Percolation transition determines protein size limit for passive transport through the nuclear pore complex

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

Potential utilization of glycerol as crosslinker in starch films for application in Regenerative Dentistry

Periodontal disease results in damage to dental insertion apparatus. Regenerative procedures are proposed to replace lost structures in the context of guided tissue regeneration (GTR), guided bone regeneration (GBR) techniques and frequently associate bone substitutes with physical barriers aiming at greater longevity and improvement of aesthetic pattern. This study evaluates the possibility of using glycerol as a starch films modifying agent, acting as a cross-linking agent, without compromising its plasticizing effect. Biodegradable cassava starch films were prepared incorporating glycerol at concentrations of 0, 15, 20, 30 and 40% aiming application at dental regenerative procedures. The characterization of films by microscopy (SEM), thermal analysis (DSC), spectroscopic (UV / Vis., FTIR, XRD), mechanical (Traction), and analysis of protein swelling, degradation and diffusion and physiological temperature) showed that the incorporation of glycerol in up to 20% attributed to the films a plasticizer character and in higher concentrations, conferred a greater interaction of glycerol (crosslinking) with the starch chains and a degradation time that allows the physical barrier in RTG and ROG. The films presented mechanical resistance, malleability and permissiveness to protein diffusion in the in vitro assays, which meet the current attributes that guide the use of these resources in biomaterials.

Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

Weak correlation between fluctuations in protein diffusion inside bacteria

A weak correlation between the diffusion-exponent fluctuations and the temperature fluctuations is discussed based on recent experimental observations for protein diffusion inside bacteria. Its existence is shown to be essential for describing the statistical properties of the fluctuations. It is also quantified how largely the fluctuations are modulated by the weak correlation.

Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of β-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

Utility of TPP-manufactured biophysical restrictions to probe multiscale cellular dynamics

Biophysical restrictions regulate protein diffusion, nucleus deformation, and cell migration, which are all universal and important processes for cells to perform their biological functions. However, current technologies addressing these multiscale questions are extremely limited. Herein, through two-photon polymerization (TPP), we present the precise, low-cost, and multiscale microstructures (micro-fences) as a versatile investigating platform. With nanometer-scale printing resolution and multiscale scanning capacity, TPP is capable of generating micro-fences with sizes of 0.5–1000 μm. These micro-fences are utilized as biophysical restrictions to determine the fluidity of supported lipid bilayers (SLB), to investigate the restricted diffusion of Src family kinase protein Lck on SLB, and also to reveal the mechanical bending of cell nucleus and T cell climbing ability. Taken together, the proposed versatile and low-cost micro-fences have great potential in probing the restricted dynamics of molecules, organelles, and cells to understand the basics of physical biology. Graphic abstract

Cytoplasmic organization promotes protein diffusion

The cytoplasm is highly organized. However, the extent to which this organization influences the dynamics of cytoplasmic proteins is not well understood. Here, we used Xenopus laevis egg extracts as a model system to study diffusion dynamics in organized versus disorganized cytoplasm. Such extracts are initially homogenized and disorganized, and will self-organize into cell-like units over the course of 20-60 min. Using fluorescence correlation spectroscopy, we observed that self-organization is accompanied by changes in protein diffusivity; as the extract organizes, proteins diffuse about twice as quickly over a length scale of a few hundred nanometers. Even though the ordered cytoplasm contained organelles and cytoskeletal elements that might be expected to interfere with diffusion, after self-organization took place, the speed of protein diffusion approached that of organelle-depleted cytosolic extracts. This finding suggests that subcellular organization optimizes protein diffusivity. The effect of organization on diffusion varies with molecular size, with the effects being largest for protein-sized molecules. These results show that cytoplasmic organization promotes the efficient diffusion of protein molecules in a densely packed environment.

Rapid Ensemble Measurement of Protein Diffusion, Probe Blinking and Photobleaching Dynamics in the Complex Cellular Space

We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.