Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins

Summary

Typical microtubule inhibitors, used widely in basic and applied research, have far-reaching effects on cells. Recently, photostatins emerged as a class of photoswitchable microtubule inhibitors, capable of instantaneous, reversible, spatiotemporally precise manipulation of microtubules. This step-by-step protocol details the application of photostatins in a 3D live preimplantation mouse embryo.

Abstract

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. Traditional pharmacological disruption of the ubiquitous microtubule network using, for instance, nocodazole can have devastating consequences for any cell. Reversibly photoswitchable microtubule inhibitors have the potential to overcome the limitations by enabling drug effects to be implemented in a spatiotemporally-controlled manner. One such family of drugs is the azobenzene-based photostatins (PSTs). These compounds are inactive in dark conditions, and upon illumination with UV light, they bind to the colchicine-binding site of β-tubulin and block microtubule polymerization and dynamic turnover. Here, the application of PSTs in the 3-dimensional (3D) live preimplantation mouse embryo is set out to disrupt the microtubule network on a subcellular level. This protocol provides instructions for the experimental setup, as well as light activation and deactivation parameters for PSTs using live-cell confocal microscopy. This ensures reproducibility and enables others to apply this procedure to their research questions. Innovative photoswitches like PSTs may evolve as powerful tools to advance the understanding of the dynamic intracellular microtubule network and to non-invasively manipulate the cytoskeleton in real-time. Furthermore, PSTs may prove useful in other 3D structures such as organoids, blastoids, or embryos of other species.

Introduction

The microtubule architecture varies widely across different cell types to support diverse functions^1,2. Its dynamic nature of growth and shrinkage allows rapid adaptation to extra- and intracellular cues and to respond to the ever-changing needs of a cell. Hence, it can be considered as the "morphological fingerprint" playing a key role in cellular identity.

Pharmacological targeting of the microtubule cytoskeleton using small molecule inhibitors has led to a plethora of fundamental discoveries in developmental biology, stem cell biology, cancer biology, and neurobiology

Protocol

Experiments were approved by the Monash Animal Ethics Committee under animal ethics number 19143. Animals were housed in specific pathogen-free animal house conditions at the animal facility (Monash Animal Research Platform) in strict accordance with ethical guidelines.

1. Preimplantation mouse embryo collection

1. Superovulate and mate mice as described previously^16,18, in compliance with the institutional an.......

Discussion

The microtubule network is integral to the fundamental inner workings of a cell. Consequently, this presents challenges in manipulating microtubule dynamics in living organisms, as any perturbation to the network tends to have widespread consequences for all aspects of cellular function. The emergence of photoswitchable microtubule-targeting compounds presents a way to precisely manipulate the cytoskeleton at a subcellular level, with superior control for the induction and reversal of microtubule growth inhibition

Materials

Name	Company	Catalog Number	Comments
Aspirator tube	Sigma-Aldrich	A5177	For mouth aspiration apparatus
Chamber slides - LabTek	Thermo Fisher Scientific	NUN155411
cRNA encoding for EB3-dTomato	N/A	N/A	Prepared according to manufacturers instructions using mMessage in vitro Transcription kit
Culture dishes - 35mm	Thermo Fisher Scientific	150560
Human chorionic growth hormone	Sigma-Aldrich	C8554
Human Tubal Fluid (HTF) medium	Cosmo-Bio	CSR-R-B071
Imaris Image Analysis Software	Bitplane
Immersion Oil W 2010	Carl Zeiss	444969-0000-000	For use with microscope immersion objective
LED torch - Red light	Celestron	93588
M2 medium	Sigma-Aldrich	M7167
Mice - wild-type FVB/N, males and females	N/A	N/A	Females 8-9 weeks old. Males 2-6 months old.
Microcapillary Pipettes - Kimble	Sigma-Aldrich	Z543306	For mouth aspiration apparatus
Microinjection buffer	N/A	N/A	5 mM Tris, 5 mM NaCl, 0.1 mM EDTA, pH 7.4
Mineral oil	Origio	ART-4008-5P
mMessage In vitro Transcription kit	Thermo Fisher Scientific	AM1340
NanoDrop Spectrophotometer	Thermo Fisher Scientific
Potassium Simplex Optimised Medium (KSOM) medium	Cosmo-Bio	CSR-R-B074
Pregnant mare serum gonadotrophin	Prospec Bio	HOR-272
PST-1P	N/A	N/A	Borowiak, M. et al., Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell. 162 (2), 403-411, doi:10.1016/j.cell.2015.06.049, (2015).
RNA purification kit	Sangon	B511361-0100
Ultrapure water	Sigma-Aldrich	W1503
ZEN Black Software	Carl Zeiss