Name: spatiotemporal subcellular manipulation of the microtubule cytoskeleton in the living preimplantation mouse embryo using photostatins
Uploaded: 2021-11-30T15:00:00
Description: Watch this Scientific Journal Video about Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins at JoVE.com

The authors would like to thank Dr. Oliver Thorn-Seshold and Li Gao for providing us with photostatins and advice on manuscript preparation, Monash Production for filming support, and Monash Micro Imaging for microscopy support.
This work was supported by the National Health and Medical Research Council (NHMRC) project grant APP2002507 to J.Z. and the Canadian Institute for Advanced Research (CIFAR) Azrieli Scholarship to J.Z. The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.

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
<ol>
	<li>Superovulate and mate mice as described previously16,18, in compliance with the institutional an...

<ol>
	<li>Hawdon, A., Aberkane, A., Zenker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-dependent+subcellular+organisation+of+pluripotent+cells.">Microtubule-dependent subcellular organisation of pluripotent cells.</a> Development. 148 (20), (2021).</li><li>Sanchez, A. D., Feldman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule-organizing+centers:+from+the+centrosome+to+non-centrosomal+sites.">Microtubule-organizing centers: from the centrosome to non-centrosomal sites.</a> Current Opinion in Cell Biology. 44, 93-101 (2017).</li><li>Galli, M., Morgan, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+size+determines+the+strength+of+the+spindle+assembly+checkpoint+during+embryonic+development.">Cell size determines the strength of the spindle assembly checkpoint during embryonic development.</a> Developmental Cell. 36 (3), 344-352 (2016).</li><li>Vazquez-Diez, C., Paim, L. M. G., FitzHarris, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-size-independent+spindle+checkpoint+failure+underlies+chromosome+segregation+error+in+mouse+embryos.">Cell-size-independent spindle checkpoint failure underlies chromosome segregation error in mouse embryos.</a> Current Biology. 29 (5), 865-873 (2019).</li><li>Baudoin, J. P., Alvarez, C., Gaspar, P., Metin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nocodazole-induced+changes+in+microtubule+dynamics+impair+the+morphology+and+directionality+of+migrating+medial+ganglionic+eminence+cells.">Nocodazole-induced changes in microtubule dynamics impair the morphology and directionality of migrating medial ganglionic eminence cells.</a> Developmental Neuroscience. 30 (1-3), 132-143 (2008).</li><li>Munz, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells+lose+their+functional+properties+after+paclitaxel+treatment.">Human mesenchymal stem cells lose their functional properties after paclitaxel treatment.</a> Scientific Reports. 8 (1), 312 (2018).</li><li>Jordan, M. A., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+as+a+target+for+anticancer+drugs.">Microtubules as a target for anticancer drugs.</a> Nature Reviews Cancer. 4 (4), 253-265 (2004).</li><li>Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P., Cassimeris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomolar+concentrations+of+nocodazole+alter+microtubule+dynamic+instability+in+vivo+and+in+vitro.">Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.</a> Molecular Biology of the Cell. 8 (6), 973-985 (1997).</li><li>Borowiak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitchable+inhibitors+of+microtubule+dynamics+optically+control+mitosis+and+cell+death.">Photoswitchable inhibitors of microtubule dynamics optically control mitosis and cell death.</a> Cell. 162 (2), 403-411 (2015).</li><li>Gaspari, R., Prota, A. E., Bargsten, K., Cavalli, A., Steinmetz, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+cis-+and+trans-Combretastatin+binding+to+tubulin.">Structural basis of cis- and trans-Combretastatin binding to tubulin.</a> Chem. 2 (1), 102-113 (2017).</li><li>Thorn-Seshold, O., Meiring, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocontrolling+microtubule+dynamics+with+photoswitchable+chemical+reagents.">Photocontrolling microtubule dynamics with photoswitchable chemical reagents.</a> ChemRxiv. , (2021).</li><li>Kopf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+control+cellular+shape+and+coherence+in+amoeboid+migrating+cells.">Microtubules control cellular shape and coherence in amoeboid migrating cells.</a> Journal of Cell Biology. 219 (6), 201907154 (2020).</li><li>Sawada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlexinD1+signaling+controls+morphological+changes+and+migration+termination+in+newborn+neurons.">PlexinD1 signaling controls morphological changes and migration termination in newborn neurons.</a> The EMBO Journal. 37 (4), 97404 (2018).</li><li>Singh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+microtubule+dynamics+directs+cell+mechanics+and+coordinates+forces+during+epithelial+morphogenesis.">Polarized microtubule dynamics directs cell mechanics and coordinates forces during epithelial morphogenesis.</a> Nature Cell Biology. 20 (10), 1126-1133 (2018).</li><li>Kepiro, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azidoblebbistatin,+a+photoreactive+myosin+inhibitor.">Azidoblebbistatin, a photoreactive myosin inhibitor.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (24), 9402-9407 (2012).</li><li>Zenker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtubule-organizing+center+directing+intracellular+transport+in+the+early+mouse+embryo.">A microtubule-organizing center directing intracellular transport in the early mouse embryo.</a> Science. 357 (6354), 925-928 (2017).</li><li>White, M. D., Zenker, J., Bissiere, S., Plachta, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instructions+for+assembling+the+early+mammalian+embryo.">Instructions for assembling the early mammalian embryo.</a> Developmental Cell. 45 (6), 667-679 (2018).</li><li>Zenker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+actin+rings+zipper+the+mouse+embryo+for+blastocyst+formation.">Expanding actin rings zipper the mouse embryo for blastocyst formation.</a> Cell. 173 (3), 776-791 (2018).</li><li>Theisen, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+and+motor+proteins+support+zebrafish+neuronal+migration+by+directing+cargo.">Microtubules and motor proteins support zebrafish neuronal migration by directing cargo.</a> Journal of Cell Biology. 219 (10), 201908040 (2020).</li><li>Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pronuclear+microinjection+of+mouse+zygotes.">Pronuclear microinjection of mouse zygotes.</a> Methods in Molecular Biology. 254, 165-194 (2004).</li><li>Greaney, J., Subramanian, G. N., Ye, Y., Homer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+in+vitro+culture+of+mouse+oocytes.">Isolation and in vitro culture of mouse oocytes.</a> Bio-protocol. 11 (15), 4104 (2021).</li><li>Subramanian, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocytes+mount+a+noncanonical+DNA+damage+response+involving+APC-Cdh1-mediated+proteolysis.">Oocytes mount a noncanonical DNA damage response involving APC-Cdh1-mediated proteolysis.</a> Journal of Cell Biology. 219 (4), 201907213 (2020).</li><li>Mihajlovic, A. I., Bruce, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+cell-fate+decision+of+mouse+preimplantation+embryo+development:+integrating+cell+position+and+polarity.">The first cell-fate decision of mouse preimplantation embryo development: integrating cell position and polarity.</a> Open Biology. 7 (11), 170210 (2017).</li><li>Roostalu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+speed+of+GTP+hydrolysis+determines+GTP+cap+size+and+controls+microtubule+stability.">The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.</a> Elife. 9, 51992 (2020).</li><li>Gao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+photocontrol+of+microtubule+dynamics+and+integrity,+migration+and+mitosis,+by+the+potent+GFP-imaging-compatible+photoswitchable+reagents+SBTubA4P+and+SBTub2M.">In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M.</a> BioRxiv. bioRxiv. , (2021).</li><li>Tichy, A. M., Gerrard, E. J., Legrand, J. M. D., Hobbs, R. M., Janovjak, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+strategy+and+vector+library+for+the+rapid+generation+of+modular+light-controlled+protein-protein+interactions.">Engineering strategy and vector library for the rapid generation of modular light-controlled protein-protein interactions.</a> Journal of Molecular Biology. 431 (17), 3046-3055 (2019).</li><li>van Haren, J., Adachi, L. S., Wittmann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+microtubule+dynamics.">Optogenetic control of microtubule dynamics.</a> Methods in Molecular Biology. 2101, 211-234 (2020).</li><li>Adikes, R. C., Hallett, R. A., Saway, B. F., Kuhlman, B., Slep, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microtubule+dynamics+using+an+optogenetic+microtubule+plus+end-F-actin+cross-linker.">Control of microtubule dynamics using an optogenetic microtubule plus end-F-actin cross-linker.</a> Journal of Cell Biology. 217 (2), 779-793 (2018).</li><li>Kogler, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extremely+rapid+and+reversible+optogenetic+perturbation+of+nuclear+proteins+in+living+embryos.">Extremely rapid and reversible optogenetic perturbation of nuclear proteins in living embryos.</a> Developmental Cell. 56 (16), 2348-2363 (2021).</li><li>Maghelli, N., Tolic-Norrelykke, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+of+the+microtubule+cytoskeleton:+setting+up+and+working+with+an+ablation+system.">Laser ablation of the microtubule cytoskeleton: setting up and working with an ablation system.</a> Methods in Molecular Biology. 777, 261-271 (2011).</li><li>Bukhari, S. N. A., Kumar, G. B., Revankar, H. M., Qin, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+combretastatins+as+potent+tubulin+polymerization+inhibitors.">Development of combretastatins as potent tubulin polymerization inhibitors.</a> Bioorganic Chemistry. 72, 130-147 (2017).</li><li>Gilazieva, Z., Ponomarev, A., Rutland, C., Rizvanov, A., Solovyeva, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promising+applications+of+tumor+spheroids+and+organoids+for+personalized+medicine.">Promising applications of tumor spheroids and organoids for personalized medicine.</a> Cancers (Basel). 12 (10), 2727 (2020).</li><li>Scherer, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+imaging+and+uptake+of+the+anticancer+drug+combretastatin+in+cell+spheroids+and+photoisomerization+in+gels+with+multiphoton+excitation.">Three-dimensional imaging and uptake of the anticancer drug combretastatin in cell spheroids and photoisomerization in gels with multiphoton excitation.</a> Journal of Biomedical Optics. 20 (7), 78003 (2015).</li></ol>

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<sup cl...

The microtubule architecture varies widely across different cell types to support diverse functions1,2. 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 &#34;morphological fingerprint&#34; 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 neurobiology3,4,5,6,7. This approach, while indispensable, presents various limitations such as toxicity and off-target effects. For example, one of the most widely used microtubule-targeting agents, nocodazole, is a powerful microtubule-depolymerizing drug8. However, small-molecule inhibitors such as nocodazole are active from the time of application and, given the essential nature of the microtubule cytoskeleton to many critical cellular functions, global depolymerization of microtubules can produce off-target effects, which may be unsuitable for many applications. Additionally, nocodazole treatment is irreversible unless samples are washed free of the drug, preventing&#160;continuous live imaging and, thus, precise tracking of individual microtubule filaments.
The development of light-activated compounds began with the creation of photouncaged molecules and has heralded a new era in targeting and monitoring the effects of microtubule growth inhibition in a precise and spatiotemporally-controlled manner. One family of reversibly photoswitchable drugs, photostatins (PSTs), were developed by replacing the stilbene component of combretastatin A-4 with azobenzene9. PSTs are inactive until illumination with UV light, whereby the inactive trans-configuration converts to the active cis-configuration by reversible isomerization. Cis-PSTs inhibit microtubule polymerization by binding to the colchicine binding site of &#946;-tubulin, blocking its interface with &#946;-tubulin and preventing dimerization required for microtubule growth10. Among a cohort of PSTs, PST-1P has emerged as a lead compound as it has the highest potency, is fully water-soluble, and shows a rapid onset of bioactivity after illumination.
The most effective trans- to cis-isomerization of PSTs occurs at wavelengths between 360-420 nm, which enables dual options for PST activation. A 405 nm laser line on a typical confocal microscope can be administered for optimal spatial targeting of microtubule growth inhibition. The ability to pinpoint the location and timing of PST activation through 405 nm laser illumination facilitates precise temporal and spatial control, allowing disruption of microtubule dynamics on a subcellular level, within sub-second response times9. Alternatively, an affordable LED UV light allows whole organism illumination to induce organism-wide disruption of the microtubule architecture. This may be a cost-effective alternative for researchers for whom the precisely-timed onset of inhibition, rather than spatial targeting, is the goal. Another feature of PSTs is their on-demand inactivation by applying green light of a wavelength in the 510-540 nm range9. This enables tracing of microtubule filaments before, during, and after PST-mediated growth inhibition.
PSTs, while still a relatively recent design, have been used in numerous in vitro applications across diverse research fields11, including investigating new mechanisms of cell migration in amoeboids12, in neurons isolated from the brain of the newborn mouse13, and wing epithelium development in Drosophila melanogaster14. Other light-reactive drugs have proven to be valuable tools in targeted disruption of cellular function. For instance, an analog of blebbistatin,&#160;azidoblebbistatin, was used for enhanced myosin inhibition under illumination15,16. This highlights the potential for new discoveries owing to the ability for spatiotemporally controlled inhibition of cellular function.
Live 3D organisms present superb yet more delicate systems to manipulate microtubule dynamics on a whole-animal, single-cell, or subcellular level under physiological conditions. In particular, the preimplantation mouse embryo offers exceptional insight into the inner workings of the cell as well as intercellular relationships within an organism17. Temporally and spatially targeted consecutive cycles of activation and deactivation of PSTs contributed to the characterization of the interphase bridge, a post-cytokinetic structure between cells, as a non-centrosomal microtubule-organizing center in the preimplantation mouse embryo16. A similar experimental setup demonstrated the involvement of growing microtubules in the sealing of the mouse embryo to allow blastocyst formation18. Furthermore, PSTs were also used in whole zebrafish embryos to investigate neuronal cell migration by inhibiting microtubule growth in a subset of cells in the hindbrain19.
This protocol describes the experimental setup and use of PST-1P in the preimplantation mouse embryo. The instructions presented here can also guide the application of PSTs for a wide array of objectives such as studying chromosome segregation and cell division, trafficking of intracellular cargo, and cell morphogenesis and migration. Furthermore, such studies will assist the implementation of PSTs&#160;in organoid systems, blastoids, and other embryo models such as Caenorhabditis elegans and Xenopus laevis, as well as potentially expand the use of PSTs for in vitro fertilization technologies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aspirator tube</td>
			<td>Sigma-Aldrich</td>
			<td>A5177</td>
			<td>For mouth aspiration apparatus</td>
		</tr>
		<tr>
			<td>Chamber slides - LabTek</td>
			<td>Thermo Fisher Scientific</td>
			<td>NUN155411</td>
			<td></td>
		</tr>
		<tr>
			<td>cRNA encoding for EB3-dTomato</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared according to manufacturers instructions using mMessage in vitro Transcription kit</td>
		</tr>
		<tr>
			<td>Culture dishes - 35mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>150560</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chorionic growth hormone</td>
			<td>Sigma-Aldrich</td>
			<td>C8554</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Tubal Fluid (HTF) medium</td>
			<td>Cosmo-Bio</td>
			<td>CSR-R-B071</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris Image Analysis Software</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil W 2010</td>
			<td>Carl Zeiss</td>
			<td>444969-0000-000</td>
			<td>For use with microscope immersion objective</td>
		</tr>
		<tr>
			<td>LED torch - Red light</td>
			<td>Celestron</td>
			<td>93588</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Sigma-Aldrich</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice - wild-type FVB/N, males and females</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Females 8-9 weeks old. Males 2-6 months old.</td>
		</tr>
		<tr>
			<td>Microcapillary Pipettes - Kimble</td>
			<td>Sigma-Aldrich</td>
			<td>Z543306</td>
			<td>For mouth aspiration apparatus</td>
		</tr>
		<tr>
			<td>Microinjection buffer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>5 mM Tris, 5 mM NaCl, 0.1 mM EDTA, pH 7.4</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Origio</td>
			<td>ART-4008-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage In vitro Transcription kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Simplex Optimised Medium (KSOM) medium</td>
			<td>Cosmo-Bio</td>
			<td>CSR-R-B074</td>
			<td></td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotrophin</td>
			<td>Prospec Bio</td>
			<td>HOR-272</td>
			<td></td>
		</tr>
		<tr>
			<td>PST-1P</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>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).</td>
		</tr>
		<tr>
			<td>RNA purification kit</td>
			<td>Sangon</td>
			<td>B511361-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Sigma-Aldrich</td>
			<td>W1503</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN Black Software</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatiotemporal subcellular manipulation of the microtubule cytoskeleton in the living preimplantation mouse embryo using photostatins

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 &#946;-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.

In line with the protocol, preimplantation mouse embryos were microinjected with cRNA for EB3, tagged with red fluorescent dTomato (EB3-dTomato). This enables the visualization of growing microtubules as EB3 binds to polymerizing microtubule plus ends24.
The experiments were performed 3 days post-fertilisation (E3) when the mouse embryo is comprised of 16 cells. Any other preimplantation developmental stage can be used, depending on the scientific question to be investi...

Watch this Scientific Journal Video about Spatiotemporal Subcellular Manipulation of the Microtubule Cytoskeleton in the Living Preimplantation Mouse Embryo using Photostatins at JoVE.com

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

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.

The microtubule cytoskeleton forms the framework of a cell and is fundamental for intracellular transport, cell division, and signal transduction. ...

This protocol is the first of its kind to apply photostatins on the living preimplantation mouse embryo, and regard uses to use light-switchable drugs on other 3D physiological systems. Light-switchable photostatins allow the manipulation of microtubule growth, in space and time, and to be converted back to an off state. Unintentionally exposing photostatins to blue light, prior to the experiment, can activate them prematurely.So we advise working in complete darkness, or red light conditions, at all times when preparing your samples. In strict dark conditions, using only red light for illumination, begin making stock and intermediate working solution of PST-1P in ultrapure water. As described in the text manuscript.Then, dilute PST-1P in fresh KSOM to achieve the final concentration of 40 M.To prepare the chamber slide for live imaging, add 10 L of PST-1P-treated KSOM into the center of one well, to form a hemispherical droplet. To prevent the droplet from evaporating, cover it with sufficient mineral oil. Then, loosely wrap the prepared culture dish in foil, or place the culture dish in a non-airtight opaque container in the incubator to ensure no light exposure.After transferring the embryos into the PST-1P-treated KSOM drop, ensure that the embryos are clustered in the center of the droplet, and settled to the bottom of the dish. Once the immersion objective lens is prepared, mount the chamber slide on the microscope, inside an environmental chamber. Set at 37 C and 5%CO2, and in complete darkness, to ensure all PST-1Ps are in the inactive trans-configuration, and that embryos can sink to the bottom of the dish.Use a red light torch to position the objective lens in contact with the immersion medium. Then, locate the edge of the droplet of the medium and position the objective directly over it. Using the live-scanning mode, and a 561 nm laser, locate the embryos within the droplet.Use stage controllers and live-scanning mode to set the start and end points for acquiring a z-stack of the whole embryo. Adjust the laser power settings up to a maximum of 5%according to your signal. And set digital offset to a maximum of 0.900, according to your signal, to optimize the appearance of the EB3-dTomato comets.Minimize background noise. Set the pinhole at 2 m. Pixel resolution at 512 by 512.And the pixel dwell time at 3.15 s. And set the zoom to 2x. Acquire a z-stack of the whole embryo with 1 m section intervals to assess the areas of microtubule growth in the whole organism.For EB3-dTomato comet tracking experiments, open the 3D z-stack image, increase the zoom to three times. And draw a rectangular ROI around the specific subcellular area of interest. Then, acquire the time lapse movie of a single z-plane, using the set parameters, with the time interval of 500 ms.To activate PST-1P, switch to a 405 nm laser, and acquire another time-lapse, with the laser set to 10%power, pinhole opened maximally, pixel resolution of 512 by 512, the pixel dwell time of 3.15 s, zoom of three times, at a time interval of 500 ms, and set 20 frames to be acquired. Immediately after activation, switch back to 561 nm laser, and repeat acquisition, as demonstrated previously, to confirm the loss of EB3-dTomato comets after activation of PST-1P. Now, to reverse PST-1P back to its inactive trans-state, engage a 514 nm laser, and acquire another time-lapse with set parameters.To visualize the recovery of EB3-dTomato comets, switch back to 561 nm laser, and repeat acquisition. In untreated control embryos, before illumination with a 405 nm laser, the EB3-dTomato signal was high within the entire cytoplasm of the cell, except the nuclear area. Post illumination, changes in the signal were not detected.Indicating that the 405 nm laser did not cause photo damage to the embryo or microtubule dynamics. During PST-1P treatment of 16-cell stage embryos, under strict dark conditions, strong EB3-dTomato expression was detected in the 3D image, demonstrating that inactive PST-1P did not elicit inhibition of microtubule polymerization under dark conditions. Before activation of PST-1P, the time-lapse imaging of a single z-plane confirmed strong EB3-dTomato expression, and microtubule polymerization in a subcellular region.After 405 nm light activation, within seconds, a reduction in the EB3-dTomato comet signal was detected. And recovery of the signal was achieved upon deactivation of PST-1P, using a 514 nm laser. The embryos micro injected with EB3-dTomato, and incubated with PST-1P, showed normal embryonic potential, developing to the blastocyst stage.And normal microtubule dynamics during mitosis. Indicating non-toxicity of an active PST-1P to the embryo. In the acquired time-lapse movies, EB3-dTomato labeling appeared as comet-like structures, and enabled tracking of growing microtubule filaments.In PST-1P treated embryos, kept in dark conditions, individual EB3-dTomato comets emanated from the interphase bridge, which acts as a non-centrosomal microtubule organizing center in the early mouse embryo. A t-stack shows EB3-dTomato comets in the region of the interphase bridge. Following activation of PST-1P, EB3-dTomato comets were reduced at the base of the interphase bridge and in the t-stack.Remember that white light can activate PST-1P prematurely. So use only red light sources for visibility. Green light also deactivates PST-1P, so please avoid using green fluorescent label markers.This technique has been used in drosophila, amoeba, mice, and multiple in vitro systems, to target microtubule growth in order to study cell migration and tissue formation.

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...