Two operational modes of atomic force microscopy reveal similar mechanical properties for homologous regions of dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3.

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin.

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Distinct mechanical properties in homologous spectrin-like repeats of utrophin.

Patients with Duchenne muscular dystrophy (DMD) lack the protein dystrophin, which is a critical molecular component of the dystrophin-glycoprotein complex (DGC). Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma of striated muscle through interaction with the cortical actin cytoskeleton via its N-terminal half and with the transmembrane protein ß-dystroglycan via its C-terminal region. Utrophin is a fetal homologue of dystrophin that can subserve many dystrophin functions and is therefore under active investigation as a dystrophin replacement therapy for DMD. Here, we report the first mechanical characterization of utrophin using atomic force microscopy (AFM). Our data indicate that the mechanical properties of spectrin-like repeats in utrophin are more in line with the PEVK and Ig-like repeats of titin rather than those reported for repeats in spectrin or dystrophin. Moreover, we measured markedly different unfolding characteristics for spectrin repeats within the N-terminal actin-binding half of utrophin compared to those in the C-terminal dystroglycan-binding half, even though they exhibit identical thermal denaturation profiles. Our results demonstrate dramatic differences in the mechanical properties of structurally homologous utrophin constructs and suggest that utrophin may function as a stiff elastic element in series with titin at the myotendinous junction.

Memory erasure using time-multiplexed potentials.

We study the thermodynamics of a Brownian particle under the influence of a time-multiplexed harmonic potential of finite width. The memory storage mechanism and the erasure protocol based on time-multiplexed potentials are utilized to experimentally realize erasure with work performed close to Landauer's bound. We quantify the work performed on the system with respect to the duty ratio of time multiplexing, which also provides a handle for approaching reversible erasures. A Langevin dynamics based simulation model is developed for the proposed memory bit and the erasure protocol, which guides the experimental realization. The study also provides insight into transport on the microscale.

Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope.

The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells [1] (Kasas and Ikai, 1995), [2] (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.

Real time reduction of probe-loss using switching gain controller for high speed atomic force microscopy.

In this article, a switching gain proportional-integral-differential controller is used to reduce probe-loss affected regions in an image, obtained during tapping mode operation. Switching signal is derived from the "reliability index" signal, which demarcates regions where the tip has lost contact with the sample (probe-loss), within couple of cantilever oscillation cycles, thereby facilitating use of higher than optimal controller gain without deteriorating on-sample performance. Efficacy of the approach is demonstrated by imaging calibration sample at tip velocity close to 240 microm/s and plasmid DNA at tip velocity of 60 microm/s indicating significant reduction of probe-loss areas and recovery of lost sample features.