Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos

Abstract

We describe a general, versatile and non-invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in C. elegans embryos. We exploit tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single particle tracking (SPT) analysis. We also introduce and validate a new method called smPReSS that infers exchange rates from quantitative analysis of single molecule photobleaching kinetics, without using SPT. Combining SPT and smPReSS allows spatially and temporally resolved measurements of protein mobility and exchange kinetics. We use these methods (a) to resolve distinct mobility states and spatial variation in exchange rates of the polarity protein Par-6 and (b) to measure spatiotemporal modulation of actin filament assembly and disassembly. The introduction of these methods in a powerful model system offers a promising new avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.

Dynamic Opposition of Clustered Proteins Stabilizes Cortical Polarity in the C. elegans Zygote

“…We used imaging conditions that allow sensitive detection of single GFP molecules (Robin et al, 2014; see Supplementary Methods). In wild type embryos during maintenance phase, PAR-3::GFP was enriched on the anterior cortex in discrete clusters, with a broad range of sizes (Figure 4A; Figure S3A; Movie S5), consistent with previous reports (Hung and Kemphues, 1999; Tabuse et al, 1998) and with the known ability of PAR-3 to oligomerize (Benton and St Johnston, 2003a; Feng et al, 2007; Li et al, 2010a; Mizuno et al, 2003)).…”

“…We used gfp(RNAi) to reduce levels of the GFP::PAR-6 transgene (Robin et al, 2014); then we imaged under conditions (low density and rapid photobleaching; see Supplementary Methods) that allow unambiguous detection of single molecule appearance events (Figure 4D, Movie S6). In wild-type embryos, the spatial distribution of average appearance rates was highly asymmetric (Figure 4D & E).…”

Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation

“…20 μm polystyrene beads (Bangs Laboratories Inc.) were added to the media and a 16 mm 2 cover slip was placed on top of 4–5 ovarioles and 5–10 beads to compress the egg chambers against the bottom coverslip 41 . Egg chambers were imaged with a 100x/1.49 NA CFI Apo oil-immersion TIRF objective (Nikon) using an Andor iXon3 897 EM-CCD camera on an inverted microscope (Ti-E; Nikon) controlled by Metamorph software (Molecular Devices).…”

Regulation of Cell Polarity by PAR-1/MARK Kinase

“…The PAR domains are persistent in the polarized zygote even though individual PAR proteins undergo both lateral diffusion within the cortex and continual exchange between the cortex and the cytoplasm (Cheeks et al, 2004; Goehring et al, 2011a; Nakayama et al, 2009; Robin et al, 2014; Sailer et al, 2015). The maintenance of stable PAR domains depends on a combination of mechanisms that control where they are recruited to the cell cortex and that counteract their lateral diffusion within the cortex.…”