Name: a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation
Uploaded: 2016-08-08T14:00:00
Description: Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

All Xenopus procedures and studies were conducted in compliance with the NRC Guide for the Care and Use of Laboratory Animals 8th edition. Protocols were approved by the University of Wyoming Institutional Animal Care and Use Committee (Assurance # A-3216-01).
1. Preparation of X. laevis Egg Extract (adapted from27,28)
<ol>
	<li>Prime female&#160;X. laevis&#160;frogs a minimum of three days and a maximum of two weeks before egg collection with a single 100 IU injection of preg...

<ol>
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Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink...

The sizes of cellular organelles typically scale with the size of the cell, and this has been perhaps best documented for the scaling of nuclear size with cell size1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis13-17. Mechanisms that contribute to nuclear size regulation are largely unknown, in part due to the complexity and essential nature of nuclear structure and function. The method described here was developed as an in vitro assay for nuclear size scaling that is amenable to biochemical manipulation and elucidation of mechanisms of nuclear size regulation.
Xenopus laevis egg extract is a well-established system to recapitulate and study complex cellular processes in an in vitro context. These extracts have revealed new fundamental information about several cellular processes including the assembly and function of the mitotic spindle, endoplasmic reticulum, and nucleus18-22. One key advantage to the extract system is that X. laevis egg extracts represent a nearly undiluted cytoplasm whose composition can be easily altered, for instance through addition of recombinant proteins or immunodepletion. Furthermore, one is able to manipulate essential processes by employing treatments that might otherwise be lethal in an in vivo context. Modifications of the egg extract procedure allow for isolation of extracts from X. laevis embryos rather than eggs, and these embryo extracts are equally amenable to biochemical manipulation23. During X. laevis development, the single-cell fertilized embryo (~1 mm diameter) undergoes a series of twelve rapid cell divisions (stages 1 - 8) to generate several thousand 50 &#181;m diameter and smaller cells, reaching a developmental stage termed the midblastula transition (MBT) or stage 824-26. The MBT is characterized by the onset of zygotic transcription, cell migration, asynchronous cell divisions, acquisition of gap phases, and establishment of nuclear steady-state sizes rather than continual nuclear expansion as in the pre-MBT embryo. From stage 4 to gastrulation (stages 10.5 - 12), the volume of individual nuclei decreases by more than 10-fold11.
Here, the goal is to identify mechanisms responsible for these reductions in nuclear size during developmental progression. The approach is to first assemble nuclei in X. laevis egg extract and to isolate those nuclei from the egg cytoplasm/extract. These nuclei are then resuspended in cytoplasm from late gastrula stage embryos. After an incubation period, the nuclei from egg extract become smaller in late stage embryo extract. We reasoned that this would be a useful assay for identifying cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. Using this assay, coupled with in vivo validation, we demonstrated that protein kinase C (PKC) contributes to developmental reductions in nuclear size in X. laevis23.

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			<td>Alexa Fluor 568 Donkey anti-mouse IgG</td>
			<td>Molecular Probes</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP disodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Sigma Aldrich</td>
			<td>E1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman</td>
			<td>J2-21M</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge rotor</td>
			<td>Beckman</td>
			<td>JS 13.1</td>
			<td></td>
		</tr>
		<tr>
			<td>chymostatin</td>
			<td>Sigma Aldrich</td>
			<td>C7268</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphate disodium</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma Aldrich</td>
			<td>C6255</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin D</td>
			<td>Sigma Aldrich</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable wipes (kimwipes)</td>
			<td>Sigma Aldrich</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>W326306</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E4378</td>
			<td></td>
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			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass crystallizing dish (150x75 mm)</td>
			<td>VWR</td>
			<td>89090-662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Macron</td>
			<td>5094-16</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst - bisBenzimide H 33342 trihydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>HCG - Human Chorionic Gonadotropin&#160;</td>
			<td>Prospec</td>
			<td>hor-250-c</td>
			<td></td>
		</tr>
		<tr>
			<td>L15 Media</td>
			<td>Sigma Aldrich</td>
			<td>L4386</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Sigma Aldrich</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysolecithin</td>
			<td>Sigma Aldrich</td>
			<td>L1381</td>
			<td></td>
		</tr>
		<tr>
			<td>mAb414</td>
			<td>Abcam</td>
			<td>ab24609</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>EMD</td>
			<td>MX0045-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma Aldrich</td>
			<td>M9397</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Sigma Aldrich</td>
			<td>M5885</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40</td>
			<td>BDH</td>
			<td>56009</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin + Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>Pp0781</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Sigma Aldrich</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic paraffin film (parafilm)</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Mallinckrodt</td>
			<td>70100</td>
			<td></td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Baker</td>
			<td>5 3140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG - Pregnant Mare Serum Gonadotropin</td>
			<td>Prospec</td>
			<td>hor-272-a</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher</td>
			<td>BP328</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHPO4</td>
			<td>EMD</td>
			<td>SX0720-1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>EMD</td>
			<td>SX0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle</td>
			<td>Thomas Scientific</td>
			<td>3411D56</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottom glass tubes, 15 ml</td>
			<td>Corex</td>
			<td>8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)</td>
			<td>ThermoFisher</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Calbiochem</td>
			<td>8550</td>
			<td></td>
		</tr>
		<tr>
			<td>thermal cycler</td>
			<td>Bio-Rad</td>
			<td>T100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman</td>
			<td>L8-80M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor</td>
			<td>Beckman</td>
			<td>SW 50.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield (anti-fade mounting medium)</td>
			<td>Vector</td>
			<td>H-1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a cell-free assay using xenopus laevis embryo extracts to study mechanisms of nuclear size regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

Assembly of Nuclei in Egg Extract
The first steps of this protocol are to prepare X. laevis egg extract (Protocol 1) and demembranated sperm nuclei (Protocol 2). These reagents are then used to assemble nuclei de novo (Protocol 3). Figure 1 shows some representative data. Addition of calcium drives the meiotically arrested egg extract into interphase, and the cycloheximide keep...

Watch this Scientific Journal Video about A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation at JoVE.com

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

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Biology

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...