Biophysical reports最新文献

In vitro motility (IVM) assays allow for the examination of the basic interaction between cytoskeletal filaments with molecular motors and the influence many physiological factors have on this interaction. Examples of factors that can be studied include changes in ADP and pH that emulate fatigue, altered phosphorylation that can occur with disease, and mutations within myofilament proteins that cause disease. While IVM assays can be analyzed manually, the main limitation is the ability to extract accurate data rapidly from videos collected without individual bias. While programs have been created in the past to enable data extraction, many are now out of date or require the use of proprietary software. Here, we report the generation of a Python-based tracking program, Philament, which automatically extracts data on instantaneous and average velocities, and allows for fully automated analysis of IVM recordings. The data generated are presented in an easily accessible spreadsheet-based, comma-separated values file. Philament also contains a novel method of quantifying the smoothness of filament motion. By fitting curves to standard deviations of velocity and average velocities, the influence of different experimental conditions can be compared relative to one another. This comparison provides a qualitative measure of protein interactions where steeper slopes indicate more unstable interactions and shallower slopes indicate more stable interactions within the myofilament. Overall, Philament's automation of IVM analysis provides easier entry into the field of cardiovascular mechanics and enables users to create a truly high-throughput experimental data analysis.

The central role of RNAs in health and disease calls for robust tools to visualize RNAs in living systems through fluorescence microscopy. Live zebrafish embryos are a popular system to investigate multicellular complexity as disease models. However, RNA visualization approaches in whole organisms are notably underdeveloped. Here, we establish our RNA tagging and imaging platform Riboglow-FLIM for complex cellular imaging applications by systematically evaluating FLIM capabilities. We use adherent mammalian cells as models for RNA visualization. Additional complexity of analyzing RNAs in whole mammalian animals is achieved by injecting these cells into a zebrafish embryo system for cell-by-cell analysis in this model of multicellularity. We first evaluate all variable elements of Riboglow-FLIM quantitatively before assessing optimal use in whole animals. In this way, we demonstrate that a model noncoding RNA can be detected robustly and quantitatively inside live zebrafish embryos using a far-red Cy5-based variant of the Riboglow platform. We can clearly resolve cell-to-cell heterogeneity of different RNA populations by this methodology, promising applicability in diverse fields.

During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).

Calcium ions (Ca²⁺) enter mitochondria via the mitochondrial Ca²⁺ uniporter, driven by electrical and concentration gradients. In this regard, transgenic mouse models, such as calsequestrin knockout (CSQ-KO) mice, with higher mitochondrial Ca²⁺ concentrations ([Ca²⁺]_mito), should display higher cytosolic Ca²⁺ concentrations ([Ca²⁺]_cyto). However, repeated measurements of [Ca²⁺]_cyto in quiescent CSQ-KO fibers never showed a difference between WT and CSQ-KO. Starting from the consideration that fluorescent Ca²⁺ probes (Fura-2 and Indo-1) measure averaged global cytosolic concentrations, in this report we explored the role of local Ca²⁺ concentrations (i.e., Ca²⁺ microdomains) in regulating mitochondrial Ca²⁺ in resting cells, using a multicompartmental diffusional Ca²⁺ model. Progressively including the inward and outward fluxes of sarcoplasmic reticulum (SR), extracellular space, and mitochondria, we explored their contribution to the local Ca²⁺ distribution within the cell. The model predicts Ca²⁺ concentration gradients with hot spots or microdomains even at rest, minor but similar to those of evoked Ca²⁺ release. Due to their specific localization close to Ca²⁺ release units (CRU), mitochondria could take up Ca²⁺ directly from high-concentration microdomains, thus sensibly raising [Ca²⁺]_mito, despite minor, possibly undetectable, modifications of the average [Ca²⁺]_cyto.

Quantifying biomolecular dynamics has become a major task of single-molecule fluorescence spectroscopy methods. In single-molecule Förster resonance energy transfer (smFRET), kinetic information is extracted from the stream of photons emitted by attached donor and acceptor fluorophores. Here, we describe a time-resolved version of burst variance analysis that can quantify kinetic rates at microsecond to millisecond timescales in smFRET experiments of diffusing molecules. Bursts are partitioned into segments with a fixed number of photons. The FRET variance is computed from these segments and compared with the variance expected from shot noise. By systematically varying the segment size, dynamics at different timescales can be captured. We provide a theoretical framework to extract kinetic rates from the decay of the FRET variance with increasing segment size. Compared to other methods such as filtered fluorescence correlation spectroscopy, recurrence analysis of single particles, and two-dimensional lifetime correlation spectroscopy, fewer photons are needed to obtain reliable timescale estimates, which reduces the required measurement time.

Viruses have a profound influence on all forms of life, motivating the development of rapid and minimally invasive methods for virus detection. In this study, we present a novel methodology that enables quantitative measurement of the interaction between individual biotic nanoparticles and antibodies in solution. Our approach employs a label-free, full-field common-path interferometric technique to detect and track biotic nanoparticles and their interactions with antibodies. It is based on the interferometric detection of light scattered by viruses in aqueous samples for the detection of individual viruses. We employ single-particle tracking analysis to characterize the size and properties of the detected nanoparticles, and to monitor the changes in their diffusive mobility resulting from interactions. To validate the sensitivity of our detection approach, we distinguish between particles having identical diffusion coefficients but different scattering signals, using DNA-loaded and DNA-devoid capsids of the Escherichia coli T5 virus phage. In addition, we have been able to monitor, in real time, the interaction between the bacteriophage T5 and purified antibodies targeting its major capsid protein pb8, as well as between the phage SPP1 and nonpurified anti-SPP1 antibodies present in rabbit serum. Interestingly, these virus-antibody interactions are observed within minutes. Finally, by estimating the number of viral particles interacting with antibodies at different concentrations, we successfully quantify the dissociation constant $K_d$ of the virus-antibody reaction using single-particle tracking analysis.