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Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca 2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca 2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca 2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca 2+ ], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies., Competing Interests: Declaration of competing interest DDT and RLC hold equity in and serve as executive officers for Photonic Pharma LLC, and OR is the sole proprietor of Editing Science LLC. Neither company had any role in this study. These relationships have been reviewed and managed by the University of Minnesota., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag-PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag-PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]; however, previous studies suggested that HTLV-1 Gag-PM interactions and therefore virus assembly are less dependent on PI(4,5)P 2 . We have recently shown that PI(4,5)P 2 binds directly to HTLV-1 unmyristoylated MA [myr(-)MA] and that myr(-)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P 2 . Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA-membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P 2 -binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P 2 . Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A phenomenon called alternans, which is a beat-to-beat alternation in action potential (AP) duration, sometimes precedes fatal cardiac arrhythmias. Alternans-suppressing electrical stimulus protocols are often represented as perturbations to the dynamics of membrane potential or AP duration variables in nonlinear models of cardiac tissue. Controllability analysis has occasionally been applied to cardiac AP models to determine whether different control or perturbation strategies are capable of suppressing alternans or other unwanted behavior. Since almost all previous cardiac controllability studies have focused on low-dimensional models, we conducted the present study to assess controllability of a higher-dimensional model, specifically the Luo Rudy dynamic (LRd) model of a cardiac ventricular myocyte. Higher-dimensional models are of interest because they provide information on the influence of a wider range of measurable quantities, including ionic concentrations, on controllability. After computing modal controllability measures, we found that larger eigenvalues of a linearized LRd model were on average more strongly controllable through perturbations to calcium-ion concentrations compared with perturbations to other variables. When only membrane potential was adjusted, the best time to apply perturbations (in the sense of maximizing controllability of the largest alternans eigenvalue) was near the AP peak time for shorter cycle lengths. Controllability results were found to be similar for both the default model parameters and for an alternans-promoting parameter set. Additionally, we developed several alternans-suppressing state feedback controllers that were tested in simulations. For the scenarios examined, our controllability measures correctly predicted which strategies and perturbation timings would lead to better feedback controller performance., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

To gain insights into cardiac arrhythmias, researchers have developed and employed various measurement techniques, such as electrocardiography, optical mapping, and patch clamping. However, there are no measurement methods that allow simultaneous recording of all cellular quantities, including intracellular ionic concentrations and gating states, that may play an important role in arrhythmia formation. To help address this shortcoming, we applied observability analysis, a method from control theory, to the Luo-Rudy dynamic (LRd) model of a cardiac ventricular myocyte. After linearizing the time-integrated LRd model about selected periodic orbits, we computed the observability properties of the model to determine whether past system states could be reconstructed from different hypothetical sets of measurements. Under the simplifying assumption that only one dynamical variable could be measured periodically, we found that intracellular potassium concentration generally yielded the largest observability values and thus contained the most information about the dominant modes of the system. The impacts on observability of measurement timings, inter-stimulus interval length, and an alternans-promoting parameter shift were also studied. Pole-placement state observer algorithms were designed and tested in simulations for several scenarios, and we found that it is possible to infer unmeasured variables from potassium-concentration measurements, and to an extent from membrane-potential measurements, both for longer periods that represent normal rhythms and shorter periods associated with tachyarrhythmias. Our results could lead to improved data assimilation algorithms that combine model predictions with measurements to estimate quantities that are difficult or impossible to measure during in vitro experiments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Analysis of fluorescence fluctuation data through the time-shifted mean-segmented Q (tsMSQ) analysis method has recently been shown to successfully identify protein oligomerization and mobility in the nuclear envelope by properly accounting for local volume fluctuations of the nuclear envelope within living cells. However, by its nature, tsMSQ produces correlated data which poses unique challenges for applying goodness of fit tests and obtaining parameter uncertainties from individual measurements. In this paper, we overcome these challenges by introducing bootstrap tsMSQ which involves randomly resampling the fluorescence intensity data to eliminate the correlations in the tsMSQ data. This analysis technique was verified in both the cytoplasm and the lumen of the nuclear envelope with well-characterized proteins that served as model systems. Uncertainties and goodness of fit tests of individual measurements were compared to estimates obtained from sampling multiple experiments. We further applied bootstrapping to fluorescence fluctuation data of the luminal domain of the SUN domain-containing protein 2 in order to characterize its self-oligomerization within the nuclear envelope. Analysis of the concentration-dependent brightness suggests a monomer-trimer transition of the protein., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Large magnetic field inhomogeneity can be a significant cause of spatial flip-angle variation when using ordinary, limited-bandwidth RF pulses. Multidimensional RF pulses are particularly sensitive to inhomogeneity due to their extended pulse length, which decreases their bandwidth. Previously, it was shown that, by breaking a 2D pulse into multiple undersampled k-space segments, the excitation bandwidth can be increased at the expense of increased imaging time. The present study shows how this increased imaging time can be offset by undersampling acquisition k-space in a phase-encoded dimension that is in the direction of excitation segmentation. Data from each segment are viewed as originating from "virtual receive coils" rather than multiple physical coils. The undersampled data are reconstructed using parallel imaging techniques (e.g. as in GRAPPA). The method was tested in vivo with brain imaging at both 3 T and 4 T, and used in conjunction with a 32-channel head coil and conventional GRAPPA on the 3 T data. Relationships with existing techniques and future applications are discussed., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Applications of multidimensional spatially-selective pulses are sometimes limited by their long pulse durations resulting from the need to execute a modulated gradient waveform in concert with RF transmission. Here, we introduce a method to design two-dimensional selective adiabatic pulses using a Cartesian k-space trajectory. The full pulse can be sampled using various undersampled segments to create a multidimensional pulse resilient to large off-resonances. Moreover, the pulse can be designed to be resilient to B 1 inhomogeneity. Experimental demonstrations of fully segmented and single-shot k-space sampling patterns are presented.+ inhomogeneity. Experimental demonstrations of fully segmented and single-shot k-space sampling patterns are presented., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The early events in the retrovirus assembly pathway, particularly the timing and nature of Gag translocation from the site of protein translation to the inner leaflet of the plasma membrane, are poorly understood. We have investigated the interrelationship between cytoplasmic Gag concentration and plasma membrane association using complementary live-cell biophysical fluorescence techniques in real time with both human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) Gag proteins. In particular, dual-color, z-scan fluorescence fluctuation spectroscopy in conjunction with total internal reflection fluorescence and conventional, epi-illumination imaging were utilized. Our results demonstrate that HTLV-1 Gag is capable of membrane targeting and particle assembly at low (i.e., nanomolar) cytoplasmic concentrations and that there is a critical threshold concentration (approaching micromolar) prior to the observation of HIV-1 Gag associated with the plasma membrane. These observations imply fundamental differences between HIV-1 and HTLV-1 Gag trafficking and membrane association., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including human immunodeficiency virus type 1 (HIV-1). Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction., (Copyright © 2013 Elsevier Ltd. All rights reserved.)