The clathrin-binding motif and the J-domain of Drosophila Auxilin are essential for facilitating Notch ligand endocytosis

<p>Abstract</p> <p>Background</p> <p>Ligand endocytosis plays a critical role in regulating the activity of the Notch pathway. The <it>Drosophila </it>homolog of auxilin (<it>dAux</it>), a J-domain-containing protein best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has recently been implicated in Notch signaling, although its exact mechanism remains poorly understood.</p> <p>Results</p> <p>To understand the role of auxilin in Notch ligand endocytosis, we have analyzed several point mutations affecting specific domains of dAux. In agreement with previous work, analysis using these stronger <it>dAux </it>alleles shows that dAux is required for several Notch-dependent processes, and its function during Notch signaling is required in the signaling cells. In support of the genetic evidences, the level of Delta appears elevated in <it>dAux </it>deficient cells, suggesting that the endocytosis of Notch ligand is disrupted. Deletion analysis shows that the clathrin-binding motif and the J-domain, when over-expressed, are sufficient for rescuing <it>dAux </it>phenotypes, implying that the recruitment of Hsc70 to clathrin is a critical role for dAux. However, surface labeling experiment shows that, in <it>dAux </it>mutant cells, Delta accumulates at the cell surface. In <it>dAux </it>mutant cells, clathrin appears to form large aggregates, although Delta is not enriched in these aberrant clathrin-positive structures.</p> <p>Conclusion</p> <p>Our data suggest that <it>dAux </it>mutations inhibit Notch ligand internalization at an early step during clathrin-mediated endocytosis, before the disassembly of clathrin-coated vesicles. Further, the inhibition of ligand endocytosis in <it>dAux </it>mutant cells possibly occurs due to depletion of cytosolic pools of clathrin via the formation of clathrin aggregates. Together, our observations argue that ligand endocytosis is critical for Notch signaling and auxilin participates in Notch signaling by facilitating ligand internalization.</p

The J-domain protein Rme-8 interacts with Hsc70 to control clathrin-dependent endocytosis in Drosophila

By screening for mutants exhibiting interactions with a dominant-negative dynamin, we have identified the Drosophila homologue of receptor-mediated endocytosis (Rme) 8, a J-domain–containing protein previously shown to be required for endocytosis in Caenorhabditis elegans. Analysis of Drosophila Rme-8 mutants showed that internalization of Bride of sevenless and the uptake of tracers were blocked. In addition, endosomal organization and the distribution of clathrin were greatly disrupted in Rme-8 cells, suggesting that Rme-8 participates in a clathrin-dependent process. The phenotypes of Rme-8 mutants bear a strong resemblance to those of Hsc70-4, suggesting that these two genes act in a common pathway. Indeed, biochemical and genetic data demonstrated that Rme-8 interacts specifically with Hsc70-4 via its J-domain. Thus, Rme-8 appears to function as an unexpected but critical cochaperone with Hsc70 in endocytosis. Because Hsc70 is known to act in clathrin uncoating along with auxilin, another J-protein, its interaction with Rme-8 indicates that Hsc70 can act with multiple cofactors, possibly explaining its pleiotropic effects on the endocytic pathway

Amplitude- and phase-resolved nano-spectral imaging of phonon polaritons in hexagonal boron nitride

Phonon polaritons are quasiparticles resulting from strong coupling of photons with optical phonons. Excitation and control of these quasiparticles in 2D materials offer the opportunity to confine and transport light at the nanoscale. Here, we image the phonon polariton (PhP) spectral response in thin hexagonal boron nitride (hBN) crystals as a representative 2D material using amplitude- and phase-resolved near-field interferometry with broadband mid-IR synchrotron radiation. The large spectral bandwidth enables the simultaneous measurement of both out-of-plane (780 cm-1) and in-plane (1370 cm-1) hBN phonon modes. In contrast to the strong and dispersive in-plane mode, the out-of-plane mode PhP response is weak. Measurements of the PhP wavelength reveal a proportional dependence on sample thickness for thin hBN flakes, which can be understood by a general model describing two-dimensional polariton excitation in ultrathin materials

Atomic ionization by sterile-to-active neutrino conversion and constraints on dark matter sterile neutrinos with germanium detectors

The transition magnetic moment of a sterile-to-active neutrino conversion gives rise to not only radiative decay of a sterile neutrino, but also its non-standard interaction (NSI) with matter. For sterile neutrinos of keV-mass as dark matter candidates, their decay signals are actively searched for in cosmic X-ray spectra. In this work, we consider the NSI that leads to atomic ionization, which can be detected by direct dark matter experiments. It is found that this inelastic scattering process for a nonrelativistic sterile neutrino has a pronounced enhancement in the differential cross section at energy transfer about half of its mass, manifesting experimentally as peaks in the measurable energy spectra. The enhancement effects gradually smear out as the sterile neutrino becomes relativistic. Using data taken with germanium detectors that have fine energy resolution in keV and sub-keV regimes, constraints on sterile neutrino mass and its transition magnetic moment are derived and compared with those from astrophysical observations

Coherent x-ray generation at 2.7nm using 25fs laser pulses

We demonstrate for the first time that coherent soft-x-ray pulses at wavelengths of 2.7nm can be generated using 25fs driving pulses. High-order harmonic generation in He is used to produce the femtosecond x-ray harmonics, which exhibit discrete individual orders up to 221, followed by a continuum of unresolved harmonics which extend up to at least the 299th order, corresponding to a wavelength of 2.7nm, or an energy of 450eV. The large ionization potential of He, together with the ultrashort nature of the driving field, results in this dramatic extension of the harmonic plateau, by approximately 200 orders more than has been observed previously. We also obtain excellent agreement with theoretical predictions. © 1998 American Institute of Physics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87449/2/296_1.pd

High-density integration of ultrabright OLEDs on a miniaturized needle-shaped CMOS backplane

This work was supported in part by the Defense Advanced Research Projects Agency (DARPA) under Contract N6600117C4012, by the National Institutes of Health under Grant U01NS090596, and by the Leverhulme Trust (RPG-2017-231). C.K.M. acknowledges funding from the European Commission through a Marie Skłodowska Curie individual fellowship (101029807). M.C.G. acknowledges funding from the Alexander von Humboldt Stiftung (Humboldt-Professorship). We thank Aaron Naden for the FIB/STEM measurements (Engineering and Physical Sciences Research Council under grant numbers EP/L017008/1, EP/R023751/1 and EP/T019298/1).Direct deposition of organic light-emitting diodes (OLEDs) on silicon-based complementary metal–oxide–semiconductor (CMOS) chips has enabled self-emissive microdisplays with high resolution and fill-factor. Emerging applications of OLEDs in augmented and virtual reality (AR/VR) displays and in biomedical applications, e.g., as brain implants for cell-specific light delivery in optogenetics, require light intensities orders of magnitude above those found in traditional displays. Further requirements often include a microscopic device footprint, a specific shape and ultrastable passivation, e.g., to ensure biocompatibility and minimal invasiveness of OLED-based implants. In this work, up to 1024 ultrabright, microscopic OLEDs are deposited directly on needle-shaped CMOS chips. Transmission electron microscopy and energy-dispersive X-ray spectroscopy are performed on the foundry-provided aluminum contact pads of the CMOS chips to guide a systematic optimization of the contacts. Plasma treatment and implementation of silver interlayers lead to ohmic contact conditions and thus facilitate direct vacuum deposition of orange- and blue-emitting OLED stacks leading to micrometer-sized pixels on the chips. The electronics in each needle allow each pixel to switch individually. The OLED pixels generate a mean optical power density of 0.25 mW mm−2, corresponding to >40 000 cd m−2, well above the requirement for daylight AR applications and optogenetic single-unit activation in the brain.Publisher PDFPeer reviewe

Drosophila Activated Cdc42 Kinase Has an Anti-Apoptotic Function

Activated Cdc42 kinases (Acks) are evolutionarily conserved non-receptor tyrosine kinases. Activating somatic mutations and increased ACK1 protein levels have been found in many types of human cancers and correlate with a poor prognosis. ACK1 is activated by epidermal growth factor (EGF) receptor signaling and functions to regulate EGF receptor turnover. ACK1 has additionally been found to propagate downstream signals through the phosphorylation of cancer relevant substrates. Using Drosophila as a model organism, we have determined that Drosophila Ack possesses potent anti-apoptotic activity that is dependent on Ack kinase activity and is further activated by EGF receptor/Ras signaling. Ack anti-apoptotic signaling does not function through enhancement of EGF stimulated MAP kinase signaling, suggesting that it must function through phosphorylation of some unknown effector. We isolated several putative Drosophila Ack interacting proteins, many being orthologs of previously identified human ACK1 interacting proteins. Two of these interacting proteins, Drk and yorkie, were found to influence Ack signaling. Drk is the Drosophila homolog of GRB2, which is required to couple ACK1 binding to receptor tyrosine kinases. Drk knockdown blocks Ack survival activity, suggesting that Ack localization is important for its pro-survival activity. Yorkie is a transcriptional co-activator that is downstream of the Salvador-Hippo-Warts pathway and promotes transcription of proliferative and anti-apoptotic genes. We find that yorkie and Ack synergistically interact to produce tissue overgrowth and that yorkie loss of function interferes with Ack anti-apoptotic signaling. Our results demonstrate how increased Ack signaling could contribute to cancer when coupled to proliferative signals

Disruption of zebrafish cyclin G-associated kinase (GAK) function impairs the expression of Notch-dependent genes during neurogenesis and causes defects in neuronal development

<p>Abstract</p> <p>Background</p> <p>The J-domain-containing protein auxilin, a critical regulator in clathrin-mediated transport, has been implicated in <it>Drosophila </it>Notch signaling. To ask if this role of auxilin is conserved and whether auxilin has additional roles in development, we have investigated the functions of auxilin orthologs in zebrafish.</p> <p>Results</p> <p>Like mammals, zebrafish has two distinct auxilin-like molecules, auxilin and cyclin <b>G-a</b>ssociated <b>k</b>inase (GAK), differing in their domain structures and expression patterns. Both zebrafish auxilin and GAK can functionally substitute for the <it>Drosophila </it>auxilin, suggesting that they have overlapping molecular functions. Still, they are not completely redundant, as morpholino-mediated knockdown of the ubiquitously expressed GAK alone can increase the specification of neuronal cells, a known Notch-dependent process, and decrease the expression of <it>Her4</it>, a Notch target gene. Furthermore, inhibition of GAK function caused an elevated level of apoptosis in neural tissues, resulting in severe degeneration of neural structures.</p> <p>Conclusion</p> <p>In support of the notion that endocytosis plays important roles in Notch signaling, inhibition of zebrafish GAK function affects embryonic neuronal cell specification and <it>Her4 </it>expression. In addition, our analysis suggests that zebrafish GAK has at least two functions during the development of neural tissues: an early Notch-dependent role in neuronal patterning and a late role in maintaining the survival of neural cells.</p