughout the cell cycle, and how they are molecularly tuned remain unknown. Here, we used optically-induced flow perturbations to determine the molecular basis of centrosome strength and ductility in C. elegans embryos. We found that both properties declined sharply at anaphase onset, long before natural disassembly. This mechanical transition required PP2A phosphatase and correlated with inactivation of PLK-1 (Polo Kinase) and SPD-2 (Cep192). In vitro, PLK-1 and SPD-2 directly protected centrosome scaffolds from force-induced disassembly. Our results suggest that, prior to anaphase, PLK-1 and SPD-2 confer strength and ductility to the centrosome scaffold so that it can resist microtubule-pulling forces. In anaphase, centrosomes lose PLK-1 and SPD-2 and transition to a weak, brittle state that enables force-mediated centrosome disassembly.

cell biology↗

bioRxiv2018

Active gelation breaks time-reversal-symmetry of mitotic chromosome mechanics

In cell division, mitosis is the phase in which duplicated sets of chromosomes are mechanically aligned to form the metaphase plate before being segregated in two daughter cells. Irreversibility is a hallmark of this process, despite the fundamental laws of Newtonian mechanics being time symmetric.\n\nHere we show experimentally that mitotic chromosomes receive the arrow of time by time-reversal-symmetry breaking of the underlying mechanics in prometaphase. By optically inducing hydrodynamic flows within prophase nuclei, we find that duplicated chromatid pairs initially form a fluid suspension in the nucleoplasm: although showing little motion on their own, condensed chromosomes are free to move through the nucleus in a time-reversible manner. Actively probing chromosome mobility further in time, we find that this viscous suspension of chromatin transitions into a gel after nuclear breakdown. This gel state, in which chromosomes cannot be moved by flows, persists even when chromosomes start moving to form the metaphase plate. Complemented by minimal reconstitution experiments, our active intra-nuclear micro-rheology reveals time-reversal-symmetry breaking of chromosome mechanics to be caused by the transition from a purely fluid suspension into an active gel.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC=\"FIGDIR/small/296566_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (36K):\norg.highwire.dtl.DTLVardef@15b00edorg.highwire.dtl.DTLVardef@199eb2dorg.highwire.dtl.DTLVardef@1f0550org.highwire.dtl.DTLVardef@95cb5a_HPS_FORMAT_FIGEXP M_FIG C_FIG One sentence summaryFlows induced in living cell nuclei reveal the rheological changes that bring chromosomes under mechanical control during mitosis.

biophysics↗