Biology subjects

Boassa, D.

Publications and source records attributed to Boassa, D..

3 recordsLinked to original sources

bioRxiv2018

Directed evolution of split APEX peroxidase

APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications, from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast display configurations, produced a 200-amino acid N-terminal fragment (with 9 mutations relative to APEX2) called \"AP\" and a 50-amino acid C-terminal fragment called \"EX\". AP and EX fragments were each inactive on their own but reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.

bioengineering↗

bioRxiv2018

CDeep3M - Plug-and-Play cloud based deep learning for image segmentation of light, electron and X-ray microscopy

As biological imaging datasets increase in size, deep neural networks are considered vital tools for efficient image segmentation. While a number of different network architectures have been developed for segmenting even the most challenging biological images, community access is still limited by the difficulty of setting up complex computational environments and processing pipelines, and the availability of compute resources. Here, we address these bottlenecks, providing a ready-to-use image segmentation solution for any lab, with a pre-configured, publicly available, cloud-based deep convolutional neural network on Amazon Web Services (AWS). We provide simple instructions for training and applying CDeep3M for segmentation of large and complex 2D and 3D microscopy datasets of diverse biomedical imaging modalities.

cell biology↗

bioRxiv2018

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native states. However, the applicability of cryofixation is limited by its incompatibilities with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation and high temporal resolution. Here we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing quality ultrastructural preservation for diverse EM applications.

biophysics↗