Soluble N-ethylmaleimide—sensitive factor attachment protein receptor (SNARE) proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here, we used optical ...tweezers to observe in a cell-free reconstitution experiment in real time a long-sought SNARE assembly intermediate in which only the membrane-distal amino-terminal half of the bundle is assembled. Our findings support the zippering hypothesis, but suggest that zippering proceeds through three sequential binary switches, not continuously, in the amino- and carboxyl-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force that mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 k B T (where k B is Boltzmann's constant and T is temperature) to mediate fusion.
Stimulated emission depletion (STED) nanoscopy allows observations of subcellular dynamics at the nanoscale. Applications have, however, been severely limited by the lack of a versatile ...STED-compatible two-colour labelling strategy for intracellular targets in living cells. Here we demonstrate a universal labelling method based on the organic, membrane-permeable dyes SiR and ATTO590 as Halo and SNAP substrates. SiR and ATTO590 constitute the first suitable dye pair for two-colour STED imaging in living cells below 50 nm resolution. We show applications with mitochondria, endoplasmic reticulum, plasma membrane and Golgi-localized proteins, and demonstrate continuous acquisition for up to 3 min at 2-s time resolution.
Myocardial infarction is a major cause of premature death in adults. Compromised cardiac function after myocardial infarction leads to chronic heart failure with systemic health complications and a ...high mortality rate
. Effective therapeutic strategies are needed to improve the recovery of cardiac function after myocardial infarction. More specifically, there is a major unmet need for a new class of drugs that can improve cardiomyocyte contractility, because inotropic therapies that are currently available have been associated with high morbidity and mortality in patients with systolic heart failure
or have shown a very modest reduction of risk of heart failure
. Microtubule detyrosination is emerging as an important mechanism for the regulation of cardiomyocyte contractility
. Here we show that deficiency of microtubule-affinity regulating kinase 4 (MARK4) substantially limits the reduction in the left ventricular ejection fraction after acute myocardial infarction in mice, without affecting infarct size or cardiac remodelling. Mechanistically, we provide evidence that MARK4 regulates cardiomyocyte contractility by promoting phosphorylation of microtubule-associated protein 4 (MAP4), which facilitates the access of vasohibin 2 (VASH2)-a tubulin carboxypeptidase-to microtubules for the detyrosination of α-tubulin. Our results show how the detyrosination of microtubules in cardiomyocytes is finely tuned by MARK4 to regulate cardiac inotropy, and identify MARK4 as a promising therapeutic target for improving cardiac function after myocardial infarction.
Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution in the depth direction (50–80 nm) ...and rapidly deteriorating resolution in thick samples, its practical biological application has been effectively limited to two dimensions and thin samples. Here, we present the development of whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), an optical nanoscope that allows imaging of three-dimensional (3D) structures at 10- to 20-nm resolution throughout entire mammalian cells. We demonstrate the wide applicability of W-4PiSMSN across diverse research fields by imaging complex molecular architectures ranging from bacteriophages to nuclear pores, cilia, and synaptonemal complexes in large 3D cellular volumes.
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•Whole-cell 4Pi single-molecule switching nanoscopy allows 10- to 20-nm 3D resolution•Refined hardware and new data analysis allow imaging of cells as thick as ∼10 μm•Using structure-averaging, the 3D shape of a bacteriophage can be resolved•Wide applicability across diverse research fields is demonstrated
A new super-resolution microscope opens a window deep into cells to image organelles and subcellular structures spanning large volumes.
The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological ...structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available numerical aperture, and coupling this with interferometric detection has demonstrated 3D resolution down to 10 nm over entire cellular volumes. The aim of this protocol is to enable interested researchers to establish 4Pi-SMS super-resolution microscopy in their laboratories. We describe in detail how to assemble the optomechanical components of a 4Pi-SMS instrument, align its optical beampath and test its performance. The protocol further provides instructions on how to prepare test samples of fluorescent beads, operate this instrument to acquire images of whole cells and analyze the raw image data to reconstruct super-resolution 3D data sets. Furthermore, we provide a troubleshooting guide and present examples of anticipated results. An experienced optical instrument builder will require ~12 months from the start of ordering hardware components to acquiring high-quality biological images.
The RSC chromatin remodeler slides and ejects nucleosomes, utilizing a catalytic subunit (Sth1) with DNA translocation activity, which can pump DNA around the nucleosome. A central question is ...whether and how DNA translocation is regulated to achieve sliding versus ejection. Here, we report the regulation of DNA translocation efficiency by two domains residing on Sth1 (Post-HSA and Protrusion 1) and by actin-related proteins (ARPs) that bind Sth1. ARPs facilitated sliding and ejection by improving “coupling”—the amount of DNA translocation by Sth1 relative to ATP hydrolysis. We also identified and characterized Protrusion 1 mutations that promote “coupling,” and Post-HSA mutations that improve ATP hydrolysis; notably, the strongest mutations conferred efficient nucleosome ejection without ARPs. Taken together, sliding-to-ejection involves a continuum of DNA translocation efficiency, consistent with higher magnitudes of ATPase and coupling activities (involving ARPs and Sth1 domains), enabling the simultaneous rupture of multiple histone-DNA contacts facilitating ejection.
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•Actin-related proteins and the P1 domain regulate Sth1 DNA translocation efficiency•The Post-HSA domain regulates Sth1 ATPase activity and DNA translocation speed•A blend of moderate-to-high ATPase activity and efficiency enable nucleosome ejection•Strong upregulation of DNA translocation confers chromatin changes and cell lethality
Clapier et al. establish that the chromatin remodeling complex RSC conducts regulated ATP-dependent DNA translocation. Distinct domains and actin-related proteins separately regulate ATPase activity and DNA translocation efficiency. These parameters combine to tune nucleosome sliding, and at higher levels, to cause the simultaneous rupture of multiple histone-DNA contacts, enabling ejection.
The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only ...been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.
mRNA localises to the
oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that
mRNA particles ...undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore,
mRNA localises normally in
, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus,
mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that
mRNA forms 110-120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring.
Au−YSZ nanocomposite films exhibited a surface plasmon resonance absorption band around 600 nm that underwent a reversible blue shift and narrowed upon exposure to CO in air at 500 °C. A linear ...dependence of the sensing signal was observed for CO concentrations ranging between 0.1 and 1 vol % in an air carrier gas. This behavior of the SPR band, upon exposure to CO, was not observed when using nitrogen as the carrier gas, indicating an oxygen-dependent reaction mechanism. Additionally, the SPR band showed no measurable signal change upon exposure to CO at temperatures below ∼400 °C. The oxygen and temperature-dependent characteristics, coupled with the oxygen ion formation and conduction properties of the YSZ matrix, are indicative of charge-transfer reactions occurring at the three-phase boundary region between oxygen, Au, and YSZ, which result in charge transfer into the Au nanoparticles. These reactions are associated with the oxidation of CO and a corresponding reduction of the YSZ matrix. The chemical-reaction-induced charge injection into the Au nanoparticles results in the observed blue shift and narrowing of the SPR band.
Coiled coils are one of the most abundant protein structural motifs and widely mediate protein interactions and force transduction or sensation. They are thus model systems for protein engineering ...and folding studies, particularly the GCN4 coiled coil. Major single-molecule methods have also been applied to this protein and revealed its folding kinetics at various spatiotemporal scales. Nevertheless, the folding energy and the kinetics of a single GCN4 coiled coil domain have not been well determined at a single-molecule level. Here we used high-resolution optical tweezers to characterize the folding and unfolding reactions of a single GCN4 coiled coil domain and their dependence on the pulling direction. In one axial and two transverse pulling directions, we observed reversible, two-state transitions of the coiled coil in real time. The transitions equilibrate at pulling forces ranging from 6 to 12 pN, showing different stabilities of the coiled coil in regard to pulling direction. Furthermore, the transition rates vary with both the magnitude and the direction of the pulling force by greater than 1000 folds, indicating a highly anisotropic and topology-dependent energy landscape for protein transitions under mechanical tension. We developed a new analytical theory to extract energy and kinetics of the protein transition at zero force. The derived folding energy does not depend on the pulling direction and is consistent with the measurement in bulk, which further confirms the applicability of the single-molecule manipulation approach for energy measurement. The highly anisotropic thermodynamics of proteins under tension should play important roles in their biological functions.
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