Small Molecule Injection into Single-Cell <i>C. elegans</i> Embryos via Carbon-Reinforced Nanopipettes

<div><p>The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of <i>Caenorhabditis elegans</i>, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into <i>C. elegans</i> embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings.</p> </div

Effect of Latrunculin A on PAR-2 localization in PAR-6-depleted embryos.

<p>(<b>a</b>) par <i>6</i> RNAi treatment for 24 hr (<i>N</i> = 5). (<b>b</b>) par <i>6</i> RNAi treatment for 24 hr combined with 90 µM LatA injection following meiosis II (<i>N</i> = 4). For each panel, columns from left to right: DIC, NMY-2::GFP/ GFP::H2B and PAR-2::mCherry.</p

Effect of Latrunculin A on PAR-6 localization.

<p>(<b>a</b>) Control with injection buffer containing 3.75% DMSO (<i>N</i> = 9). (<b>b</b>) Injection of 60 µM (<i>N</i> = 10) and (<b>c</b>) 90 µM (<i>N</i> = 5) LatA. In each panel, DIC is on the left and PAR-6::mCherry is on the right.</p

CRNP injection characterization and experimental configuration.

<p>(<b>a</b>) Characterization of injection volume. A differential interference contrast (DIC) image of a CRNP and fluorescence images before injection of dextran-TxRed into a droplet of glycerol and immediately after the injection. (<b>b</b>) Histogram of injection volume from multiple injections using one representative CRNP. (<b>c</b>) A cartoon and a (<b>d</b>) DIC image of the experimental configuration for embryo injection. A quartz holding pipette was used to immobilize the embryo during injection. Light suction applied through the holding pipette also allowed for the withdrawal of the CRNP. In all figures, unless otherwise stated, <i>t</i> = 0 is defined as the beginning of meiosis II, injection occurs at t ^≈ 0: 15, anterior is to the left, and scale bars are 10 µm.</p

Effect of Latrunculin A concentration on PAR-2 localization.

<p>(<b>a</b>) Control with injection buffer containing 3.75% DMSO in 0.8x EB (<i>N</i> = 10). (<b>b</b>) Injection of 60 µM LatA (<i>N</i> = 8). Shown is an example embryo in which PAR-2 localized at both the cortex and the centrosomes. (<b>c</b>) Injection of 90 µM LatA (<i>N</i> = 13). Shown is an example embryo with PAR-2 localization at the centrosomes only. For each panel, columns from left to right: DIC; NMY2::GFP/GFP::H2B, and mCherry::PAR-2. In some images, only one centrosome is in focus. (<b>d</b>) Distribution of PAR-2 localization phenotypes after injection of 60 µM or 90 µM LatA. In this and all subsequent figures, rows of images are aligned by nuclear dynamics, as assessed by GFP::H2B.</p

Carbon-reinforced nanopipette (CRNP) fabrication.

<p>(<b>a</b>) CRNP fabrication procedure. Quartz capillaries were filled with Fe(NO₃)₃ catalyst and left to dry, then pulled into pipettes of desired geometry. Carbon was grown within the pipette using chemical vapor deposition (CVD). (<b>b</b>) A SEM image of the tip of a CRNP, scale bar is 20 nm.</p

Injection of YOYO-1 into multi-cell embryos.

<p>(a) Two-cell embryo in which the P1 blastomere has been injected with 1 µM YOYO-1. (<b>b</b>) Four-cell embryo in which the ABa blastomere has been injected with 1 µM YOYO-1.</p