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 non-specific 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.
Stretch activation (SA), a delayed increase in force production after rapid muscle lengthening, is critical to the function of vertebrate cardiac muscle and insect asynchronous indirect flight muscle. SA enables or increases power generation in muscle types used in a cyclical manner. Recently, myosin isoform expression has been implicated as a mechanism for varying the amplitude of SA in some muscle types. For instance, we found that expressing a larval Drosophila myosin isoform in a muscle type with minimal SA, the Drosophila jump muscle, substantially increased SA amplitude and enabled positive cyclical power generation. To test whether other myosin isoforms could increase SA amplitude and whether the Drosophila heart benefits from SA, we identified two Drosophila cardiac myosin isoforms, CardM1 and CardM2, and expressed them in Drosophila jump muscle. CardM1, CardM2, and control jump muscle fibers all displayed the characteristic phase 3 of SA, with CardM2 SA amplitude ∼60% greater than that of CardM1 and control fibers. Increasing [Pi] from 0 to 16 mM increased CardM2 SA tension amplitude by 74%, yet had minimal or no effect on CardM1 or control muscle SA amplitude. CardM2 displayed the most prominent phase 3 dip when we induced shortening deactivation, a delayed decrease in force after muscle shortening. The magnitude of CardM2 shortening deactivation tension was ∼50% greater than control or CardM1 fibers. This, along with its greater stretch-activated tension, caused CardM2 to be the only isoform to produce positive power when its fiber length was sinusoidally oscillated. The results support our hypotheses that some myosin isoforms enable greater SA tension levels and suggest that the Drosophila heart is benefiting from SA and shortening deactivation in a manner similar to vertebrate hearts.
Binuclear ruthenium complexes have been investigated for potential DNA-targeted therapeutic and diagnostic applications. Studies of DNA threading intercalation, in which DNA base pairs 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 semi-rigid 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 semi-rigid linker provides enough molecular spacing to allow a single monomer to fully stack with base pairs, comparable to the monomeric parent ruthenium complex. The DNA base pairs 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 rational design of therapeutic and diagnostic small molecules to target and modify the DNA duplex.
The ability of biological systems to withstand and recover from various disruptions, such as spontaneous genetic mutations and environmental damage, largely relies on intricate feedback mechanisms. We theoretically study the mechanical response of an epithelial tissue facing damage in the form of a circular wound. Our model describes a feedback loop between the generation of active forces in the actomyosin and tissue mechanics, described by the vertex model. While the exact dynamics of wound closure may be influenced by several biophysical mechanisms that interplay in a nontrivial way, our findings suggest that the closure may initiate as an active instability, triggered by a reduced myosin turnover rate at the wound's perimeter. We explore the interplay between myosin dynamics and the elastic properties of the tissue, elucidating their collective role in determining a wound's loss of stability, leading to the initiation of the closure process.
Supraphysiological shear rates (>2000 s-1) amplify von Willebrand factor unfurling and increase platelet activation and adhesion. These elevated shear rates and shear rate gradients also play a role in shear-induced platelet aggregation (SIPA). The primary objective of this study is to investigate the contributions of major binding receptors to platelet deposition and SIPA in a stenotic model. Microfluidic channels with stenotic contractions ranging from 0% to 75% are fabricated and coated with human type I collagen at 100 μg/mL. Fresh human blood is reconstituted to 40% hematocrit and treated to stain platelets. Platelet receptors αIIbβ3, GPIb, or GPVI are blocked with inhibitory antibodies or proteins to reduce platelet function under flow at 500, 1000, 5000, or 10,000 s-1 over 5 min of perfusion. Additional validation experiments are performed by dual-blocking receptors and performing coagulability testing by rotational thromboelastometry. Control samples exhibit SIPA correlating to increasing shear rate and increasing stenotic contraction. Inhibition of αIIbβ3 or GPIb receptors causes a nearly total reduction in platelet adhesion and a loss of aggregation at >1000 s-1. GPVI inhibition does not notably reduce platelet adhesion at 500 or 1000 s-1 but affects microthrombus stability at 5000-10,000 s-1 following aggregation formation in 50%-75% stenotic channels. Inhibition of von Willebrand factor-binding receptors completely blocks adhesion and aggregation at shear rates >1000 s-1. Inhibition of GPVI reduces platelet adhesion at 5000-10,000 s-1 but renders thrombi susceptible to fragmentation. This study yields further insight into mechanisms regulating rapid growth and stabilization of arterial thrombi at supraphysiological shear rates.
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