Nuclear envelope formation in vitro: a sea urchin egg cell-free system.
ABSTRACT The formation of the nuclear envelope (NE) typically occurs once during every mitotic cycle in somatic cells, and also around the sperm nucleus following fertilization. Much of our understanding of NE assembly has been derived from systems modeling the latter event in vitro. In these systems, demembranated sperm nuclei are combined with fertilized egg cytoplasmic extracts and an ATP-regenerating system and in a multistep process they form the functional double bilayer of the NE. Using a system that we developed from sea urchin gametes, we have demonstrated that NE assembly is regulated by membrane vesicles in a spatial and temporal fashion, emphasizing the roles of phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), diacylglycerols (DAG), and lipid-modifying enzymes in NE assembly.
- SourceAvailable from: Dominic L Poccia[Show abstract] [Hide abstract]
ABSTRACT: Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCgamma), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P(2)) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.PLoS ONE 01/2010; 5(8):e12208. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.PLoS ONE 01/2012; 7(7):e40669. · 3.73 Impact Factor
Nuclear Envelope Formation In Vitro:
A Sea Urchin Egg Cell-Free System
Richard D. Byrne, Vanessa Zhendre, Banafshé Larijani,
and Dominic L. Poccia
Keywords Nuclear envelope; Sea urchin; Phosphatidylinositol; Phosphatidyl-
choline; Cholesterol; Phospholipase C; PtdIns(4;5)P2; Diacylglycerol; Liquid NMR;
Liquid nuclear magnetic resonance
Abstract The formation of the nuclear envelope (NE) typically occurs once during
every mitotic cycle in somatic cells, and also around the sperm nucleus following fer-
tilization. Much of our understanding of NE assembly has been derived from systems
modeling the latter event in vitro. In these systems, demembranated sperm nuclei are
combined with fertilized egg cytoplasmic extracts and an ATP-regenerating system and
in a multistep process they form the functional double bilayer of the NE. Using a system
that we developed from sea urchin gametes, we have demonstrated that NE assembly
is regulated by membrane vesicles in a spatial and temporal fashion, emphasizing
the roles of phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2), diacylglycerols (DAG), and lipid-modifying enzymes in NE assembly.
Cell-free systems derived from the gametes of a number of species have been used to
elucidate the mechanisms of nuclear envelope assembly. The sea urchin has a number
of advantageous features (1–3). First, sea urchins produce millions of gametes when
fertile, allowing the purification of large amounts of protein and lipids. Second, the in
vitro system is an open one, allowing easy manipulation with pharmacological inhibi-
tors and recombinant proteins (4–6). Third, sea urchin eggs have completed meiosis,
and after fertilization they progress through the first mitotic cycles with a high degree
of synchrony, allowing extracts of various cell cycle stages to be prepared. Finally, the
components of the cell-free system, the egg cytoplasmic extract (S10) and demem-
branated nuclei, can be further fractionated (7, 8), the former to membrane vesicle
fractions (MVs) and cytosol (S150) and the latter to chromatin and detergent-resistant
membranous structures (corresponding to sperm nuclear envelope remnants also seen
in vivo (1) and previously referred to as lipophilic structures [LSs] (2)).
R. Hancock (ed.) The Nucleus: Volume 2: Chromatin, Transcription, Envelope,
Proteins, Dynamics, and Imaging,
© Humana Press 2008
208 R.D. Byrne et al.
The sea urchin cell-free system has proven to be suited to nuclear envelope stud-
ies due to the stepwise assembly of the envelope (3). Following addition of nuclei
to egg cytoplasmic extracts and an ATP-regenerating system, MVs initially bind to
the surface of decondensing chromatin. In the presence of GTP, in a step requiring
an endogenous phospholipase C (PLC) activity and the hydrolysis of PtdIns (4,5)P2
to form the fusogenic lipid DAG, these vesicles fuse to form a double bilayer (4, 5).
In a final step, the pronucleus expands approximately twofold in diameter in the
presence of additional ATP (4), incorporating more membrane and functional
nuclear pores. Nuclei at these different steps of assembly can be isolated as
required, and their lipid contents determined and quantified (4, 5).
We have previously described membrane domains within both the demem-
branated nuclei and the egg cytoplasmic extracts that contribute in an obligatory
manner to the formation of the nuclear envelope in vitro (7, 8). For example, MV1
is a polyphosphoinositide-rich membrane fraction (5) containing very high levels
of phosphatidylinositol (PI)–PLC and PtdIns (4,5)P2(6), which can be separated
from other egg cytoplasmic membranes on sucrose gradients by virtue of its low
buoyant density (7). Also, detergent resistant membrane structures (sperm nuclear
envelope remnants) on demembranated sperm can be isolated (2). Analysis of these
fractions has revealed novel features that indicate how they participate in nuclear
envelope assembly (8).
2.1 Gamete Collection, Preparation of Egg Cytoplasmic
Extracts, and Sperm Nuclei Isolation
1. 0.5 M KCl. Stored at room temperature.
2. 22-gauge, 1.5-inch needles (Sigma-Aldrich, Dorset, UK or St. Louis, MO,
3. Medium-size weighing boats.
4. Millipore-filtered artificial seawater (MPSW). Reef salt (Instant Ocean; Aqua-
Medic Commercial, Telford, UK) is dissolved at a concentration of 33 g/L and
stirred overnight. This is filtered through a 0.22-µm membrane filter (500 mL
GP Express PLUS; Millipore, Watford, UK or Billerica, MA, USA). The fil-
tered seawater is hereafter referred to as MPSW (see Note 1).
5. 3-amino-1,2,4-triazole (ATA), 30 mM: prepared fresh as a 10× stock in distilled
water no more than 30 min before use and stored on ice.
6. Nylon mesh of 64, 100, 120, and 210 µm (Sefar, Bury, UK or Kansas City, MO,
7. Lysis buffer (LB): 10 mM Hepes, pH 8.0, 250 mM NaCl, 5 mM MgCl2, 110 mM
glycine, 250 mM glycerol, and 1 mM DTT, supplemented with 1 mM PMSF
immediately before use.
12 Nuclear Envelope Formation In Vitro 209
8. Nuclei extraction buffer (SXN): 50 mM Hepes, pH 7.2, 250 mM sucrose,
150 mM NaCl, 0.5 mM spermidine, 0.15 mM spermine, and 300 mM glucose,
aliquot in 50-mL volumes and store at −80°C.
9. Bath sonicator: Ultrawave U50 230 V 50 Hz, 500 mL (Ultrawave, Cardiff, UK
or Fischer Scientific, Fair Lawn, NJ, USA).
10. Nuclei freezing buffer: 10 mL of SXN, 2 mL of 3% (w/v) BSA in SXN, 6 mL
of glycerol. Prepare fresh on day of use.
2.2 Nuclear Envelope Assembly Assay
1. TN buffer: 10 mM Tris-HCl, pH 7.2, 150 mM NaCl in distilled water, aliquot
in 50-mL volumes and store at −80°C.
2. 100 mM ATP, 5 mM GTP (Sigma), 1 M creatine phosphate, and 2.5 mg/mL
creatine phosphokinase. All are made up in LB, aliquoted in 10-µL amounts
and stored at −80°C.
3. CaCl2 stock solutions: 500 nM for L. pictus and 1.25 µM for P. lividus, and
250 mM EGTA stock solution, all in LB.
4. TN supplemented with 5 mM MgCl2 and 0.5 M sucrose, frozen at −20°C in 1-
5. Microscope slides, circular coverslips (13-mm diameter), and nail varnish.
6. 3′3-dihexyloxacarbocyanine iodide (DiOC6) and 1,1′-didodecyl-3,3,3′,3′-
tetramethylindocarbocyanine perchlorate (DiIC12; Invitrogen, Paisley, UK or
Carlsbad, CA, USA) are dissolved in methanol at 10 mg/mL. These dye solu-
tions are stored in the dark at room temperature. Immediately before use they
are diluted 10-fold in LB (for DiOC6) or methanol (for DiIC12; see Note 2).
2.3 Preparation of Fertilized Egg Soluble Protein (S150)
and Subfractionation of Egg MV Fractions
1. Membrane wash buffer (MWB): 50 mM Hepes, pH 7.5, 250 mM sucrose,
50 mM KCl, 1 mM DTT, 1 mM ATP, and 1 mM PMSF in distilled water, frozen
in 50-mL aliquots at −80°C.
2. Soniprep 150 probe sonicator (Sanyo-Gallenkamp, Leicester, UK or Palisades
Park, NJ, USA).
3. 2 M sucrose in TN, frozen in 25-mL aliquots at −20°C.
4. Gradient maker (Hoefer, San Francisco, CA, USA) and gradient fractionator
(Auto Densi-flow; GRI, Essex, UK or Buchler Instruments, Fort Lee, NJ,
5. Mineral oil.
6. Ultracentrifuge tubes (Beckman, Buckinghamshire, UK or Fullerton, CA,
USA; see Note 3).
210 R.D. Byrne et al.
7. Ultracentrifuge and SW40 rotor (Beckman).
8. 22-gauge, 1.5-inch needles.
2.4 Isolation of Detergent-Resistant Membranes
from 0.1% Triton X-100-Treated Nuclei
1. Triton X-100 solution: 10% v/v in distilled water, stored at room temperature.
2. Bath sonicator: Ultrawave U50 230 V, 50 Hz, 500 mL (Ultrawave).
2.5 Detection of Sea Urchin Proteins by Western Analysis
1. 10 mL LB supplemented with 0.1% Triton X-100 and a Complete Mini pro-
tease inhibitor cocktail tablet (Roche, Mannheim, Germany or Indianapolis,
IN, USA; cat. 1836153).
2. 4× sodium dodecyl sulfate (SDS) sample buffer: 250 mM Tris, pH 6.8, 20% (v/v)
glycerol, 4% SDS, 0.01% bromophenol blue, 50 mM β-mercaptoethanol.
3. Heating block.
4. NuPAGE Novex 4–12% bis-tris pre-cast gels and NuPAGE MOPS SDS
running buffer (Invitrogen).
5. Mini-electrophoresis cell (XCell Surelock; Invitrogen).
6. Blotting paper and Immobilon-P transfer membrane (Millipore, Watford, UK
or Billerica, MA, USA).
8. Transfer buffer: 25 mM Tris base, 192 mM glycine, and 20% (v/v) methanol.
9. Semi-dry transfer cell (Trans-Blot SD; Bio-Rad, Hertfordshire, UK or
Hercules, CA, USA).
10. Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
and 1.76 mM KH2PO4, pH 7.2), supplemented with 0.2% (v/v) Tween-20 to
11. Milk powder and ultrapure 30% w/v BSA solution (Sigma). These are added
to PBST to make 5% (w/v) and 3% (v/v) solutions respectively.
12. Anti-goat HRP (Perbio, Northumberland, UK or Rockford, IL, USA), anti-
mouse HRP serum and ECL detection reagents (both from GE Healthcare,
Amersham, UK or Piscataway, NJ, USA).
13. X-ray film (Cronex-5; AGFA, Mortsel, Belgium or Greenville, SC, USA).
2.6 Lipid Extraction Procedure—Modified Folch Method
1. 3% (v/v) solution of dimethyldichlorosilane in HPLC-grade toluene (Fischer
Scientific, Fair Lawn, NJ). This solution is made fresh on the day of silanation.
12 Nuclear Envelope Formation In Vitro 211
2. HPLC-grade methanol and HPLC-grade water (Fischer Scientific).
3. Chloroform/methanol 2.5:1 (Fischer Scientific). This is made by first acidifying
the methanol with a drop of 10 N HCl (1 drop per 100–200 mL methanol) and
subsequently adding the required volume of chloroform. The solution is stored
at room temperature.
4. Probe sonicator (Soniprep 150; Sanyo-Gallenkamp, UK or Palisades Park, NJ,
5. Filter apparatus: 0.22-µm GV Durapore membrane filters, conical flask with
side arm, funnel, and clamp (Millipore).
6. K4EDTA, pH 6.0 (5): 5.84 g of EDTA (purified grade, Sigma) is dissolved in
100 mL distilled water. Thirty pellets of KOH (Sigma) are added periodically
7. Nitrogen source, sample incubator (Techne Dri-block DB3), and sample concen-
trator (Barloworld Scientific, Staffordshire, UK).
2.7 Solution Nuclear Magnetic Resonance (NMR)
1. Deuterated solvents (chloroform [CDCl3], methanol [CD3OD], and
0.2 M EDTA in D2O (Sigma)) made up as CDCl3/CD3OD/K4 EDTA
2. 5-mm NMR tubes (Wilmad NMR tubes, Royal Imperial grade, Sigma-
3. Bruker AMX-2 500 MHz NMR spectrometer with a Bruker 5-mm
broadband indirect probe tuned to 202 MHz and 500.13 Hz, stabilized
at 298 K.
4. Dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylserine
(DPPS), dipalmitoyl phosphatidylethanolamine (DPPE), and phosphati-
dylinositol (PI) sodium salt (Sigma; or Avanti Polar Lipids, Alabaster,
5. XWIN-NMR software (Bruker, Billerica, MA, USA).
This section details the methods we have developed to isolate fractions from sea
urchin egg and sperm that contribute to the assembly of the nuclear envelope.
We emphasize additions and corrections to detailed methods previously published.
We present a typical Western blotting procedure useful for localization of protein
antigens to individual membrane fractions. We have used 2-D NMR and mass
spectrometry methods to quantify phospholipid species in sea urchin extracts.
A procedure for 2-D solution NMR is presented here. Lipid mass spectrometry of
phospholipids is described elsewhere in this series (9).
212 R.D. Byrne et al.
3.1 Collection of Gametes
1. Lytechinus pictus, Strongylocentrotus purpuratus, and Paracentrotus lividus
sea urchins are injected twice intracoelomically (100 µL each injection for L.
pictus, approximately 300 µL for the latter two species) with 0.5 M KCl fol-
lowed by swirling the animals to distribute the KCl (see Note 4).
2. Eggs (orange) are collected by placing the inverted sea urchin over a full
beaker of MPSW. Sperm (white/pale brown) are collected “dry” by placing
an inverted sea urchin into a plastic weighing boat. Female sea urchins are
kept moist during this procedure by the regular application of MPSW;
males are covered with MPSW-soaked tissue to avoid dilution of the
3.2 Egg Lysis and Preparation of Fertilized Egg
1. All the following steps take place at 16°C unless otherwise stated.
2. Eggs from individual animals are first checked to ensure they fertilize cor-
rectly. Sperm is diluted approximately 500-fold in MPSW and a few drops are
added to eggs before viewing under a light microscope (preferably inverted,
×10 objective). Fertilized eggs (90% of eggs raising a fertilization envelope
within 30 sec of sperm addition) are pooled and concentrated by centrifugation
at 100×g for 1 min.
3. Eggs are resuspended in 5 volumes of MPSW, pH 5.0 and collected by centrif-
ugation as in step 2. Residual acidic MPSW is removed by two washes in
MPSW as described in step 2 (see Note 5).
4. Approximately 10 mL of eggs are resuspended in 100 mL MPSW supple-
mented with 3 mM ATA (see Note 6).
5. Sperm is diluted approximately 500-fold and added to the eggs to give an
approximate final sperm/egg ratio of 10–100:1. Eggs are viewed as above to
ensure correct fertilization.
6. Two minutes after fertilization, eggs are filtered through a nylon filter
(L. pictus, 100 µm filter; S. purpuratus, 64-µm filter) and washed twice in 10
volumes of MPSW as described in step 2 (see Note 7). These steps take place
10–15 min after fertilization to produce a G1-phase extract.
7. Eggs are washed three times in 10 volumes of ice-cold LB as described in step
2 (one more wash than previously described in ref. (10)) to thoroughly remove
contamination of the extracts by MPSW.
8. Eggs are finally resuspended in 1 volume of LB and homogenized by twice
vigorously drawing the suspension into a 10-mL syringe fitted with a 22-
gauge, 1.5-inch needle, followed by vigorous expulsion. See Notes 8 and 9,
and Figs. 12.1 and 12.2.
12 Nuclear Envelope Formation In Vitro 213
9. The homogenate is centrifuged at 10,000×g for 10 min at 4°C to produce three
layers: an upper yolky layer, a lower pigmented pellet, and in the middle the
egg cytoplasmic extract (referred to hereafter as S10; see Note 10).
10. The S10 is removed with a P200 pipette and snap-frozen in liquid nitrogen in
0.2-, 0.5-, or 1.0-mL aliquots.
3.3 Sperm Nuclei Isolation and Membrane Removal
1. Before preparation, samples of sperm from each male are collected and
observed under ×40 magnification to ensure that the sperm are active and
fertilize eggs (as described in Section 3.2).
2. Sperm are concentrated in a microcentrifuge tube for 10 min at 500×g, 4°C
(see Note 11).
3. 250 µL of concentrated sperm are resuspended in 10 mL of ice-cold SXN
buffer in a 15-mL centrifuge tube, using a plastic transfer pipette.
Fig. 12.1 Rate of nuclear decondensation
with varying pH of the LB buffer added. The
pH of the resultant extract is lower
Fig. 12.2 Variation of the rate of nuclear decondensation with the concentration of extract in
milligrams protein per milliliter. Dilute extracts may not support decondensation or nuclear enve-
214 R.D. Byrne et al.
4. Sperm are centrifuged at 2,600×g for 5 min at 4°C.
5. The supernatant is aspirated off and the pellet resuspended in 1.5 mL of SXN.
6. The sample is bath sonicated for 6 min with continual mixing with a plastic
transfer pipette (see Note 12).
7. Sperm nuclei are centrifuged at 2,600×g, 4°C for 90 sec. The supernatant is
removed and each pellet is resuspended in 990 µL SXN and 10 µL of 10% (v/v)
Triton X-100 (final concentration 0.1%).
8. Samples are initially shaken vigorously to allow the detergent to react, and
further mixing with plastic pipettes takes place every 5 min for 15 min at room
temperature (see Note 13).
9. The Triton-treated nuclei are pelleted at 2,600×g, 4°C for 1 min and washed
twice with 1 mL of SXN as above.
10. The final pellet is resuspended in 500 µL of SXN and 500 µL of freezing solu-
tion. This suspension is thoroughly mixed with progressively smaller pipette
tips (P1000–P200 size), and aliquoted in 250-µL volumes. Samples are cryo-
genically frozen in liquid nitrogen and stored at −80°C. Previously, nuclei were
only stored for short periods at 4°C (10).
11. We no longer use the lysolecithin method of sperm nuclei permeabilization
3.4 Nuclear Envelope Assembly Assay
1. 0.1% Triton-treated nuclei, S10, and ATP-GS components (ATP, creatine
kinase, and creatine phosphate) are thawed on ice.
2. Nuclei are pelleted at 1,500×g, 4°C for 2 min in a microcentrifuge and gently
resuspended in 50 µL of TN buffer (see Note 14).
3. In a 1.5-mL microcentrifuge tube, 3 µL of nuclear suspension are added to
20µL of S10 and supplemented with 1.2 µL of ATP-GS (final concentration
approximately 6×105 nuclei/µL).
4. This mixture is incubated at room temperature for 1 h with periodic agitation.
Decondensation of nuclei from a conical to spherical shape is confirmed under
a ×100 oil-immersion objective. It is of note that we often see full decondensa-
tion after 1 h and not 40 min as we previously reported (Fig. 12.2) (10). This is
probably due to variations in the ratio of nuclei to cytoplasm concentration.
5. At this point, samples may be additionally treated with 5 µL of GTP stock
(1 mM final concentration) for 2 h to induce fusion of the bound membrane
vesicles. The fusion step has been lengthened from 40 min (10) to 120 min to
maximize the number of nuclei displaying nuclear envelopes (Fig. 12.3). The
rate of completion of nuclear envelope formation varies with the extract, but
the reaction is usually complete by 90 min (6).
6. To control variations of free [Ca2+], samples are supplemented to a final con-
centration of 12.5 mM EGTA and either 20 nM (for L. pictus) or 50 nM (for
P. lividus) CaCl2 at the same time as GTP is added. We have optimized these
12 Nuclear Envelope Formation In Vitro 215
calcium conditions, as well as calculating the free calcium in the reaction mix
using the program WinmaxC32 2.50 (http://www.stanford.edu/ cpatton/down-
loads.htm) as illustrated in Fig. 12.4.
7. To remove unbound membranes, samples from the reactions are underlain with
1 volume of TN containing 5 mM MgCl2 and 0.5 M sucrose. The nuclei are
pelleted for 15 min at 750×g, 4°C and the pellet is gently resuspended in 20 µL
of LB using a 200-µL pipette.
8. To visualize chromatin-bound membrane vesicles, 3 µL of either diOC6 or
diIC12 is added to each sample yielding either green or red emission.
9. 3 µL of dye-stained sample is spotted on a slide and covered with a coverslip,
which is attached to the slide with nail varnish at 2 points. Visualization of
decondensed nuclei takes place by phase contrast microscopy as in Step 4.
Fig. 12.3 Progress of complete nuclear
envelope formation. The maximal
percentage of nuclei exhibiting complete
nuclear envelope formation is usually
achieved by 90 min after addition of GTP
to the system
Fig. 12.4 Buffering of free Ca2+ in the cell-free system. The optimum free Ca2+ can vary between
species. a Various concentrations of Ca2+ were added to LB containing 12.5 mM EGTA.
b Concentrations of free Ca2+ were calculated using the WinmaxC32 program
216 R.D. Byrne et al.
Alternatively, dyes are visualized under a ×100 oil immersion objective with
excitation with an argon/krypton laser and the resulting fluorescence is sepa-
rated using a combination of a dichroic beamsplitter (Q495LP; Chroma) and a
HQ510/20 nm emission filter. Images are captured with a Hamamatsu Orca
camera and processed in OpenLab. Binding of membrane vesicle fractions to
decondensed chromatin is shown in Fig. 12.5.
10. The integrity of the envelope can be additionally confirmed by the addition of
2µL of ATP-GS to the reaction mix for 30 min after the fusion step. Nuclei are
subsequently visualized as above. Under these conditions, nuclei will swell
from a diameter of 4 to 8 µm only if the nuclear envelope is fully formed (11).
11. We have reported several manipulations of the steps described above using
a variety of tools. In particular, we have demonstrated that the MV fusion
step is dependent upon the hydrolysis of PtdIns(4,5)P2 to DAG by an endog-
enous PLC activity (4, 5). For example, the addition of PtdIns(4,5)P2-con-
suming PI 5-phosphatases prior to GTP inhibits MV fusion, as does the
specific PLC inhibitor U73122, but not its inactive analogue U73343. We
can bypass the need for GTP in fusion by using recombinant human PLCγ2
protein, or the functional analog of its DAG product, phorbol 12-myristate
3.5 Preparation of Egg Cytosolic Extracts (S150)
and Purified MV Subfractionation
1. S10 is centrifuged at 150,000×g for 2 h at 4°C, after which the supernatant is
removed and centrifuged again for a further 1 h as above. The cleared superna-
tant (S150) contains soluble egg proteins. S150 is snap-frozen in liquid nitrogen
in 100- and/or 500-µL aliquots and stored at −80°C.
Fig. 12.5 Binding of membrane vesicle fractions MV1 and MV2beta to decondensed sea urchin
sperm chromatin. a MV1 is labeled with DiIC18 (red), sperm remnant membranes with DiOC6
(green), and nuclear DNA with Hoechst 33342 (blue). b MV2 labeled with DiIC18 (red), sperm
remnant membranes with DiOC6 (green), and nuclear DNA with Hoechst 33342 (blue). (Adapted
from ref. (7)). To view this figure in color, see COLOR PLATE 6
12 Nuclear Envelope Formation In Vitro 217
2. The resulting pellets from the above centrifugations (MV0) are gently resus-
pended in MWB (200 µL per 2 mL starting material) using a P1000 pipette and
probe sonicated for 3 sec at power 22 to produce a uniform suspension.
3. MV0 in MWB is diluted to 1 mL with 800 µL of TN (see Note 15).
4. 1.5 mL of 2 M sucrose in TN (“heavy” solution) is diluted 20-fold in TN to
produce a 0.1 M sucrose in TN (“light” solution).
5. The heavy and light sucrose solutions are loaded into the gradient maker
(5.5 mL of each) with a stirring bar added to the heavy chamber (see Note 16).
With stirring, the plug between the columns is opened, allowing the two solu-
tions to mix. The 0.1–2 M sucrose gradient is layered using the Densi-flow into
an ultracentrifuge tube at a speed (∼2.5) such that it takes approximately
12 min to pour a gradient.
6. The resuspended MV0 is slowly layered onto the top of the gradient, and
topped off with mineral oil.
7. The gradient is centrifuged at 150,000×g for 20 h at 4°C.
8. MV subfractions appear in the gradient as pale bands that are removed from
the centrifuge tubes by side puncture with a 22-gauge, 1.5-inch needle on a
1- or 2-mL syringe.
9. Collected MV subfractions are washed once in 4 volumes of MWB at
150,000×g for 30 min at 4°C, and finally resuspended in 250 µL of MWB and
washed as above.
10. The final pellets are resuspended in an appropriate volume of MWB (100 µL
per 5 mL S10) and aliquoted in 10-µL amounts. These are snap-frozen in liquid
nitrogen and stored at −80°C.
11. The four fractions (MV1, MV2, MV3, and MV4) formed have densities of
1.02, 1.04–1.08, 1.13, and 1.18, respectively.
12. The binding profile of the isolated MVs can be assessed in the nuclear assem-
bly assay (see Section 3.4) by combining 20 µL of S150, 1.2 µL of ATP-GS,
and 2 µL of the relevant MV fraction prestained with a lipophilic dye (see
3.6 Isolation of Detergent-Resistant Membranes
from 0.1% Triton X-100-Treated Nuclei
1. 0.1% Triton X-100-extracted nuclei are thawed on ice, pelleted at 1,500×g for
2 min at 4°C and resuspended in 45 µL of TN (see Note 18).
2. 5 µL of 10% Triton X-100 is added and the sample is bath sonicated for
3. The nuclei are collected by centrifugation at 1,500×g for 10 min at 4°C. The
pellet containing these 1% Triton X-100-treated nuclei is washed twice in TN
(as in Section 3.6.1) and resuspended in 50 µL of TN.
4. The supernatant containing the sperm nuclear membrane material is diluted
70-fold in TN and stirred for 1.5 h at 4°C to allow reassembly. This dilution
218 R.D. Byrne et al.
factor is larger than we have previously described (10) in order to bring Triton
X-100 below its critical micelle concentration (CMC), preventing it from per-
turbing the reassembly reaction.
5. The suspension is pelleted at 150,000×g for 1 h at 4°C and the pellet is finally
resuspended in 50 µL of TN buffer.
3.7 Detection of Sea Urchin Proteins by Western Blot Analysis
1. S10 is separated into its constituent fractions as described in Section 3.5.
2. The MV0 pellet is resuspended in LB/0.1% Triton X-100/protease inhibitors
to the same volume as the S150 from which it was obtained.
3. Protein concentration of samples is determined by the Bradford assay (Biorad),
then 4× SDS buffer is added and samples are heated for 10 min at 95°C.
4. Equal volumes of samples are loaded onto a NuPAGE 4–12% gel. Gels are run
at 75 V to allow samples to concentrate in the stacking gel followed by 125 V
until the dye front leaves the bottom of the gel.
5. Gels are removed from their plastic casts and assembled on the semi-dry trans-
fer cell as follows: four pieces of transfer buffer-soaked blotting paper, metha-
nol-activated Immobilon-P transfer membrane (Millipore), the gel, and four
pieces of transfer buffer-soaked blotting paper. Care is taken to ensure air
pockets are not trapped between the sandwich layers.
6. Transfer takes place for 90 min at 12 V.
7. The membrane is removed from the sandwich and air-dried, protein side up,
until white in color.
8. The membrane is incubated with agitation at room temperature for 1 h in PBST
supplemented with 3% BSA (w/v).
9. The buffer is removed and replaced by the same solution supplemented with the
desired primary antibody. We have used antibodies against phosphoinositide
monophosphate kinase I (PIPK) and PLCγ at dilutions of 1:1,000 and 1:2,500,
respectively. The membrane and antibody in buffer are agitated overnight at 4°C.
10. The membrane is washed 5× ∼10 min at room temperature in PBST (see Note
11. The membrane is incubated with agitation in PBST supplemented with 5%
(w/v) milk powder and the appropriate secondary antibody (anti-goat for PIPK,
1:10,000; anti-mouse for PLCγ, 1:5,000) for 1–2 h at room temperature.
12. Wash the membrane as in step 10.
13. The membrane is finally incubated with ECL detection reagent, wrapped in
Saran wrap, and exposed to X-ray film for an appropriate time. Films are
subsequently developed in an automated developer.
14. We have used this protocol to detect a PIPK in sea urchin extracts, and have
been able to show that it is localized both in the soluble S150 and MV0 mem-
brane fractions. By contrast, PLCγ is virtually entirely membrane localized (6)
(Fig. 12.6). This methodology could equally well be applied to probing
subfractionated MV1–4 for proteins, or detecting proteins in 0.1% Triton-
treated nuclei and extracted sperm nuclear membrane material.
12 Nuclear Envelope Formation In Vitro 219
3.8 Lipid Extraction Procedure—Modified Folch Method (12)
1. Prior to the extraction all glassware is silanated to enhance the recovery of
phospholipids, particularly the phosphoinositides. Glassware is silanated by
immersion into a 3% (v/v) solution of dimethyldichlorosilane (toxic! work
under a fume hood) in HPLC-grade toluene for 1 h. The glassware is subse-
quently rinsed twice in HPLC-grade methanol, twice in HPLC-grade water,
and dried in a heat cabinet or under a fume hood before use.
2. Biological samples are added to ice-cold acidified chloroform/methanol in
glass screw-cap universal tubes (see Note 20). Four mL of solvent is used per
200µL sample (if the sample size is smaller than 200 µL, a minimum of 4 mL
solvent is used). If necessary, lipid standards for further applications are added
at this point.
3. The samples are probe sonicated for 10 sec at power 22, and left for 1 h at room
temperature. Samples can be stored at −80°C after sonication if necessary.
4. The samples are removed from the sample tubes with a glass Pasteur pipette,
and filtered through a 0.22-µm GV Durapore membrane filter into another
glass sample tube.
5. Sample phase separation is induced by adding 0.2 volumes of K4EDTA in
distilled water followed by centrifugation at 800×g for 15 min at 4°C.
6. The lower phase is removed with a glass Pasteur pipette, transferred to a new
glass tube, and dried under nitrogen at 30°C. Dried lipid extracts can be stored
at −80°C for a number of months.
3.9 Solution NMR Lipid Analysis
1. Extracted lipids (see Section 3.7) are dissolved in the CDCl3/CD3OD/K4 EDTA
solvent mix and left to equilibrate in a glass tube for 1 h.
2. The samples are transferred to 5-mm NMR tubes (see Note 21).
3. Samples are scanned using an indirectly detected 2D 31P–1H method (see Note
22) established via multiple quantum coherence (HMQC) giving a single
Fig. 12.6 Detection of phosphatidylinosi-
tol 5-monophosphate kinase (PIPK) and
PLCγ in sea urchin cytoplasmic (S10),
cytosolic (S150), and total membrane
(MV0) fractions. PIPK was detected in
S. purpuratus and PLCγ in L. pictus
220 R.D. Byrne et al.
phosphorus resonance for each phospholipid in the projection along the F1 axis
and proton resonances along the F2 axis.
4. Scanning is for 2 h (48 scans) on concentrated samples or 20 h (480 scans)
where lipid is present at a less than micromolar concentration. During scan-
ning, the field is locked to the CDCl3 signal to aid reproducibility.
5. The 2D spectra scanning parameters are as follows: 1H and 31P 90-degree
pulses of 8.0 and 9.5 µsec, respectively, relay delay 2 sec, acquisition time
0.68 sec, sweep width F2 2,008 Hz in 2,750 real points, sweep width F1 607 Hz
in 64 slices, Malcolm Levitt decoupling scheme (MLEV) 17 mixing at 7.1 kHz
power for 101 msec (45 cycles), globally optimized alternating rectangular
pulse (GARP) 31P decoupling during acquisition at 2.4 k Hz power, refocus
delay 0.071 sec (JP−H = 7 Hz).
6. Data sets are processed with zero-shifted sine-bell window functions in both
directions and displayed in magnitude mode for 2-D integration.
7. To allow the quantification of phospholipids, three sets of standards are made
(see Note 23). The first is comprised of DPPC, DPPE, DPPS, and PI each at
3.9µM; the second set of 0.40 µM DPPC, 0.40 µM DPPE, and 0.39 µM PI; and
the third set of 0.04 µM DPPC, 0.04 µM DPPE, and 0.039 µM PI.
8. The standards are dissolved in 700 µL of the CDCl3/CD3OD/K4 EDTA solvent
mix and analyzed as described in Step 5, with XWIN-NMR used to integrate
9. Using the known phospholipid molar concentrations [PL] and the integration
values of peaks (A), the efficiency coefficient of each phospholipid (K) relative
to DPPC can be calculated from each of the standard sets using Eq. 12.1:
10. The molarities of individual phospholipids are calculated as follows. The ratio
of A1/A2 is multiplied by its K value. Thus the molarity of PI relative to DPPC
is calculated by Eq. 12.2 (see Note 24):
. (Eq. 12.2)
1. Overnight stirring ensures complete solvation of the salt, especially when pre-
pared in large batches. Approximately 2 L of MPSW can be prepared using one
filter. Other salt sources work well, and large amounts of unfiltered sea water can
be prepared in a cylindrical container with a circulating pump at the bottom that
12 Nuclear Envelope Formation In Vitro 221
mixes the solution overnight (Rolf C Hagen Ltd, Castlefield, UK or Mansfield,
MA, USA). The pH and density are checked after final preparation.
2. DiIC12 is more sensitive than DiOC6 at equal concentrations. However, DiIC12
will form crystals in aqueous solution, interfering with visualization of nuclei
under the microscope and is best diluted in methanol.
3. We have found 14×95-mm centrifuge tubes (Beckmann #344060) to be the
most suited to the MV0 subfractionation.
4. Injection of animals with an excessive volume of KCl can lead to inhibition of
5. These washes should be performed as quickly as possible to ensure that eggs
are exposed to an acidic environment for a short period of time only. We have
also dejellied eggs by passing them through a nylon filter (210 µm for L. pictus,
and 120 µm for S. purpuratus), as previously described (13). We find that this
is just as efficient as the pH 5 wash.
6. ATA prevents hardening of the fertilization envelope, and thus aids the homog-
enization step to produce extracts. We add ATA to the eggs a few minutes prior
7. The nylon filter strips the eggs of the fertilization envelope, and is size adjusted
to the egg diameter of the species.
8. It is useful to occasionally monitor the pH of the homogenate with pH paper.
The final pH should be above 7.3, since the eggs become alkaline after fertiliza-
tion. The homogenate pH is always lower than the pH of the LB used. The
effect of altering the initial pH of LB on the rate of decondensation is shown in
9. It is important to keep extracts concentrated. Usually the protein concentration
of the extract is around 6 mg/mL. The effect of diluting extracts to lower protein
concentrations on the rate of decondensation is shown in Fig. 12.2.
10. The S10 contains membrane vesicles (MVs), soluble proteins, ribosomes, and
some mitochondria. It is free of female pronuclei, yolk granules, the majority
of the mitochondria, and large cytoplasmic structures.
11. After concentration, three layers are visible, an upper aqueous phase, the mid-
dle “concentrated viable sperm” phase, and a small black pellet. Only the sperm
12. This step removes most of the sperm tails. The anchor of the tail in the deepest
section of the centriolar fossa may remain in some sea urchin species. For more
thorough removal of tails, sonication has been extended to 2 min from the previ-
ously described method (10).
13. This process is monitored with lipophilic dyes to confirm that 0.1% Triton
removes the sperm head lateral membranes. We perform this step at room tem-
perature instead of 4°C as described previously, to allow more stringent removal
of lateral nuclear membranes (10).
14. After resuspension, the concentration of nuclei is around 5×109 nuclei/mL (con-
firm by counting in a hemocytometer).
15. If not used immediately, this MV0 can be snap-frozen in liquid nitrogen and
stored at −80°C.