Biology subjects

Kad, N. M.

Publications and source records attributed to Kad, N. M..

12 recordsLinked to original sources

bioRxiv2025

Molecular mechanisms of altered contraction with the β-myosin R403Q mutation in porcine ventricular muscle and a human stem cell-derived cardiomyocyte model.

I.The R403Q mutation in the sarcomere protein beta-myosin heavy chain ({beta}-MHC) is a known genetic cause of hypertrophic cardiomyopathy (HCM), associated with ventricular hypercontractility, impaired relaxation, and cardiac arrhythmias. Despite extensive research, the mutations impact on myosin contractile properties remains unclear, likely due, at least in part, to discrepancies cross different model systems. In this study, we used a multidisciplinary approach to explore mutational effects using two distinct heterozygous R403Q systems: a Yucatan minipig model and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). X-ray diffraction of R403Q minipig ventricular muscle demonstrated reduced order of the thick filament, suggesting destabilization of the inhibited OFF (vs. ON) state of myosin in relaxed muscle, which correlated with elevated force at submaximal calcium. Super-resolution, single-molecule fluorescence microscopy indicated elevated ATPase activity in thick filament zones lacking cMyBP-C. Furthermore, R403Q myofibrils exhibited slower activation and relaxation kinetics, with reduced sensitivity to ADP. Molecular dynamics simulations suggested that altered interactions at the actomyosin interface contribute to these effects, rather than changes at the nucleotide binding pocket, typically associated with ADP release. Human engineered heterozygous R403Q hiPSC-CMs exhibited reduced maximal myofibril force, slowed contractile kinetics, and hypercontraction in engineered heart tissue constructs-consistent with HCM phenotypes observed in the heterozygous porcine model. Our results demonstrate that the R403Q mutation induces early and persistent contractile dysfunction, and that hypercontractility and slower contractile kinetics may result from a combination of an increased population of activated (ON) myosin heads and delayed detachment during cross-bridge cycling, respectively.

biophysics↗

bioRxiv2025

Direct measurement of mavacamten and deoxyATP perturbation of the SRX/DRX ratio in porcine cardiac myofibrils using a simple, accessible and multiplexed approach

Cardiac muscle adapts to varying physiological demands by modulating the number of active myosin II motors available for contraction. These motors are organized into thick filaments in the sarcomere and generate force through an ATP-dependent interaction with thin filaments that contain actin. To conserve energy, when demand is low myosin can occupy the super-relaxed (SRX) state, which acts as a reserve. Here, we build upon the earlier studies from the Cooke lab to quantify the size of this cardiac reserve using fluorescence imaging of Cy3-ATP directly in myofibrils. This approach employs a pulse-chase method and exploits the high permeability of isolated myofibrils to monitor nucleotide release in situ. By preserving sarcomeric architecture while enabling rapid reagent exchange, this method bridges the gap between complex single-molecule imaging and traditional stopped-flow bulk assays using MANT-ATP. Using this approach we have studied biochemical perturbation of the SRX reserve with deoxyATP and mavacamten. DeoxyATP caused large depletion of the cardiac reserve, and mavacamten increased its size, consistent with its clinical application. Our results demonstrate the utility of this technique and the potential for further enhancement using multiplexing, holding promise for future applications in health and disease.

biophysics↗

bioRxiv2025

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. MethodsPermeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. ResultsIn contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. ConclusionsWe used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

biophysics↗

bioRxiv2024

The mitotic chromosome periphery: a fluid coat that mediates chromosome mechanics

Mitotic chromosomes are specialised packets of condensed genetic material with dynamic mechanical properties. Each chromosome is coated by a sheath of proteins and RNA, called the mitotic chromosome periphery (MCP). The MCP is widely considered as an essential chromosome compartment where its multiple functions bestow material properties important for successful cell division. However, the details of the micromechanical properties of mitotic chromosomes, and specifically if and how the MCP contributes to these features, remain poorly understood. In this study, we present the most comprehensive characterisation of single-chromosome mechanics to date spanning a broadband frequency range, using optical tweezers and a novel microrheology technique. We extend this analysis to the first direct measurements of MCP micromechanics by manipulating levels of Ki-67, the chief organiser of this compartment, and apply a rheological model to isolate its contribution to chromosome dynamics. We report that the MCP governs high-frequency self-reorganisation dynamics and acts as a structural constraint, providing force-damping properties that mitigate mitotic stress. This work significantly advances our understanding of chromosome micromechanics and how the MCP contributes to the fundamental properties of chromosomes.

biophysics↗

bioRxiv2024

Spatially resolving how phosphorylation affects β-cardiac myosin activity in porcine myofibril sarcomeres with single molecule resolution

Cardiac muscle contraction is mediated by myosin binding from the thick filament of the sarcomere to the thin filament in an ATP powered reaction. This process is highly regulated on a beat-to-beat basis by calcium interactions with the thin filament. Additionally, the number of heads available for participation in contraction is also regulated, resulting in a dynamically variable reserve of heads for controlling contractile force. We aimed to discover the size of this reserve and how it is modulated by phosphorylation. Using single molecule imaging of fluorescently labelled ATP molecules binding and releasing myosins within porcine cardiac sarcomeres, we could determine myosin activity with high spatial resolution. We find three kinetic species when examining the myosin ATPase. The fastest is consistent with non-specific ATP binding to myosins surface, and the slower two species are consistent with the previously identified DRX and SRX states. The former is thought to represent myosins in an ON state, ready to interact with the thin filament and the latter an OFF state with slowed ATPase that constitutes the cardiac reserve. We find that the cardiac reserve is 50% in the sarcomere and this can be sub-divided into the P-, C- and D-zones, with the D-zone having the least population of OFF heads (44%). Treatment with PKA phosphorylates cardiac myosin binding protein-C (cMyBP-C) leading to a 16% reduction in reserve in the C-zone (where cMyBP-C is found), a 10% reduction in the P-zone, and an unexpected 8% increase in the D-zone. By contrast, myosin regulatory light chain (RLC) phosphorylation with myosin light chain kinase (MLCK) resulted in a large 24% decrease in reserve myosins, interestingly the least affected area of the sarcomere was the C-zone. Altogether these data suggest that cMyBP-Cs interaction with RLC governs the degree of activation due to RLC phosphorylation.

biophysics↗

bioRxiv2023

Combining cancer chemotherapeutics with bacterial DNA repair inhibitors to develop novel antimicrobials

Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. Here we have exploited the cytotoxicity of the anti-cancer compound cisplatin to screen for FDA-approved drugs that impair bacterial nucleotide excision DNA repair (NER), the primary mechanism bacteria use to repair cisplatin lesions. Five compounds have emerged of which three possess ideal antimicrobial properties including cell penetrance, specific activity for NER, and the ability to kill a multi-drug resistant clinically relevant E. coli strain. Targeting NER offers a new therapeutic approach for infections in cancer patients by combining antimicrobial activity with cancer chemotherapy.

biochemistry↗

bioRxiv2022

Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)

Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes (koff), events per second ([~]kon), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.

molecular biology↗

bioRxiv2022

Culture media, DMSO and efflux affect the antibacterial activity of cisplatin and oxaliplatin

Cisplatin was originally discovered through its antibacterial action, and subsequently has found use as a potent broad spectrum anticancer agent. This study determines the effect of growth media and solvent on the antibacterial activity of cisplatin and its analogue, oxaliplatin. E. coli MG1655 or MG1655{Delta} tolC were treated with the platinum compounds under different conditions and susceptibility was determined. Our results showed that DMSO reduced the activity of cisplatin by 4-fold (MIC 12.5 mg/L) compared with 0.9% NaCl-solubilized cisplatin (MIC 3.12 mg/L) when tested in MOPS. Surprisingly, complete loss of activity was observed in Mueller Hinton Broth II (MHB II). By supplementing MOPS with individual components of MHB II such as the sulphur containing amino acids, L-cysteine and L-methionine, individually or in combination reduced activity by [≥]8-fold (MIC [≥]25 mg/L). Oxaliplatin was less active against E. coli (MIC 100 mg/L) but exhibited similar inactivation in the presence of DMSO, MHBII or MOPS spiked with L-cysteine and L-methionine (MIC [≥]400 mg/L). Our data suggest that the antibacterial activity of cisplatin and oxaliplatin is modulated by both choice of solvent and composition of growth media. We demonstrate that this is primarily due to sulphur-containing amino acids cysteine and methionine, an essential component of the recommended media for testing antimicrobial susceptibility, MHBII. Significance and impact of the studyAs well as an anticancer treatment, cisplatin possesses antibacterial activity and is active against AMR resistant persister cells, opening the possibility of renewed use against resistant bacterial strains. Our findings provide evidence on how the composition of growth media and choice of solvent modulate the antibacterial activity of cisplatin and its analogue, oxaliplatin. These observations provide a necessary, consistent standard for assessing the antibacterial activity of platinum-based compounds, as a precursor towards their application against bacterial infection. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2020

Single molecule imaging reveals cFos is capable of binding to and diffusing on DNA tightropes independently of cJun

AP-1 proteins are members of the basic leucine zipper (bZIP) family of dimeric transcription factors, which facilitate a multitude of cellular processes, but are primarily known for their oncogenic potential in several cancer types. The oncogenic transcription factor AP-1 binds a specific DNA target site (5TCA[G/C]TGA), however the physical mechanism of how this is achieved has not been determined. The archetypal AP-1 complex is formed by cFos and cJun, which heterodimerize via their leucine zipper domains. We investigated the DNA-binding bZIP domains of AP-1 interacting with DNA tightropes using real-time single molecule fluorescence imaging in vitro. We find that AP-1 bZIP domains comprising cFos:cJun and cJun:cJun rapidly scan DNA using a 1D diffusional search with average diffusion constants of 0.14 m2s-1 and 0.26 m2s-1 respectively. We also report for the first time that cFos is able to bind to and diffuse on DNA (0.29 m2s-1) as a mixed population of monomers and homodimers, despite previous studies suggesting that it is incapable of independent DNA binding. Additionally, we note increased pause lifetimes for the cFos:cJun heterodimer compared to the cJun:cJun homodimer, and were able to detect distinct pausing behaviours within diffusion data. Understanding how cFos:cJun and other transcription factors identify their targets is highly relevant to the development of new therapeutics which target DNA binding proteins using these search mechanisms.

biochemistry↗

bioRxiv2019

The TFIIH components p44/p62 act as a damage sensor during nucleotide excision repair

Nucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in mammalian NER genes lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and five structural subunits. Two of these structural TFIIH proteins, p44 and p62 remain relatively unstudied; p44 is known to regulate the helicase activity of XPD during NER whereas p62s role is thought to be structural. However, a recent cryo-EM structure shows that p44, p62, and XPD make extensive contacts within TFIIH, with part of p62 occupying XPDs DNA binding site. This observation implies a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPDs ATPase but upon encountering DNA damage, further stimulation is only observed when p62 is part of the ternary complex; suggesting a role for the p44/p62 heterodimer in TFIIHs mechanism of damage detection. Using single molecule imaging, we demonstrate that p44/p62 independently interacts with DNA; it is seen to diffuse, however, in the presence of UV-induced DNA lesions the complex stalls. Combined with the analysis of a recent cryo-EM structure we suggest that p44/p62 acts as a novel DNA-binding entity within TFIIH that is capable of recognizing DNA damage. This revises our understanding of TFIIH and prompts more extensive investigation into the core subunits for an active role during both DNA repair and transcription.

biochemistry↗

bioRxiv2018

Single Molecule Visualization of Cardiac Myosin-Binding Protein C N-terminal Fragments Interacting with Thin Filaments: Mechanisms of Calcium Sensitization

Cardiac muscle contraction is activated by calcium binding to troponin and the consequent motion of tropomyosin on actin within the sarcomere. These movements permit myosin binding, filament sliding and motion generation. One potential mechanism by which the N-terminal domains of cardiac myosin-binding protein C (cMyBP-C) play a modulatory role in this activation process is by cMyBP-C binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently-labelled cMyBP-C N-terminal fragments and GFP-labelled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound cooperatively, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that utilizes a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium and thus, enhance myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.

biophysics↗

bioRxiv2018

Single molecule imaging reveals the concerted release of myosin from regulated thin filaments

Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which in turn shifts the position of tropomyosin on actin to expose myosin binding sites. The binding of the first myosin holds tropomyosin in a position such that more myosin binding sites on actin are available, resulting in cooperative activation. Troponin and tropomyosin also act to turn off the thin filament; however, this is antagonized by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation we use the RTF tightrope assay, in which single RTFs are suspended between pedestals above a microscope coverslip surface. Single molecule imaging of GFP tagged myosin-S1 (S1-GFP) is used to follow the activation of RTF tightropes. In sub-maximal activation conditions, S1-GFP molecules bind forming metastable clusters, from which release and rebinding of S1-GFP leads to prolonged activation in these regions. Because the RTFs are not fully active we are able to directly observe deactivation in real time. Using a Reversible Jump Markov Chain Monte Carlo model we are able to dynamically assess the fate of active regions. This analysis reveals that myosin binding occurs in a stochastic stepwise fashion; however, an unexpectedly large probability of multiple simultaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated, active process.

biophysics↗