Biophysical journal最新文献

Cardiomyopathies, often caused by mutations in genes encoding muscle proteins, are traditionally treated by phenotyping hearts and addressing symptoms post irreversible damage. With advancements in genotyping, early diagnosis is now possible, potentially introducing earlier treatment. However, the intricate structure of muscle and its myriad proteins make treatment predictions challenging. Here we approach the problem of estimating therapeutic targets for a mutation in mouse muscle using a spatially explicit half sarcomere muscle model. We selected 9 rate parameters in our model linked to both small molecules and cardiomyopathy-causing mutations. We then randomly varied these rate parameters and simulated an isometric twitch for each combination to generate a large training dataset. We used this dataset to train a Conditional Variational Autoencoder (CVAE), a technique used in Bayesian parameter estimation. Given simulated or experimental isometric twitches, this machine learning model is able to then predict the set of rate parameters which are most likely to yield that result. We then predict the set of rate parameters associated with twitches from control mice with the cardiac Troponin C (cTnC) I61Q variant and control twitches treated with the myosin activator Danicamtiv, as well as model parameters that recover the abnormal I61Q cTnC twitches.

Most kinesin molecular motors dimerize to move processively and efficiently along microtubules; however, some can maintain processivity even in a monomeric state. Previous studies have suggested that asymmetric potentials between the motor domain and microtubules underlie this motility. In this study, we demonstrate that the kinesin-3 family motor protein KLP-6 can move forward along microtubules as a monomer upon release of autoinhibition. This motility can be explained by a change in length between the head and tail, rather than by asymmetric potentials. Using mass photometry and single-molecule assays, we confirmed that activated full-length KLP-6 is monomeric both in solution and on microtubules. KLP-6 possesses a microtubule-binding tail domain, and its motor domain does not exhibit biased movement, indicating that the tail domain is crucial for the processive movement of monomeric KLP-6. We developed a mathematical model to explain the biased Brownian movements of monomeric KLP-6. Our model concludes that a slight conformational change driven by neck-linker docking in the motor domain enables the monomeric kinesin to move forward if a second microtubule-binding domain exists.

Microtubule stability is known to be governed by a stabilizing GTP/GDP-Pi cap, but the exact relation between growth velocity, GTP hydrolysis and catastrophes remains unclear. We investigate the dynamics of the stabilizing cap through in vitro reconstitution of microtubule dynamics in contact with micro-fabricated barriers, using the plus-end binding protein GFP-EB3 as a marker for the nucleotide state of the tip. The interaction of growing microtubules with steric objects is known to slow down microtubule growth and accelerate catastrophes. We show that the lifetime distributions of stalled microtubules, as well as the corresponding lifetime distributions of freely growing microtubules, can be fully described with a simple phenomenological 1D model based on noisy microtubule growth and a single EB3-dependent hydrolysis rate. This same model is furthermore capable of explaining both the previously reported mild catastrophe dependence on microtubule growth rates and the catastrophe statistics during tubulin washout experiments.

It is interesting to find pathologically that leukocytes, especially neutrophils, tend to adhere in the liver sinusoids dominantly but not in the post-sinusoidal venules. While both views of receptor-ligand interactions and physical trapping are proposed for mediating leukocyte adhesion in liver sinusoids, integrated investigations for classifying their respective contributions are poorly presented. With a combination of Monte Carlo simulation and immersed boundary method (IBM), this study explored numerically the effects of molecular interaction kinetics and sinusoidal mechanical properties on leukocyte adhesion in liver sinusoid jointly. Results showed that, within the range of biological limitations, the lumen stenosis ratio, leukocyte stiffness, Disse space stiffness and endothelium permeability regulate the comprehensive adhesion process in a descending order of significance in the presence of receptor-ligand interactions. While leukocyte adhesions could be mutually promoted with proper combinations of leukocyte stiffness, lumen stenosis, and molecular interaction, the binding affinity is insensitive under the conditions with low leukocyte stiffness in normal lumen stenosis and high leukocyte stiffness in high lumen stenosis. This work deepened the understanding of recruitment mechanism of leukocyte in liver sinusoids.

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 like 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 like that seen in many simulations is consistent with the relatively moderate fluorescence changes seen in experiments.

Recent developments of single-molecule and super-resolution microscopies reveal novel spatial-temporal features of various cellular processes with unprecedented details, and greatly facilitate the development of theoretical models. In this review, we synthesize our view of how to meaningfully integrate these experimental approaches with theoretical modeling to obtain deeper understanding of the physical mechanisms of cell biology.

Hirudin is a bioactive small protein that binds thrombin to interrupt the blood clotting cascade. It contains an ordered and a disordered (IDR) region. Conjugating with polyethylene glycol (PEGylation) is an important modification of biopharmaceuticals to improve their lifetime and retention. Here we studied by molecular dynamics simulation (MD) how hirudin P18 and its PEGylated variant differ in their structural flexibility depending on binding to thrombin and charge screening by NaCl. We also compare with glycated hirP18 and the hirV1 variant to assess effects of different polar attachments and sequence variability. First, we synthesized unlabeled and PEG-labeled hirP18 followed by an activity assay to ascertain that the peptide-PEG conjugate retains anticoagulant activity. Next, we carried 16 different microsecond MD simulations of the different proteins, bound and unbound, for two sequences and different salt conditions. Simulations were analyzed in terms of scaling exponents to study the effect of ionic strength on hirudin size and solvent-exposed surface area. We conclude that charge patterning of the sequence and the presence of arginine are two important features for how PEG interacts with the protein folded and intrinsically disordered regions. Specifically, PEG can screen end-to-end electrostatic interactions by 'hiding' a positively charged region of hirudin, whereas hirV1 is less sticky than hirP18 due to different PEG-hirudin hydrophobic interactions and the presence of an arginine in hirP18. Conjugation with either PEG or a glycan significantly reduces solvent-exposed area of hirudin, but PEG interacts more efficiently with surface residues than does glycan due to its narrower chain that can fit in surface grooves, and alternation of polar (oxygen) and non-polar (CH₂-CH₂) groups that interact favorably with charged and hydrophobic surface patches.

The nuclear pore complex (NPC) is responsible for the selective transport of biomolecules in and out of the nucleus. This selective feature is achieved through intrinsically disordered proteins, FG-nups, that are anchored to the inner wall of the NPC. Cargo smaller than approximately 5 nm can rapidly diffuse through the NPC whereas larger cargo is increasingly slowed down. Larger cargos bound to chaperone proteins (from the karyopherin or Kap family) can still be transported due to non-specific interactions with the FG-Nups. Although various mechanisms for the transport of Kaps have been proposed, a consensus has still to be reached. Here we conducted a coarse-grained molecular dynamics study to shed light on Kap translocation through NPCs. We investigated the effect of Kap surface charge and hydrophobicity on the transport rate. We found that the negative charge of the Kaps is essential for transport whereas Kap hydrophobicity of the transport particle aids in the translocation. Interestingly, our results indicate that the positive net charge of the nuclear Nups (especially Nup1) is instrumental for the transport of Kaps, revealing a (previously proposed) gradient of increasing binding affinity of the Kaps with FG-Nups from the cytoplasm to the nucleus.

T cell-based immunotherapy has recently emerged a promising strategy to treat cancer, which requires the activation of antigen-directed cytotoxicity to kill cancer cells. Mechanical signaling, although often overshadowed by its biochemical counterpart, play a crucial role in T cell anti-cancer responses, from activation to cytolytic killing. Rapid advancements in the fields of chemistry, biomaterial, and micro/nanoengineering offer an interdisciplinary approach to incorporate mechano- and immuno-modulatory ligands, including but not limited to synthetic peptides, small molecules, cytokines, artificial antigens, onto the biomaterial-based platforms to modulate mechanotransducive processes in T cells. Surface engineering of these immunomodulatory ligands with optimization of ligand density, geometrical arrangement, and mobility are proven to better mimic natural ligation between immunoreceptor-ligand to directly enhance or inhibit mechanotransduction pathways in T cells, through triggering upstream mechanosensitive channels, adhesion molecules, cytoskeletal components, or downstream mechano-immunological regulators. Despite its tremendous potential, however, current research on this new biomaterial surface engineering approach for mechano-modulatory of T cell activation and effector functions remains in a nascent stage. This review highlights the recent progress in this new direction, focusing on achievements in mechano-modulatory ligand-based surface engineering strategies and underlying principles, and outlooks the further research in the rapidly evolving field of T cell mechanotransduction engineering for efficient immunotherapy.