Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72-ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator.DOI: http://dx.doi.org/10.7554/eLife.23063.001

We have used ion beam analysis and scanning near-field optical microscopy to characterize the three-dimensional domain structure of a thin film of a phase-separating polymer mixture. In the initially mixed film, there first occurs coverage of one of its surfaces by an unbroken layer of one phase; between this -wet. surface and the substrate, layers of domains form. Eventually, the layered domain structure becomes unstable, and a transformation occurs to the equilibrium wetting state where both phases are in contact with both surfaces, causing a phase separation to be purely two-dimensional at later times. During this <

We report on scanning far- and near-field two-photon microscopy of cell nuclei stained with DAPI and bisbenzimidazole Hoechst 33342 (BBI-342) with the 647-nm laser line of a cw ArKr mixed-gas laser. Two-photon-excited fluorescence images are obtained for 50-200 mW of average power at the sample. A nearly quadratic dependence of fluorescence intensity on laser power confirmed the two-photon effect. The nonlinearity was further supported by evidence of three-dimensional sectioning in a scanning far-field microscope. We find that the cw two-photon irradiation sufficient for imaging within typically 5 s does not significantly impair cell cycling of BBI-342-labeled live cells. Finally, high-resolution imaging in scanning near-field microscopy with good contrast is demonstrated.

We have implemented continuous-wave two-photon excitation of near-UV absorbing fluorophores in a scanning near-field optical microscope (SNOM). The 647-nm emission of an Ar-Kr mixed gas laser was used to excite the UV-absorbing DNA dyes DAPI, the bisbenzimidazole Hoechst 33342, and ethidium bromide in a shared aperture SNOM with uncoated fiber tips. Polytene chromosomes of Drosophila melanogaster and the nuclei of 3T3 Balb/c cells labeled with these dyes were readily imaged. The fluorescence intensity showed the expected nonlinear (second order) dependence on the excitation power in the range of 8-180 mW. We measured the fluorescence intensity as a function of the tip-sample displacement in the direction normal to the sample surface in the single- and two-photon excitation modes (SPE, TPE). The fluorescence intensity decayed faster in TPE than in SPE.

No abstract