Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of familial Parkinson’s disease. LRRK2 is a multi-domain protein containing a kinase and GTPase. Using correlative light ...and electron microscopy, in situ cryo-electron tomography, and subtomogram analysis, we reveal a 14-Å structure of LRRK2 bearing a pathogenic mutation that oligomerizes as a right-handed double helix around microtubules, which are left-handed. Using integrative modeling, we determine the architecture of LRRK2, showing that the GTPase and kinase are in close proximity, with the GTPase closer to the microtubule surface, whereas the kinase is exposed to the cytoplasm. We identify two oligomerization interfaces mediated by non-catalytic domains. Mutation of one of these abolishes LRRK2 microtubule-association. Our work demonstrates the power of cryo-electron tomography to generate models of previously unsolved structures in their cellular environment.
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•Cryo-electron tomography and integrative modeling reveal LRRK2’s structure in situ•Parkinson’s disease-linked LRRK2 forms double-helical filaments around microtubules•Homotypic interactions between the WD40 and COR domains form the filaments•The GTPase and kinase domains face the microtubule and cytoplasm, respectively
Cryo-electron tomography combined with integrative modeling yields the structure of LRKK2 in a cellular context and shows how filaments of the protein embrace microtubules.
The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G ...protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.
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•PKA RIα undergoes liquid-liquid phase separation in response to cAMP dynamics•RIα condensates dynamically sequester cAMP and retain high PKA activity•RIα phase separation is necessary for effective cAMP compartmentation•A PKA oncoprotein disrupts RIα bodies, leading to aberrant signaling and growth
cAMP-responsive condensate formation by PKA’s RIα subunit controls local signaling, and disruption of phase separation in this context contributes to tumorigenesis.
When the J-domain of the heat shock protein DnaJB1 is fused to the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), replacing exon 1, this fusion protein, J-C subunit (J-C), becomes the ...driver of fibrolamellar hepatocellular carcinoma (FL-HCC). Here, we use cryo-electron microscopy (cryo-EM) to characterize J-C bound to RIIβ, the major PKA regulatory (R) subunit in liver, thus reporting the first cryo-EM structure of any PKA holoenzyme. We report several differences in both structure and dynamics that could not be captured by the conventional crystallography approaches used to obtain prior structures. Most striking is the asymmetry caused by the absence of the second cyclic nucleotide binding (CNB) domain and the J-domain in one of the RIIβ:J-C protomers. Using molecular dynamics (MD) simulations, we discovered that this asymmetry is already present in the wild-type (WT) RIIβ2C2 but had been masked in the previous crystal structure. This asymmetry may link to the intrinsic allosteric regulation of all PKA holoenzymes and could also explain why most disease mutations in PKA regulatory subunits are dominant negative. The cryo-EM structure, combined with small-angle X-ray scattering (SAXS), also allowed us to predict the general position of the Dimerization/Docking (D/D) domain, which is essential for localization and interacting with membrane-anchored A-Kinase-Anchoring Proteins (AKAPs). This position provides a multivalent mechanism for interaction of the RIIβ holoenzyme with membranes and would be perturbed in the oncogenic fusion protein. The J-domain also alters several biochemical properties of the RIIβ holoenzyme: It is easier to activate with cAMP, and the cooperativity is reduced. These results provide new insights into how the finely tuned allosteric PKA signaling network is disrupted by the oncogenic J-C subunit, ultimately leading to the development of FL-HCC.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In this report, we propose an idea of connecting 1D photonic crystal (PhC) nanocavity with novel waveguide-like strain shapers in a polydimethylsiloxane substrate. In theory, the induced film-edge ...strain by the strain shapers can significantly enhance the wavelength tunability of PhC nanocavity under different applied stress. By our nanofabrication process, we realize this idea in the form of a tunable nanolaser. In experiments, the nanolaser shows the enhanced wavelength tunability both under stretching and compressive stress. By investigating the effects of PhC periods and length of the strain shapers, we obtain an optimized lasing wavelength tunability of −13.9 nm under every percentage strain variation in experiments. When further taking the 2 nd -order mode in the nanocavity into consideration, we observe a wide lasing wavelength tuning range over 160 nm, which takes only the applied strain <inline-formula><tex-math notation="LaTeX">\Delta \xi _{tot}</tex-math></inline-formula> of ±0.05. In addition to the large wavelength response beneficial for tunable nanolasers and optical strain sensors, we also believe that this waveguide-like strain shapers can serve as the optical interconnections at the same time in the flexible photonic circuits.
We investigate the optical properties of gold nanoring (NR) dimers in both simulation and experiment. The resonance peak wavelength of gold NR dimers is strongly dependent on the polarization ...direction and gap distance. As the gold NR particles approach each other, exponential red shift and slight blue shift of coupled bonding (CB) mode in gold NR dimers for longitudinal and transverse polarizations are obtained. In finite element method analysis, a very strong surface plasmon coupling in the gap region of gold NR dimers is observed, whose field intensity at the gap distance of 10 nm is enhanced 23% compared to that for gold nanodisk (ND) dimers with the same diameter. In addition, plasmonic dimer system exhibits a great improvement in the sensing performance. Near-field coupling in gold NR dimers causes exponential increase in sensitivity to refractive index of surrounding medium with decreasing the gap distance. Compared with coupled dipole mode in gold ND dimers, CB mode in gold NR dimers shows higher index sensitivity. This better index sensing performance is resulted form the additional electric field in inside region of NR and the larger field enhancement in the gap region owing to the stronger coupling of collective dipole plasmon resonances for CB mode. These results pave the way to design plasmonic nanostructures for practical applications that require coupled metallic nanoparticles with enhanced electric fields.
In this report, using two-dimensional photonic crystals (PhC) and a one-dimensional PhC nano-beam cavity, we realized the development of all-polymeric dye-lasers on a dye-doped, suspended ...poly-methylmethacrylate film with a wavelength-scale thickness. In addition to the characterization of basic lasing properties, we also evaluated its capacity to serve as an attachable strain sensor. Through experimentation, we confirmed the stable lasing performances of the dye-laser attaching on a rough surface. Moreover, we also theoretically studied the wavelength responses of the utilized PhC resonators to stretching strain and further improved them via the concept of strain shaping. The attachability and high strain sensing response of the presented thin film PhC dye-lasers demonstrate their potential as attachable strain sensors.
We propose and demonstrate a trapping configuration integrating coupled waveguides and gold bowtie structures to form near-field plasmonic tweezers. Compared with excitation from the top, waves ...coupled through the waveguide can excite specific bowties on the waveguide and trap particles precisely. Thus this scheme is more efficient and compact, and will assist the circuit design on a chip. With lightning rod and gap effects, the gold bowtie structures can generate highly concentrated resonant fields and induce trapping forces as strong as 652 pN W(-1) on particles with diameters as small as 20 nm. This trapping capability is investigated numerically and verified experimentally with observations of the transport, trapping, and release of particles in the system.
We demonstrate an elliptical gold nanodisk array (GNA) for engineering the spectral profile of surface lattice resonance (SLR). The nanodisk's shape has a great impact on SLR. Small linewidth of 20 ...nm at an aspect ratio of 1.17, as well as large wavelength tuning of 64 nm within 4% strain via different orientations and polarizations, are achieved experimentally. The enhanced wavelength response of 6.93 nm per 1% strain variation for elliptical GNA is 2.4 times better than that for general circular GNA. Furthermore, the strain sensing for elliptical GNA approaches is 5.7 times greater than that for circular GNA.
It is difficult to imagine where the signaling community would be today without the Protein Data Bank. This visionary resource, established in the 1970s, has been an essential partner for sharing ...information between academics and industry for over 3 decades. We describe here the history of our journey with the protein kinases using cAMP-dependent protein kinase as a prototype. We summarize what we have learned since the first structure, published in 1991, why our journey is still ongoing, and why it has been essential to share our structural information. For regulation of kinase activity, we focus on the cAMP-binding protein kinase regulatory subunits. By exploring full-length macromolecular complexes, we discovered not only allostery but also an essential motif originally attributed to crystal packing. Massive genomic data on disease mutations allows us to now revisit crystal packing as a treasure chest of possible protein:protein interfaces where the biological significance and disease relevance can be validated. It provides a new window into exploring dynamic intrinsically disordered regions that previously were deleted, ignored, or attributed to crystal packing. Merging of crystallography with cryo-electron microscopy, cryo-electron tomography, NMR, and millisecond molecular dynamics simulations is opening a new world for the signaling community where those structure coordinates, deposited in the Protein Data Bank, are just a starting point!
A switch in time: Gel–sol transitions of a urea‐based 2rotaxane gelator are controlled by the degree of solvent exposure of the hydrogen‐bond‐donating urea station and the orientation of the ...hydrogen‐bond‐accepting CO groups of the interlocked ethylene glycol based macrocycle (N blue, O and macrocycle red, phenyl green). Both acid/base and anion‐exchange control can be used to reversibly transition the rotaxane in 1‐pentanol between solution and gel states.
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