Biophysical journal最新文献

Ca²⁺ blinks measure the exit of Ca²⁺ from the junctional sarcoplasmic reticulum (JSR) in a cardiac myocyte during a Ca²⁺ spark. Here, the relationship between experimental blink fluorescence measurements and the [Ca²⁺] in the JSR is explored using long 3D simulations of diastolic Ca²⁺ release. For a fast intra-SR Ca²⁺-activated fluorophore such as Fluo-5N, we show that a simple mathematical formula relates the two for an ideal blink (i.e., when fluorescence signals come only from the JSR). The formula shows that normalized JSR [Ca²⁺] is much lower than the normalized fluorescence and that JSR Ca²⁺ depletes ∼40-50% more than previously inferred from blink fluorescence measurements. In addition, we show that stray fluorescence signals (e.g., from other parts of the sarcoplasmic reticulum network) can mask even deeper Ca²⁺ depletion. Overall, the simulations show that strong JSR Ca²⁺ depletion such as that seen in many simulations is consistent with the relatively moderate fluorescence changes seen in experiments.

Experimentally monitoring the kinematics of branching network growth is a tricky task, given the complexity of the structures generated in three dimensions. One option is to drive the network in such a way as to obtain two-dimensional growth, enabling a collection of independent images to be obtained. The density of the network generates ambiguous structures, such as overlaps and meetings, which hinder the reconstruction of the chronology of connections. In this paper, we propose a general method for global network reconstruction. Each network connection is defined by a unique label, enabling it to be tracked in time and space. In this work, we distinguish between lateral and apical branches on the one hand, and extremities on the other. Finally, we reconstruct the network after identifying and eliminating overlaps. This method is then applied to the model filamentous fungus Podospora anserina to reconstruct its growing thallus. We derive criteria for differentiating between apical and lateral branches. We find that the outer ring is favorably composed of apical branches, while densification within the network comes from lateral branches. From this, we derive the specific dynamics of each of the two types. Finally, in the absence of any latency phase during growth initiation, we can reconstruct a time based on the equality of apical and lateral branching collections. This makes it possible to directly compare the growth dynamics of different thalli.

During the active transport of cellular cargo, forces generated by cargo-associated molecular motors propel the cargo along cytoskeletal tracks. However, the forces impact not only the cargo, but also the underlying cytoskeletal filaments. To better understand the interplay between cargo transport and the organization of cytoskeletal filaments, we employ coarse-grained computer simulations to study actin filaments interacting with cargo-anchored myosin motors in a confined domain. We show that cargo transport can lead to the segregation of filaments into domains of preferred filament polarity separated by clusters of aggregated cargoes. The formation of polarity-sorted filament domains is enhanced by larger numbers of cargoes, more motors per cargo, and longer filaments. Analysis of individual trajectories reveals dynamic and heterogeneous behavior, including locally stable aggregates of cargoes that undergo rapid coalescence into larger clusters when sufficiently close. Our results provide insight into the impact of motor-driven organelle transport on actin filaments, which is relevant both in cells and in synthetic environments.

Microtubules (MTs) constitute the largest components of the eukaryotic cytoskeleton and play crucial roles in various cellular processes, including mitosis and intracellular transport. The property allowing MTs to cater to such diverse roles is attributed to dynamic instability, which is coupled to the hydrolysis of guanosine-5'-triphosphate (GTP) to guanosine-5'-diphosphate (GDP) within the β-tubulin monomers. Understanding the dynamics and structural features of both GDP- and GTP-complexed MT tips, especially at an all-atom level, remains challenging for both experimental and computational methods because of their dynamic nature and the prohibitive computational demands of simulating large, many-protein systems. This study employs the "equation-free" multiscale computational method to accelerate the relaxation of all-atom simulations of MT tips toward their putative equilibrium conformation. Using large MT lattice systems (14 protofilaments × 8 heterodimers) comprising ∼21-38 million atoms, we applied this multiscale approach to leapfrog through time and nearly double the computational efficiency in realizing relaxed all-atom conformations of GDP- and GTP-complexed MT tips. Commencing from an initial 4 μs unbiased all-atom simulation, we interleave coarse-projective equation-free jumps with short bursts of all-atom molecular dynamics simulation to realize an additional effective simulation time of 1.875 μs. Our 5.875 μs of effective simulation trajectories for each system expose the subtle yet essential differences in the structures of MT tips as a function of whether β-tubulin monomer is complexed with GDP or GTP, as well as the lateral interactions within the MT tip, offering a refined understanding of features underlying MT dynamic instability. The approach presents a robust and generalizable framework for future explorations of large biomolecular systems at atomic resolution.

In this work we present a minimal structure-based model of protein diffusional search along local DNA amid protein binding and unbinding events on the DNA, taking into account protein-DNA electrostatic interactions and hydrogen-bonding (HB) interactions or contacts at the interface. We accordingly constructed the protein diffusion-association/dissociation free energy surface and mapped it to 1D as the protein slides along DNA, maintaining the protein-DNA interfacial HB contacts that presumably dictate the DNA sequence information detection. Upon DNA helical path correction, the protein 1D diffusion rates along local DNA can be physically derived to be consistent with experimental measurements. We also show that the sequence-dependent protein sliding or stepping patterns along DNA are regulated by collective interfacial HB dynamics, which also determines the ruggedness of the protein diffusion free energy landscape on the local DNA. In comparison, protein association or binding with DNA are generically dictated by the protein-DNA electrostatic interactions, with an interaction zone of nanometers around DNA. Extra degrees of freedom (DOFs) of the protein such as rotations and conformational fluctuations can be well accommodated within the protein-DNA electrostatic interaction zone. As such we demonstrate that the protein binding or association free energy profiling along DNA smoothens over the 1D diffusion free energy landscape, which leads to population variations for an order of magnitude upon a marginal free energetic smoothening around the specific or consensus sites. We further show that the protein unbinding or dissociation from a comparatively high-binding affinity DNA site is dominated by lateral diffusion to the flanking low-affinity sites. The results predict that experimental characterizations on the relative protein-DNA binding affinities or population profiling on the DNA are systematically and physically impacted by the extra DOFs of protein motions aside from 1D translation or helical tracking, as well as from flanking DNA sequences due to protein 1D diffusion and nonspecific binding/unbinding.

Dense-core vesicles (DCVs) are found in various types of cells, such as neurons, pancreatic β- cells, and chromaffin cells. These vesicles release transmitters, peptides, and hormones to regulate diverse functions, such as the stress response, immune response, behavior, and blood glucose levels. In traditional electron microscopy after chemical fixation, it is often reported that the dense cores occupy a portion of the vesicle toward the center and are surrounded by a clear halo. With electron microscopy after cryofixation in adrenal chromaffin cells, we report here that we did not observe halos, but dense cores filling up the entire vesicles suggesting that halos are likely the product of chemical fixation. More importantly, we observed that a fraction of DCVs contained 36-168 nm clear-core vesicles. A similar fraction of DCVs labeled with fluorescent false neurotransmitter FFN 511 or the dense-core matrix protein chromogranin A (CGA) were colocalized with fluorescently labeled or endogenous CD63 or ALIX, the membrane or lumen marker of ∼40-160 nm exosomes. These results suggest that DCVs contain exosomes. Since exosomes are generally thought to reside within multivesicular bodies in the cytosol and are released to the extracellular space to mediate diverse cell-to-cell communications, our findings suggest that DCV fusion from many cell types is a new source for releasing exosomes to mediate intercellular communications. Given that DCV fusion mediates many physiological functions, such as stress responses, immune responses, behavior regulation, and blood glucose regulation, exosome release from DCV fusion might contribute to mediating these important functions.

Neuropeptides are inter-cellular signaling molecules occurring throughout animals. Most neuropeptides bind and activate G-protein-coupled receptors, but some also activate ionotropic receptors (or "ligand-gated ion channels"). This is exemplified by the tetra-peptide H-Phe-Met-Arg-Phe-NH₂ (FMRFamide (FMRFa)), which activates mollusk and annelid FMRFa-gated sodium channels (FaNaCs) from the trimeric degenerin/epithelial sodium channel superfamily. Here, we explored the structure-activity relationships determining FMRFa potency at mollusk and annelid FaNaCs in the light of emerging structural data, using synthetic neuropeptide analogs, heterologous expression, and two-electrode voltage clamp. Substitutions of the FMRFa N-terminal phenylalanine residue (F1) and methionine residue (M2) decreased or abolished FMRFa potency at mollusk Aplysia kurodai FaNaC but had little effect at annelid Malacoceros fuliginosus FaNaC1. Conversely, F4 substitutions had little effect on FMRFa potency at A. kurodai FaNaC but either abolished, strongly decreased, or slightly increased potency at M. fuliginosus FaNaC1. Accordingly, recently published high-resolution FaNaC structures show that F1 and F4 residues orient deep into the neuropeptide-binding pockets of A. kurodai FaNaC and M. fuliginosus FaNaC1, respectively. We also use noncanonical amino acid substitutions in A. kurodai FaNaC to describe the physico-chemical determinants of FMRFa F1 binding to A. kurodai FaNaC aromatic side chains. Our results show that the "deeper" of the two FMRFa phenylalanine residues in the binding pocket is crucial for FMRFa potency despite the peptide orienting very differently into the homologous binding sites of two closely related receptors.

Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA basepairs must be broken for intercalation, have revealed means of optimizing a model binuclear ruthenium complex to obtain reversible DNA-ligand assemblies with the desired properties of high affinity and slow kinetics. Here, we used single-molecule force spectroscopy to study a binuclear ruthenium complex with a longer semirigid linker relative to the model complex. Equilibrium results suggest a DNA affinity that is an order of magnitude higher than the parent binuclear ruthenium complex, likely due to a sterically relieved DNA threading intercalation mechanism. Notably, kinetics analysis shows that less DNA elongation is required for threading intercalation compared to the parent complex, and the association rate is two orders of magnitude faster. The ruthenium complex elongates the DNA duplex by ∼0.3 nm per bound ligand to reach the equilibrium intercalated state, with a significantly different energy landscape relative to the parent complex. Mechanical properties of the ligand-saturated DNA duplex show a higher persistence length, indicating that the longer semirigid linker provides enough molecular spacing to allow a single monomer to fully stack with basepairs, comparable to the monomeric parent ruthenium complex. The DNA basepairs in the equilibrium threading intercalated state are likely intact, and the ruthenium complex is shielded from the polar solution, providing measurable single-molecule confocal fluorescence signals. The obtained confocal fluorescence imaging of the bound dye confirms mostly uniform intercalation along the tethered DNA, consistent with other intercalators. The results of this study, along with previously examined ruthenium complex variants, illustrate tunable intercalation mechanisms guided by the rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.