Functional divergence of Plexin B structural motifs in distinct steps of Drosophila olfactory circuit assembly.
Plexins exhibit multitudinous, evolutionarily conserved functions in neural development. How Plexins employ their diverse structural motifs in vivo to perform distinct roles is unclear. We previously reported that Plexin B (PlexB) controls multiple steps during the assembly of the Drosophila olfactory circuit (Li et al., 2018). Here, we systematically mutagenized structural motifs of PlexB and examined the function of these variants in these multiple steps: axon fasciculation, trajectory choice, and synaptic partner selection. We found that the extracellular Sema domain is essential for all three steps, the catalytic site of the intracellular RapGAP is engaged in none, and the intracellular GTPase-binding motifs are essential for trajectory choice and synaptic partner selection, but are dispensable for fasciculation. Moreover, extracellular PlexB cleavage serves as a regulatory mechanism of PlexB signaling. Thus, the divergent roles of PlexB motifs in distinct steps of neural development contribute to its functional versatility in neural circuit assembly.
View details for DOI 10.7554/eLife.48594
View details for PubMedID 31225795
Atlas of Subcellular RNA Localization Revealed by APEX-Seq.
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
View details for DOI 10.1016/j.cell.2019.05.027
View details for PubMedID 31230715
Proximity labeling: spatially resolved proteomic mapping for neurobiology.
Current opinion in neurobiology
2018; 50: 17–23
Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.
View details for PubMedID 29125959
- Beyond Immunoprecipitation: Exploring New Interaction Spaces with Proximity Biotinylation. Biochemistry 2017
Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells.
Cell chemical biology
A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches.
View details for DOI 10.1016/j.chembiol.2017.02.002
View details for PubMedID 28238724
RNA targeting with CRISPR-Cas13.
2017; 550 (7675): 280–84
RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.
View details for PubMedID 28976959
View details for PubMedCentralID PMC5706658
Rapid Removal of Matrices from Small-Volume Samples by Step-Voltage Nanoelectrospray
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2013; 52 (42): 11025-11028
Matrix unloaded: By changing from fixed-voltage (left) to step-voltage nanoelectrospray (right), the mass-spectrometric analysis of small-volume physiological samples is possible. Separation and ionization are achieved in one process, which avoids sample loss and dilution and prevents interference by the matrix. The result is high sensitivity even for samples at the nanoliter level.
View details for DOI 10.1002/anie.201302870
View details for Web of Science ID 000328812600015
View details for PubMedID 24038751