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

Summary

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.

Abstract

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.

Introduction

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 size^1-10. This is particularly true during embryogenesis and cell differentiation, when dramatic reductions in both cell and nuclear size are often observed^11,12. Furthermore, altered nuclear size is a key parameter in cancer diagnosis and prognosis^13-17. 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 as....

Protocol

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 from^27,28)

1. Prime female X. laevis frogs a minimum of three days and a maximum of two weeks before egg collection with a single 100 IU injection of pregnant mare serum gonadotropin (PMSG).
2. One day before the experiment, inject primed frogs with 800 IU of human chorionic gonadotropin (HCG) an....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 568 Donkey anti-mouse IgG	Molecular Probes	A10037
ATP disodium salt	Sigma Aldrich	A2383
Benzocaine	Sigma Aldrich	E1501
Bovine Serum Albumin	Sigma Aldrich	A3059
CaCl2	Sigma Aldrich	C3306
Centrifuge	Beckman	J2-21M
Centrifuge rotor	Beckman	JS 13.1
chymostatin	Sigma Aldrich	C7268
creatine phosphate disodium	Calbiochem	2380
cycloheximide	Sigma Aldrich	C6255
cytochalasin D	Sigma Aldrich	C8273
disposable wipes (kimwipes)	Sigma Aldrich	Z188956
L-cysteine	Sigma Aldrich	W326306
EGTA	Sigma Aldrich	E4378
Formaldehyde	Sigma Aldrich	F8775
Glass crystallizing dish (150 x 75 mm)	VWR	89090-662
Glycerol	Macron	5094-16
HEPES	Sigma Aldrich	H4034
Hoechst - bisBenzimide H 33342 trihydrochloride	Sigma Aldrich	B2261
HCG - Human Chorionic Gonadotropin	Prospec	hor-250-c
L15 Media	Sigma Aldrich	L4386
leupeptin	Sigma Aldrich	L2884
Lysolecithin	Sigma Aldrich	L1381
mAb414	Abcam	ab24609
MgCl2	EMD	MX0045-2
MgSO4	Sigma Aldrich	M9397
Maltose	Sigma Aldrich	M5885
NP40	BDH	56009
Paraformaldehyde	Electron Microscopy Sciences	15710
Penicillin + Streptomycin	Sigma Aldrich	Pp0781
pepstatin	Sigma Aldrich	P5318
PIPES	Sigma Aldrich	P6757
Plastic paraffin film (parafilm)	Sigma Aldrich	P7793
KCl	Sigma Aldrich	P9541
KH2PO4	Mallinckrodt	70100
KOH	Baker	5 3140
PMSG - Pregnant Mare Serum Gonadotropin	Prospec	hor-272-a
NaCl	Sigma Aldrich	S3014
NaHCO3	Fisher	BP328
Na2HPO4	EMD	SX0720-1
NaOH	EMD	SX0590
Pestle	Thomas Scientific	3411D56
Round bottom glass tubes, 15 ml	Corex	8441
Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG)	ThermoFisher	A10037
sucrose	Calbiochem	8550
thermal cycler	Bio-Rad	T100
Ultracentrifuge	Beckman	L8-80M
Ultracentrifuge rotor	Beckman	SW 50.1
Vectashield (anti-fade mounting medium)	Vector	H-1000