Small Molecule Injection into Single-Cell C. elegans Embryos via Carbon-Reinforced Nanopipettes

Lucy D Brennan,Shanna M Fellman,Paul M Mceuen,Kenneth J Kemphues,Mohammad Soltani,Thibault Roland,Sueyeon Chung,Michelle D Wang,Joshua W Kevek,Diane G Morton,Bob Goldstein

doi:10.1371/journal.pone.0075712

Abstract

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 Caenorhabditis elegans, 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 C. elegans 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.

Highlights

The ability to introduce chemical inhibitors into single cells has been an important approach for understanding signaling pathways in many organisms
We have developed an injection technique using carbon-reinforced nanopipettes (CRNPs) to introduce a chemical inhibitor into the single-celled C. elegans embryo and have used this approach to reinvestigate early polarity establishment
The use of carbon-reinforced nanopipettes to penetrate the durable eggshells of single-celled C. elegans embryos has provided a new technique with which to examine a very specific developmental event: the earliest stage of polarity establishment

Summary

Introduction

The ability to introduce chemical inhibitors into single cells has been an important approach for understanding signaling pathways in many organisms. Some cells, such as those of the Caenorhabditis elegans embryo, are surrounded by an outer layer that provides protection from the environment and makes application of drugs challenging. To address this difficulty, we have developed an injection technique using carbon-reinforced nanopipettes (CRNPs) to introduce a chemical inhibitor into the single-celled C. elegans embryo and have used this approach to reinvestigate early polarity establishment. It is clear that actomyosin contractility plays a significant role in anteroposterior (A-P) polarization [2,10,13] and there is evidence that microtubules can direct polarity initiation in the early embryo [14,15,16,17]

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Results

Conclusion