Cell Biology Protocols

5.844 g of NaCl, 0.6099 g of MgCl2· 6H2O and 0.3804 g of EGTA. Titrate to pH 6.8 with 1 M NaOH. Freeze in aliquots at −20 ◦C.

Before use VRC is added to a final concentration of 2 mM from the 100× stock solution, and AEBSF is added to a final concentration of 1 mM from the 100× stock solution. Additionally, for

some experiments Triton X-100 is added to a final concentration of 0.5% from the 20× stock solution.

Extraction buffer: (10 mM PIPES, pH 6.8, 250 mM ammonium sulfate, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA)

To make 1 l of this stock solution use 3.024 g of PIPES, 102.69 g of sucrose,

222 IN VITRO TECHNIQUES

33.035 g of ammonium sulfate, 0.6099 g of MgCl2·6H2O, and 0.3804 g of EGTA. Titrate to pH 6.8 with 1 M NaOH. Freeze in aliquots at −20 ◦C.

Prior to use, Triton X-100 is added to a final concentration of 0.5% from the 20× stock solution, VRC is added to a final concentration of 2 mM from the 100× stock solution, and AEBSF is added to a final concentration of 1 mM from the 100× stock solution.

Digestion buffer: (10 mM PIPES, pH 6.8, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, 1 mM EGTA)

To make 1 l of this stock solution use 3.024 g of PIPES, 102.69 g of sucrose, 2.922 g of NaCl, 0.6099 g of MgCl2· 6H2O and 0.3804 g of EGTA. Titrate to pH 6.8 with 1 M NaOH. Freeze in aliquots at −20 ◦C.

Before use, Triton X-100 is added to a final concentration of 0.5% from the 20× stock solution, VRC is added to a final concentration of 2 mM from the 100× stock solution, and AEBSF is added to a final concentration of 1 mM from the 100× stock solution.

2M NaCl buffer: (10 mM PIPES, pH 6.8, 300 mM sucrose, 2 M NaCl, 3 mM MgCl2, 1 mM EGTA)

To make 1 l of this stock solution use 3.024 g of PIPES, 102.69 g of sucrose, 116.88 g of NaCl, 0.6099 g of MgCl2· 6H2O and 0.3804 g of EGTA. Titrate to pH 6.8 with 1 M NaOH. Freeze in aliquots at −20 ◦C.

Before using, VRC is added to a final concentration of 2 mM from the 100× stock solution and AEBSF is added to a final concentration of 1 mM from the 100× stock solution.

Triton stock: (10% (w/v) Triton X-100)

This is a 20× stock solution frozen in aliquots at −20 ◦C.

VRC stock: (200 mM Vanadyl riboside complex)

This is a 100× stock solution frozen in aliquots at −20 ◦C.

AEBSF Stock: (100 mM 4-(2-Aminoethyl)-benzenesulfonyl fluoride, hydrochloride)

This is a 100× stock solution frozen in aliquots at −20 ◦C.

To prepare the 100× stock solution, dissolve 100 mg in 4 ml water. Other protease inhibitors can be added if proteolysis is suspected. Do not use EDTA since divalent ions are necessary for structural integrity.

Phosphate buffered saline: (10 mM Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl)

To make 1 l use 1.420 g of Na2HPO4, 0.136 g of KH2PO4, 8.006 g of NaCl and 0.2013 g of KCl. Autoclave and store in aliquots at room temperature.

Formaldehyde fixative: (4% solution)

The 4% formaldehyde fixative is prepared fresh, just before use in cytoskeletal buffer from a stock solution of 16% formaldehyde (EM-grade) stored under an inert gas. Alternatively, fresh formaldehyde can be prepared from paraformaldehyde powder.

Equipment

Tissue culture incubator

Low-speed centrifuge (for suspension cells)

Procedure

Cells grown in monolayers, either on coverslips or in dishes, can be extracted by exchanging solutions. Suspension cells or cells removed from a growth surface can be sequentially processed by centrifugation, removal of the supernatant and resuspension in the next solution. 1 These procedures, as presented, uncover the nuclear matrix in whole cells without prior nuclear isolation. This allows the best preservation of ultrastructure and reveals the nuclear matrix–intermediate filament scaffold [7]. This consists of an internal nuclear matrix and the intermediate filaments of the cytoskeleton integrated into a single cellwide structure by their attachments to the nuclear lamina. The same procedures can, however, be used to extract isolated nuclei in suspension and this is sometimes preferable for biochemical fractionation. One compatible method for nuclear isolation is that of Penman [8, 9].

A. Cross-link stabilized nuclear matrix

This method affords the best conservation of the nuclear matrix, as judged by the ultrastructural preservation of the nuclear RNP network [6]. This preparation is excellent for microscopy, but the crosslinking can make biochemical analysis difficult.

1.Wash cells in phosphate buffered saline at 4 ◦C. 1

2.Permeabilize cells in cytoskeletal buffer

with 0.5% Triton X-100 at 4 ◦C for 2–5 min. This step will remove soluble proteins, both cytoplasmic and nucleoplasmic, and prevent their cross-linking.

3.Wash briefly in cytoskeletal buffer at 4 ◦C.

4.Cross-link structures using 4% formaldehyde in cytoskeletal buffer at 4 ◦C for 40 min.

three times for 2 min each to remove formaldehyde.

6.Cross-linked chromatin is removed by

digestion with 400 units of RNase-free DNase I in digestion buffer at 32 ◦C for 50 min. Most DNA is removed from the structure at this step. Residual DNA can be removed by washing:

7.Wash 1: Wash cells with extraction buffer (which contains 0.25 M ammonium sulfate) at room temperature for 5 min.

8.Wash 2: Wash cells with 2 M NaCl buffer at room temperature for 5 min.

9.The structure can be processed for microscopy after a wash with cytoskeletal buffer.

B. Classical nuclear matrix

This method is more suitable for molecular analysis. The spatial distribution of nuclear components, for example those involved in transcription, RNA splicing and DNA replication, is well preserved as compared to the unextracted nucleus. The procedure can be stopped after the DNase I digestion and 0.25 M ammonium sulfate extraction, but further extraction in 2 M NaCl removes some proteins of the RNP fibres, uncovering a core structure of branched 10 nm filaments, the core filaments of the nuclear matrix [5].

1.Wash cells with phosphate buffered saline at 4 ◦C. 1

2.Permeabilize cells in cytoskeletal buffer with Triton X-100 at 4 ◦C for 2–5 min. This will remove soluble proteins, both cytoplasmic and nucleoplasmic.

3.Digest chromatin with 400 units of

RNase-free DNase I in digestion buffer for 30–50 min at 32 ◦C. This step

224 IN VITRO TECHNIQUES

will remove DNA and the nucleosomal histones. The nuclear structure at this point is the nuclear matrix. 2 3

4.Extract cells with extraction buffer at 4 ◦C for 3–5 min. This will remove histone H1 and will strip the cytoskeleton except for the intermediate filaments

which remain tightly anchored to the outside of the nuclear lamina. 2

5.Optional: Extract the structure in 2 M NaCl buffer at 4 ◦C for 3–5 min. Better preservation is obtained by increasing the NaCl concentration slowly–or in

steps. This step strips some proteins from the nuclear matrix uncovering a highly branched network of 10 nm filaments that form the core structure of the nuclear matrix. 4

6.For microscopy, fix immediately after fractionation. Incubate the nuclei in 4%

formaldehyde in cytoskeletal buffer at 4 ◦C for 30–50 min.

Notes

1 Cells in suspension are most conveniently processed for biochemical experiments following trypsinization or scraping. For suspended cells, we use about 1 ml for each 107 cells until the digestion step and then halve the volume. Suspension processing can be done by centrifuging at 1000 × g for 3 min at 4 ◦C and sequentially resuspending cell pellets in the next wash or extraction solution between steps. The supernatant fractions can be saved for biochemical analysis. The extracted cell structure at each step is in the pellet.

2 Steps 3 and 4 may be reversed,

composition of the resulting nuclear matrix is also the same. An easy alternative is to perform the DNase I digestion first and then add ammonium sulfate slowly from a 1 M stock solution to a final concentration of 0.25 M.

3 DNA release can be evaluated microscopically by staining with a fluorescent DNA-binding dye such as 4 -6- diamidino-2-phenylindole (DAPI; 1– 10 ug/ml in phosphate buffered saline for 5 min), or by pulse-labelling cells in 3H-thymidine before fractionation and then measuring radioactivity.

4 Nuclear matrix proteins that are not part of this core filament network should be in the supernatant fraction.

References

1.Monneron, A. and Bernhard, W. (1969) Fine structural organization of the interphase nucleus in some mammalian cells. J. Ultrastruct. Res., 27, 266–288.

2.Nickerson, J. (2001) Experimental observations of a nuclear matrix. J. Cell Sci., 114, 463–474.

3.Nash, R. E., Puvion, E. and Bernhard, W. (1975) Perichromatin fibrils as components of rapidly labeled extranucleolar RNA. J. Ultrastruct. Res., 53, 395–405.

4.Berezney, R. and Coffey, D. S. (1974) Identification of a nuclear protein matrix. Biochem. Biophys. Res. Commun., 60, 1410–1417.

5.Nickerson, J. A., Krockmalnic, G., Wan, K.

M.and Penman, S. (1997) The nuclear matrix revealed by eluting chromatin from a crosslinked nucleus, Proc. Nat. Acad. Sci. USA, 94, 4446–4450.

6.He, D. C., Nickerson, J. A. and Penman, S. (1990) Core filaments of the nuclear matrix.

J.Cell. Biol., 110, 569–580.

7.Fey, E. G., Wan, K. M. and Penman, S. (1984) Epithelial cytoskeletal framework and nuclear matrix–intermediate filament scaffold: threedimensional organization and protein composition. J. Cell Biol., 98, 1973–1984.

8. Penman, S. (1966) RNA metabolism in the HeLa cell nucleus. J. Mol. Biol., 17, 117–130.

9.Capco, D. G., Krockmalnic, G. and Penman, S. (1984) A new method of preparing

embedment-free sections for transmission electron microscopy: applications to the cytoskeletal framework and other three-dimensional networks. J. Cell Biol., 98, 1878–1885.

Background

The nuclear envelope of the eukaryotic cell nucleus is composed of the outer and inner nuclear membranes, nuclear pore complexes, and a protein filament meshwork underlying the inner nuclear membrane, termed the nuclear lamina (see Figure 6.3). The lamina contains nucleusspecific (type V) intermediate filament proteins, the lamins, plus numerous integral and peripheral proteins of the inner membrane, which bind lamins [1, 2]. Two different types of lamins can be distinguished according to their expression patterns and biochemical properties. B- type lamins are constitutively expressed throughout development and are essential for cell viability. A-type lamins, which are expressed only in differentiated cells, are not essential for development, but serve yet unknown functions in tissue homeostasis. More recent studies have also identified lamins in the nuclear interior, where they form complexes with nucleoplasmic lamin binding proteins, such as lamina-associated polypeptide 2α (LAP2α) [3].

The discovery of novel binding partners of lamins and of lamina-associated polypeptides has significantly changed our view of the functions of lamin complexes in recent years. Apart from the long-known function of the lamina as a scaffolding framework providing mechanical stability

for the nucleus, recent studies suggested that lamin complexes are also involved in chromatin organization and chromosome segregation as well as in DNA replication and RNA processing [1, 4].

The hypothesis that many diverse nuclear functions are directly or indirectly linked to the nuclear lamina is supported by recent findings revealing that mutations in A- type lamins or in some of their binding partners give rise to an increasing scope of diverse genetic diseases known as laminopathies [5]. Mutations in the LMNA gene that encodes A-type lamins result in a broad range of disease phenotypes, affecting skeletal and heart muscle, adipose, nerve and bone tissue. The molecular mechanisms of these diseases are completely unknown. At least some of the pathological phenotypes may be the result of impaired interactions of mutant lamins with chromatin proteins and with proteins in transcriptional complexes. The identification of novel (tissue-specific) lamin binding partners is therefore an essential first step in unravelling lamin functions and has to be followed by a detailed molecular analysis of the interactions, including analysis of interaction domains, binding strengths, assembly dynamics and molecular structure.

Here we describe two in vitro techniques, which have been used to analyse the interaction of lamins with LAP2α

and with chromatin: the in vitro overlay binding assay, which allows identification of interaction domains, and the in vitro

nuclear assembly assay, which reveals the dynamic properties of lamin–chromatin interactions during the cell cycle.

Introduction

Transblotted proteins immobilized on a nitrocellulose membrane are incubated with a radioactively labelled binding partner. If various fragments representing different domains of the protein are immobilized onto the membrane, this assay identifies interaction domains and may also reveal a rough determination of relative binding strengths of different domains. A major pitfall of this assay is the denaturation of immobilized proteins during SDSPAGE and blotting, which may, despite partial renaturation of proteins on the membrane during incubation in a binding buffer, cause unspecific interactions. Therefore negative controls, applying noninteracting proteins, which have similar structure and/or properties as analysed proteins, should always be included.

Reagents

Blocking buffer: 2% BSA in overlay buffer

Dilution buffer: 1% BSA in overlay buffer with 1 mM PMSF (phenylmethylsulfonylfluoride)

Overlay buffer: 10 mM Hepes/KOH (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 2 mM EGTA, 0.1% Triton X-100

PBST (0.05% Tween 20 in PBS)

Phosphate-buffered saline (PBS): 2.6 mM KCl, 137 mM NaCl, 1.5 mM KH2PO4, 8 mM Na2HPO4.7H2O, pH 7.4

Ponceau S: 0.2% (w/v) Ponceau S, 3%

(w/v) trichloroaceticacid

Triton X-100, 1 mM dithiothreitol (DTT)

Procedure

1.Separate bacterially expressed recombinant lamina proteins (bacterial cell lysates are usually fine) by SDS-PAGE

and transblot onto nitrocellulose membranes (0.2 µm). 1

2.Stain nitrocellulose membranes with

immobilized proteins using Ponceau S to visualize proteins and estimate protein quantity, followed by thorough washing in PBST. 2

3.Incubate in overlay buffer for 1 h with three changes (to allow renaturation of proteins), then block the membranes for 30 min in blocking buffer.

4.In the meantime, the interaction part-

ner to be tested is transcribed and translated in vitro and [35S]-labelled, using the TNT Quick coupled transcription/translation system (Promega, Madison, WI), according to the manufacturer’s protocol.

5.Probe the membranes with 100 µl of the standard in vitro translation reaction mixture (diluted 1 : 50 in overlay buffer) containing the [35S]-labelled protein for 3 h at room temperature, while gently moving the incubation chamber.

6.Wash extensive in overlay buffer and air-dry the nitrocellulose.

Introduction

Metaphase cell lysates containing metaphase chromosomes and lamina proteins, or isolated chromosomes and chromosomefree metaphase cell lysates, are incubated at 37 ◦C for different time periods in order to investigate the dynamic interaction of different lamina proteins with chromatin during nuclear reassembly after mitosis. Furthermore, dominant negative mutants interfering with the assembly can be tested in this assay.

Reagents

Complete culture medium: Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 µg/ml streptomycin

KHM buffer: 50 mM Hepes/KOH (pH 7.0), 78 mM KCl, 4 mM MgCl2, 10 mM EGTA, 8.4 mM CaCl2, 1 mM dithiothreitol (DTT), 20 µM cytochalasin B

3× SDS-PAGE sample buffer: 186 mM Tris/HCl (pH 6.8), 300 mM dithiothreitol (DTT), 6% sodium dodecyl sulfate (SDS), 0.1% bromphenol blue, 30% glycerol

Equipment

Heraeus Megafuge 1.0 Incubator (37 ◦C, 8.5% CO2)

Metal ball cell cracker (EMBL, Heidelberg)

Plastic cell culture flasks 175 cm2 (Nunc)

Sterile hood

Thermoblock for Eppendorf tubes

Procedure (adapted from ref. 6)

A. Synchronization of cells

1. Plate NRK (normal rat kidney) cells in ten 175 cm2 cell culture flasks and allow to grow to approximately 60–70% confluency in complete medium at 37 ◦C and 8.5% CO2.

2.Replace the medium with complete medium plus 2 mM thymidine in order to arrest cells at the G1-S phase boundary.

3.After 10 h incubation in thymidine medium, wash the monolayer of cells three times with sterile PBS and incubate in complete medium for 4 h.

DMSO) and incubate cells for a further

10 h.

5.Harvest the loosely attached prometaphase cells using a mechanical shakeoff.

B. Preparation of mitotic lysates

1.Pellet mitotic cells from ten flasks at 1000 rpm (Heraeus Megafuge, 1.0R) for 3 min.

2.Wash twice in PBS and incubate in 30 ml complete medium containing