In neurons, the distinct molecular composition of axons and dendrites is established through polarized targeting mechanisms, but it is currently unclear how nonpolarized cargoes, such as mitochondria, become uniformly distributed over these specialized neuronal compartments. Here, we show that TRAK family adaptor proteins, TRAK1 and TRAK2, which link mitochondria to microtubule-based motors, are required for axonal and dendritic mitochondrial motility and utilize different transport machineries to steer mitochondria into axons and dendrites. TRAK1 binds to both kinesin-1 and dynein/dynactin, is prominently localized in axons, and is needed for normal axon outgrowth, whereas TRAK2 predominantly interacts with dynein/dynactin, is more abundantly present in dendrites, and is required for dendritic development. These functional differences follow from their distinct conformations: TRAK2 preferentially adopts a head-to-tail interaction, which interferes with kinesin-1 binding and axonal transport. Our study demonstrates how the molecular interplay between bidirectional adaptor proteins and distinct microtubule-based motors drives polarized mitochondrial transport.

The fundamental and applied physics of noble-metal nanoparticles is currently attracting much attention. To a great extent this is due to promising new applications of noble-metal colloidal nanoparticles in fields such as materials science, [1] biophysics, [2] molecular electronics, and fluorescence-spectral engineering based on surface-enhancement effects. [3] In particular the nanoparticles have promising applications as bright fluorescent markers with enhanced photostability in fluorescence microscopy, sensor technology, and microarrays. The enhancement of the fluorescence emission of molecules near a metal surface arises from interactions with surface plasmon (SP) resonances in the metal particles. [4][5][6] These interactions may also result in shortening of the excited-state lifetime thus improving the photostability of the dye.[7]The optical properties of a fluorescent molecule located near a metal nanoparticle are affected by the near-field electrodynamical environment. [4][5][6] This can cause an enhancement or quenching of the fluorescence depending on the distance between the molecule and the metal surface. In the case of fluorescent molecules located at very short distances from a metal surface, non-radiative energy transfer to SPs in the metal takes place. [8,9] Electromagnetic-field enhancement due to SPs, however, still occurs at longer distances from the metal core. As a result, there is an optimal fluorescent molecule to metal-core distance for fluorescence enhancement. Important factors affecting the strength of the fluorescence enhancement are the size and shape of the nanoparticle, the orientation of the dye dipole moments relative to the nanoparticle surface normal, the overlap of the absorption and emission bands of the dye with the plasmon band of the metal, and the radiative decay rate and quantum yield (Q) of the fluorescent molecules.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ϳ40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or preoligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.The EGF receptor (EGFR 2 ; ErbB1) has an essential role in the regulation of growth and differentiation of a large range of cell types. The EGFR belongs to the ErbB family, all four members of which have been implicated in the development of different cancers (1). The first step in the signal transduction cascade is the binding of its ligand such as EGF or TGF-␣ to the ectodomain, which provokes receptor dimerization and oligomerization. Deletion of the dimerization domain, which is present in domain II of the EGFR ectodomain, blocks receptor activation completely, demonstrating that receptor dimerization is critical for the allosteric activation of the tyrosine kinase (2, 3). Activation of the receptor tyrosine kinase results in cross-phosphorylation of the receptors, and the phosphotyrosines in the intracellular domain serve subsequently as docking sites for adaptor proteins such as Grb2 and Shc and enzymes such as phospholipase C␥, which contain phosphotyrosine-specific SH2 (Src homology 2) or phosphotyrosine-binding domains. Eventually, the active ligand-receptor complex becomes internalized via both clathrin-dependent and clathrin-independent pathways, followed by the intracellular transport to lysosomes, where the ligand-receptor complexes are degraded (4).Although EGF binding and dimerization seem to be strictly connected, both microscopic and biochemical studies have demonstrated that, in resting cells, the receptor is already found on the cell surface as non-active dimers, the so-called predimers. This phenomenon was initially discovered using electron microscopy and immunogold labeling of the EGFR: in the resting cell, ϳ35% of the total receptor population was present as receptor predimers (5). Th...

The temperature-sensitive luminescence of nanoparticles enables their application as remote thermometers. The size of these nanothermometers makes them ideal to map temperatures with a high spatial resolution. However, high spatial resolution mapping of temperatures >373 K has remained challenging. Here, we realize nanothermometry with high spatial resolutions at elevated temperatures using chemically stable upconversion nanoparticles and confocal microscopy. We test this method on a microelectromechanical heater and study the temperature homogeneity. Our experiments reveal distortions in the luminescence spectra that are intrinsic to high-resolution measurements of samples with nanoscale photonic inhomogeneities. In particular, the spectra are affected by the high-power excitation as well as by scattering and reflection of the emitted light. The latter effect has an increasing impact at elevated temperatures. We present a procedure to correct these distortions. As a result, we extend the range of high-resolution nanothermometry beyond 500 K with a precision of 1–4 K. This work will improve the accuracy of nanothermometry not only in micro- and nanoelectronics but also in other fields with photonically inhomogeneous substrates.

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.