Biology subjects

Mitra, G.

Publications and source records attributed to Mitra, G..

6 recordsLinked to original sources

bioRxiv2026

Building synthetic cell-cell synapses using engineered scaffold protein condensates

Synaptic assemblies that form at cell-cell junctions are often organized by multivalent scaffold proteins with condensate forming properties. An emerging hypothesis is that these scaffolds provide a thresholding mechanism whereby small increases in the density of cell-cell recognition receptors can trigger all-or-none assembly selectively at the correct partner cell interface. We test this hypothesis by engineering synthetic scaffolded synapses. Coarse-grained simulations suggest that properly-tuned scaffold interactions can yield a sharp condensation threshold. Guided by these simulations, we designed modular scaffold proteins consisting of PDZ domains and intrinsically disordered regions (IDRs) of different molecular grammars. Within a specific region of parameter space, controlled by valency, receptor binding affinity and IDR-driven condensation, the synthetic scaffold proteins exhibit switch-like condensation behaviors, where co-condensates of receptor and scaffold selectively form only at interfaces with the proper partner cell. Notably, orthogonal receptor-scaffold pairs enable a single receiver cell to form multiple distinct interfaces, each associated with a different partner cell. We demonstrate that these synthetic synapse assemblies can recruit different client proteins to enhance intercellular signaling, orient cell division, or sculpt complex synaptic morphologies. Our results illustrate how the ultrasensitive condensation properties of modular scaffold proteins can be harnessed to create novel and highly specific cell-cell interfaces.

synthetic biology↗

bioRxiv2025

Distinguishing near- versus off-critical phase behaviors of intrinsically disordered proteins

Intrinsically disordered prion-like low complexity domains (PLCDs) drive phase transitions that underlie the biogenesis of different biomolecular condensates. The mapping of critical points is essential for generating quantitative assessments of driving forces and for distinguishing phase behaviors in the critical versus off-critical regimes. Computations play an important role in connecting the molecular-scale interactions to mesoscale phase behaviors of PLCDs and other intrinsically disordered proteins. We report results from accurate mapping of the critical regime for an archetypal PLCD. This is achieved by combining large-scale simulations with computations of Binder cumulants and the use of rigorous finite-size scaling approaches. The computed binodal, defined by knowledge of the critical point and intersection of the left arm of the binodal by the overlap and percolation lines, can be demarcated into three distinct regimes. Regime I is farthest from the critical point, with the coexisting dilute phase being akin to a gas of dispersed polymers. Regime II lies above the intersection of the overlap line and the dilute arm of the binodal. Here, coexisting dilute phases are characterized by heterogeneous cluster-size distributions with heavy tails. In Regimes I and II, dense phases are confined percolated networks. Regime III is closest to the critical point. Here, the dense phase becomes unconfined and the percolated network swells to become system-spanning. Thus, Regime III comprises two interconnected, system-spanning networks. In addition to accurately mapping the critical point, we also evaluated methods for identifying the theta temperature. We find that conventional scaling approaches lead to erroneously low estimates of the theta temperature. Instead, accurate estimation of the theta temperature requires direct calculation of the temperature dependence of the two-body interaction coefficient. We discuss the broader implications of these findings for inferring solvent quality from scaling analyses.

biophysics↗

bioRxiv2025

Computational rheometry of viscoelastic networks: From random graphs to biomolecular condensates

Multivalent biomacromolecules including multi-domain and intrinsically disordered proteins form biomolecular condensates via reversible phase transitions. Condensates are viscoelastic materials that display composition-specific rheological properties and responses to mechanical forces. Graph-based descriptions of microstructures can be combined with computational rheometry to model the outcomes of passive and active mechanical measurements. We consider two types of network models for microstructures. In the Jeffreys model, each edge in the network is a Jeffreys element. In the Stokes-Maxwell model, each edge is a Maxwell element that is embedded in an incompressible viscous fluid that can undergo Stokes flow. We describe results from comparative assessments of the two models for individual elements, ordered lattices, random geometric graphs, structured graphs, and graphs for condensates that are extracted from coarse-grained simulations of disordered proteins. Results from deformation and relaxation tests and flow field analysis reveal how distinct length and time scales contribute to the responses of different types of networks. No single test provides definitive assessments of the connections between material properties and microstructures. Instead, a range of active and passive rheometric tests are essential for distinguishing the responses of different types of networks. Our work establishes computational rheometry as a framework for bridging disparate length and timescales to assess how molecular-scale interactions and dynamics give rise to viscoelastic responses on the mesoscale.

biophysics↗

bioRxiv2023

Mesoscale molecular assembly is favored by the active, crowded cytoplasm

The mesoscale organization of molecules into membraneless biomolecular condensates is emerging as a key mechanism of rapid spatiotemporal control in cells1. Principles of biomolecular condensation have been revealed through in vitro reconstitution2. However, intracellular environments are much more complex than test-tube environments: They are viscoelastic, highly crowded at the mesoscale, and are far from thermodynamic equilibrium due to the constant action of energy-consuming processes3. We developed synDrops, a synthetic phase separation system, to study how the cellular environment affects condensate formation. Three key features enable physical analysis: synDrops are inducible, bioorthogonal, and have well-defined geometry. This design allows kinetic analysis of synDrop assembly and facilitates computational simulation of the process. We compared experiments and simulations to determine that macromolecular crowding promotes condensate nucleation but inhibits droplet growth through coalescence. ATP-dependent cellular activities help overcome the frustration of growth. In particular, actomyosin dynamics potentiate droplet growth by reducing confinement and elasticity in the mammalian cytoplasm, thereby enabling synDrop coarsening. Our results demonstrate that mesoscale molecular assembly is favored by the combined effects of crowding and active matter in the cytoplasm. These results move toward a better predictive understanding of condensate formation in vivo.

biophysics↗

bioRxiv2023

A deep catalog of protein-coding variation in 985,830 individuals

Coding variants that have significant impact on function can provide insights into the biology of a gene but are typically rare in the population. Identifying and ascertaining the frequency of such rare variants requires very large sample sizes. Here, we present the largest catalog of human protein-coding variation to date, derived from exome sequencing of 985,830 individuals of diverse ancestry to serve as a rich resource for studying rare coding variants. Individuals of African, Admixed American, East Asian, Middle Eastern, and South Asian ancestry account for 20% of this Exome dataset. Our catalog of variants includes approximately 10.5 million missense (54% novel) and 1.1 million predicted loss-of-function (pLOF) variants (65% novel, 53% observed only once). We identified individuals with rare homozygous pLOF variants in 4,874 genes, and for 1,838 of these this work is the first to document at least one pLOF homozygote. Additional insights from the RGC-ME dataset include 1) improved estimates of selection against heterozygous loss-of-function and identification of 3,459 genes intolerant to loss-of-function, 83 of which were previously assessed as tolerant to loss-of-function and 1,241 that lack disease annotations; 2) identification of regions depleted of missense variation in 457 genes that are tolerant to loss-of-function; 3) functional interpretation for 10,708 variants of unknown or conflicting significance reported in ClinVar as cryptic splice sites using splicing score thresholds based on empirical variant deleteriousness scores derived from RGC-ME; and 4) an observation that approximately 3% of sequenced individuals carry a clinically actionable genetic variant in the ACMG SF 3.1 list of genes. We make this important resource of coding variation available to the public through a variant allele frequency browser. We anticipate that this report and the RGC-ME dataset will serve as a valuable reference for understanding rare coding variation and help advance precision medicine efforts.

genetics↗

bioRxiv2021

Free spermidine evokes superoxide radicals that manifest toxicity

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Besides, a specific mechanism of SpeG function conferring pathogenic fitness to Staphylococcus aureus USA300 strain is unknown. Here, we provide evidence that although spermidine mitigates oxidative stress by lowering hydroxyl radical and hydrogen peroxide levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the E. coli {Delta}speG strain as well as naturally SpeG-deficient S. aureus RN4220 strain. However, wild-type E. coli and S. aureus USA300 with a horizontally-acquired speG gene tolerate applied exogenous spermidine stress. Furthermore, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Superoxide radicals thus generated, further affects redox balance and iron homeostasis. Since iron acquisition and metabolism in the host tissues is a challenging task for S. aureus, the current findings helped us explain that the evolutionary gain of SpeG function by S. aureus USA300 strain allows it to neutralize exogenous spermidine- and spermine-mediated toxicity towards iron metabolism inside the host tissues.

biochemistry↗