Biology subjects

Dickinson, D. J.

Publications and source records attributed to Dickinson, D. J..

3 recordsLinked to original sources

bioRxiv2018

Snapshots of single particles from single cells using electron microscopy

Cryo-electron microscopy has become an indispensable tool for structural studies of biological macromolecules. There are two predominant methods for studying the architectures of multi-protein complexes: (1) single particle analysis of purified samples and (2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, while the latter can capture proteins in their native state but is hindered by a low signal-to-noise ratio and results in lower-resolution structures. Here, we present a method combining microfluidic single cell extraction with single particle analysis by electron microscopy to characterize protein complexes from individual C. elegans embryos. Using this approach, we uncover three-dimensional structures of ribosomes directly from single embryo extracts. In addition, we investigate structural dynamics during development by counting the number of ribosomes per polysome in early and late embyros. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary and disease contexts.

biochemistry↗

bioRxiv2018

Optogenetic dissection of mitotic spindle positioning in vivo

The position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of G-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through G-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the G regulators RIC-8Ric-8A and RGS-7Loco/RGS3, and replacement of G with a light-inducible membrane anchor demonstrated that G-GTP signaling is dispensable for pulling force generation. In the absence of G, cortical recruitment of GPR-1/2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment of LIN-5 overruled normal cell-cycle and polarity regulation, and provided experimental control over the spindle and cell cleavage plane. Our results define G*GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle positioning forces. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.

cell biology↗

bioRxiv2018

C. elegans RAP-1 reinforces LET-60/Ras induction of cell fate

The notoriety of the small GTPase Ras as the most mutated oncoprotein has led to a well-characterized signaling network largely conserved across metazoans. Yet the role of its close relative Rap1 (Ras Proximal), which shares 100% identity between their core effector binding sequences, remains unclear. A long-standing controversy in the field is whether Rap1 also functions to activate the canonical Ras effector, the S/T kinase Raf. We used the developmentally simpler Caenorhabditis elegans, which lacks the extensive paralog redundancy of vertebrates, to examine the role of RAP-1 in two distinct LET-60/Ras-dependent cell fate patterning events: induction of 1{ring} vulval precursor cell (VPC) fate and of the excretory duct cell. Fluorescently tagged endogenous RAP-1 is localized to plasma membranes and is expressed ubiquitously, with even expression levels across the VPCs. RAP-1 and its activating GEF PXF-1 function cell autonomously and are necessary for maximal induction of 1{ring} VPCs. Critically, mutationally activated endogenous RAP-1 is sufficient both to induce ectopic 1{ring}s and duplicate excretory duct cells. Like endogenous RAP-1, before induction GFP expression from the pxf-1 promoter is uniform across VPCs. However, unlike endogenous RAP-1, after induction GFP expression is increased in presumptive 1{ring}s and decreased in presumptive 2{ring}s. We conclude that RAP-1 is a positive regulator that promotes Ras-dependent inductive fate decisions. We hypothesize that PXF-1 activation of RAP-1 serves as a minor parallel input into the major LET-60/Ras signal through LIN-45/Raf.

genetics↗