RESUMO
The Dual-beam ion irradiation facility for Fusion materials (DiFU) has been developed and installed at the Ruder Boskovic Institute with the purpose to perform irradiation of samples of fusion materials by one or two ion beams. Ion beams are delivered to the DiFU chamber by a 6 MV EN Tandem Van de Graaff and a 1 MV HVE Tandetron accelerator, enabling irradiation of areas up to 30 × 30 mm2. The sample holder enables the three-dimensional positioning of samples that can be irradiated while being heated, cooled, or kept at room temperature. Ion fluxes are measured indirectly by the insertion of two large Faraday cups. Besides, the ion flux is monitored continuously by two sets of horizontal and vertical slits, which, in turn, define the limits of the irradiation area on the sample. Sample temperature and conditions during irradiation are additionally monitored by a set of thermocouples, an IR camera, and a video camera. Particular care is dedicated to the mitigation of carbon contamination during ion irradiation.
RESUMO
TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].