Biology subjects

Machta, B.

Publications and source records attributed to Machta, B..

6 recordsLinked to original sources

bioRxiv2024

Prewetting couples membrane and protein phase transitions to greatly enhance coexistence in models and cells.

Biopolymers that separate into condensed and dilute phases in solution also prewet membranes when one or more components couple to membrane lipids. Here we demonstrate that this prewetting transition becomes exquisitely sensitive to lipid composition when membranes have compositions near the boundary of liquid-ordered/liquid-disordered phase coexistence. In simulation and reconstitution, we couple polyelectrolytes to membranes of saturated lipids, unsaturated lipids, and cholesterol, and find that the coexistence of prewet and dry surface phases is dramatically potentiated by proximity to the membrane phase transition. In cells, we employ an optogenetic tool to characterize prewetting both at the plasma membranes (PM) and the endoplasmic reticulum (ER), and find that prewetting is potentiated or inhibited by perturbations of membrane composition. Prewetting can also mediate membrane adhesion, with avidity dependent on membrane composition. This effect is demonstrated in cells through the potentiation or inhibition of ER-PM contact sites. The strong correspondence between results in simulation, reconstitution, and cells demonstrates a new role for membrane lipids in regulating the recruitment and assembly of soluble proteins.

biophysics↗

bioRxiv2024

Pseudo-perplexity in One Fell Swoop for Protein Fitness Estimation

Protein language models trained on the masked language modeling objective learn to predict the identity of hidden amino acid residues within a sequence using the remaining observable sequence as context. They do so by embedding the residues into a high dimensional space that encapsulates the relevant contextual cues. These embedding vectors serve as an informative context-sensitive representation that not only aids with the defined training objective, but can also be used for other tasks by downstream models. We propose a scheme to use the embeddings of an unmasked sequence to estimate the corresponding masked probability vectors for all the positions in a single forward pass through the language model. This One Fell Swoop (OFS) approach allows us to efficiently estimate the pseudo-perplexity of the sequence, a measure of the models uncertainty in its predictions, that can also serve as a fitness estimate. We find that ESM2 OFS pseudo-perplexity performs nearly as well as the true pseudo-perplexity at fitness estimation, and more notably it defines a new state of the art on the ProteinGym Indels benchmark. The strong performance of the fitness measure prompted us to investigate if it could be used to detect the elevated stability reported in reconstructed ancestral sequences. We find that this measure ranks ancestral reconstructions as more fit than extant sequences. Finally, we show that the computational efficiency of the technique allows for the use of Monte Carlo methods that can rapidly explore functional sequence space.

bioinformatics↗

bioRxiv2022

Chemical potential measurements constrain models of cholesterol-phosphatidylcholine interactions

Bilayer membranes composed of cholesterol and phospholipids exhibit diverse forms of non-ideal mixing. In particular, many previous studies document macroscopic liquid-liquid phase separation as well as nanometer-scale heterogeneity in membranes of phosphatidylcholine (PC) lipids and cholesterol. Here, we present experimental measurements of cholesterol chemical potential (c) in binary membranes containing dioleoyl PC (DOPC), 1-palmitoyl-2-oleoyl PC (POPC), or dipalmitoyl PC (DPPC), and in ternary membranes of DOPC and DPPC, adapting a calibrated experimental protocol developed to measure c in cells (Ayuyan and Cohen, Biophys. J. 114:904-918). c is the thermodynamic quantity that dictates the availability of cholesterol to bind other factors, and notably must be equal between coexisting phases of a phase-separated mixture. It is simply related to concentration under conditions of ideal mixing but is found to be far from ideal for the majority of lipid mixtures investigated. Here we perform experimental measurements of c, constraining thermodynamic models of membrane interactions. Our measurements are consistent with models involving cholesterol-phospholipid complexes, but only if complexes are more weakly bound than has been assumed in previous reports. Experimental measurements are also well described by regular solution theory and lattice models with pairwise interactions between components. We find that c can vary by ~1.5 kBT at constant cholesterol mole-fraction implying a more than five-fold change in its availability for binding receptors and other reactions. These findings reinforce that c depends on membrane composition overall, suggesting avenues for cells to alter the availability of cholesterol without varying cholesterol concentration. SIGNIFICANCEThe chemical potential of cholesterol (c) reflects its availability to interact with other molecules. In a complex mixtures this chemical potential can vary dramatically even at fixed cholesterol concentration. In this report, we present measurements of c in bilayer membranes composed of mixtures of cholesterol with one or two phospholipids. We find that c in these mixtures depends strongly on the phospholipids that are present, with activity varying by a factor of more than five at fixed cholesterol concentration. This suggests that the availability of cholesterol in biological membranes could be tuned without altering cholesterol concentration directly, by adjusting the concentration of other lipid or protein components.

biophysics↗

bioRxiv2021

Rapid propagation of membrane tension at a presynaptic terminal

Many cellular activities, such as cell migration1, cell division2,3, signaling4,5, infection6, phagocytosis7 and exo-endocytosis8-11, generate membrane tension gradients that in turn regulate them12. Moreover, membrane flows, which are driven by tension gradients, can limit exo-endocytosis coupling in space and time, as net membrane flow from exocytic to endocytic sites is required to maintain membrane homeostasis13. However, there is controversy over how rapidly plasma membrane flows can relax tension gradients; contrary to the common view12,14,15, recent work showed membrane tension does not equilibrate in several cell types16. Here we show membrane tension can propagate rapidly or slowly, spanning orders of magnitude in speed, depending on cell type. In a neuronal terminal specialized for rapid synaptic vesicle turnover and where exo-endocytosis events occur at distinct loci, membrane tension equilibrates within seconds. By contrast, membrane tension does not propagate in neuroendocrine adrenal chromaffin cells secreting catecholamines. Thus, slow membrane flow and tension equilibration may confine exo- and exocytosis to the same loci17. Stimulation of exocytosis causes a rapid, global decrease in the synaptic terminal membrane tension, which recovers slowly due to endocytosis. Our results demonstrate membrane tension propagates rapidly at neuronal terminals and varies during synaptic activity, likely contributing to exo-endocytosis coupling.

biophysics↗

bioRxiv2021

Surface Densities Prewet a Near-Critical Membrane

Recent work has highlighted roles for thermodynamic phase behavior in diverse cellular processes. Proteins and nucleic acids can phase separate into three-dimensional liquid droplets in the cytoplasm and nucleus and the plasma membrane of animal cells appears tuned close to a two-dimensional liquid-liquid critical point. In some examples, cytoplasmic proteins aggregate at plasma membrane domains, forming structures such as the post-synaptic density and diverse signaling clusters. Here we examine the physics of these surface densities, employing minimal simulations of co-acervating polymers coupled to an Ising membrane surface in conjunction with a complementary Landau theory. We argue that these surface densities are a novel phase reminiscent of pre-wetting, in which a molecularly thin three-dimensional liquid forms on a usually solid surface. However, in surface densities the solid surface is replaced by a membrane with an independent propensity to phase separate. We show that proximity to criticality in the membrane dramatically increases the parameter regime in which a pre-wetting-like transition occurs, leading to a broad region where coexisting surface phases can form even when a bulk phase is unstable. Our simulations naturally exhibit three surface phase coexistence even though both the membrane and the polymer bulk can only display two phase coexistence on their own. We argue that the physics of these surface densities enables diverse functions seen in Eukaryotic cells.

biophysics↗

bioRxiv2020

The Bacterial Cytoskeleton Spatially Confines Functional Membrane Microdomains

Cell membranes laterally segregate into microdomains enriched in certain lipids and scaffold proteins. Membrane microdomains modulate protein-protein interactions and are essential for cell polarity, signaling and membrane trafficking. How cells organize their membrane microdomains, and the physiological importance of these microdomains, is unknown. In eukaryotes, the cortical actin cytoskeleton is proposed to act like a fence, constraining the dynamics of membrane microdomains. Like their eukaryotic counterparts, bacterial cells have functional membrane microdomains (FMMs) that act as platforms for the efficient oligomerization of protein complexes. In this work, we used the model organism Bacillus subtilis to demonstrate that FMM organization and movement depend primarily on the interaction of FMM scaffold proteins with the domains protein cargo, rather than with domain lipids. Additionally, the MreB actin-like cytoskeletal network that underlies the bacterial membrane was found to frame areas of the membrane in which FMM mobility is concentrated. Variations in membrane fluidity did not affect FMM mobility whereas alterations in cell wall organization affected FMM mobility substantially. Interference with MreB organization alleviates FMM spatial confinement whereas, by contrast, inhibition of cell wall synthesis strengthens FMM confinement. The restriction of FMM lateral mobility by the submembranous actin-like cytoskeleton or the extracellular wall cytoskeleton appears to be a conserved mechanism in prokaryotic and eukaryotic cells for localizing functional protein complexes in specific membrane regions, thus contributing to the organization of cellular processes.

microbiology↗