No abstract

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.

We recently demonstrated that the expression of the importin α subtype is switched from α2 to α1 during neural differentiation in mouse embryonic stem cells (ESCs) and that this switching has a major impact on cell differentiation. In this study, we report a cell-fate determination mechanism in which importin α2 negatively regulates the nuclear import of certain transcription factors to maintain ESC properties. The nuclear import of Oct6 and Brn2 was inhibited via the formation of a transport-incompetent complex of the cargo bound to a nuclear localization signal binding site in importin α2. Unless this dominant-negative effect was downregulated upon ESC differentiation, inappropriate cell death was induced. We propose that although certain transcription factors are necessary for differentiation in ESCs, these factors are retained in the cytoplasm by importin α2, thereby preventing transcription factor activity in the nucleus until the cells undergo differentiation.

A blazing technique using electron-beam lithography to achieve higher efficiency of gratings and Fresnel lenses is described. Transmission-type blazed gratings have been formed in polymethyl methacrylate films. As a result of measurement, we found that their diffraction efficiency of the first order in these gratings amounts to as much as 60 to 70% at 0.633 microm. Fresnel lenses of 1-mm diameter and 5-mm focal length, which have a sawtooth relief profile, have been also fabricated, and the experimental results showed high-efficiency performance (about 50%) and nearly diffraction-limited focusing.

Micro lenses are basic components of micro optics. We have proposed a new fabrication technique for micro lenses that uses electron-beam lithography and have developed an electron-beam lithography system that is specially designed for this purpose. To demonstrate the feasibility of this technique, ordinary-type Fresnel (zone-plate) lenses and a special-type lens for converting an incident Gaussian intensity distribution into a uniform one were designed and fabricated. It was found that these Fresnel (zone-plate) lenses have near-diffraction-limited performance. The fabrication technique and experimental results are discussed.