a-semi-high-throughput-imaging-method-data-visualization-toolkit-to

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze <em>C. elegans</em> Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Ludwig Institute for Cancer Research, San Diego

University of California, San Francisco

Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of  proteins.

A toolkit for GFP-mediated tissue-specific protein degradation in .

Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.

Article	Year
A toolkit for GFP-mediated tissue-specific protein degradation in . Development (Cambridge, England)\| PubMed ID: 28619826	2017
The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Morphogenesis. Developmental cell\| PubMed ID: 30827898	2019

Zhiling Zhao

Biography

JoVE Journal Publications

Other Publications