Cell Biology Protocols

PROTOCOL 6.34

Vimentin production, purification, assembly and study by EPR

John F. Hess, John C. Voss and Paul G. FitzGerald

Introduction

The hallmark of all intermediate filament (IF) proteins is the central rod domain, a region long predicted to form an alpha helical coiled coil. Although full-length IF proteins have not been crystallized, the alpha helical coiled nature of subdomains within the central rod has been confirmed by crystallization of vimentin fragments [1, 2] and EPR spectroscopy [3]. We use the following methods to produce, purify, spin label and then assemble filaments for EPR studies of vimentin structure and assembly. The process of spin labelling a protein requires that a single cysteine be substituted at the position where the spin label is required. Therefore, we have removed the single endogenous cysteine present in human vimentin and substituted cysteines where required. All of our constructs contain a serine at position 328, the original location of the endogenous cysteine. Thus, vimentin cys342 contains a cysteine at position 342 and a serine at position 328.

Procedures

is introduced into any of several E. coli BL21 (DE3) strains for IPTG induction of vimentin expression followed by isolation and purification of IBs (inclusion bodies).

Reagents and equipment

For bacterial growth:

37 ◦C Shaking incubator for liquid culture

37 ◦C Incubator for bacterial plates

Bacterial culture media including antibiotics

IPTG (isopropyl-B-D-galactopyranoside)

For preparation of bacterial lysates and gel electrophoresis:

Microcentrifuge

SDS gels and apparatus for electrophoresis

Protein standards

Centrifuge and rotors

Procedure

General plasmid transformation and bacterial growth protocols can be found in references such as Molecular Cloning [4]. Protocols related to bacterial induction using

A.Production of vimentin in E. coli IPTG can be obtained from companies

Expression of vimentin in E. coli is performed using a pET7 expression plasmid generously provided by Roy Quinlan (Durham University, UK). This construct

such as Novagen, Invitrogen, Stratagene and others.

1.Transform competent E. coli BL21 (DE3) (Novagen, Invitrogen or other

332 IN VITRO TECHNIQUES

suppliers), with plasmid DNA and plate on LB plates containing 100 µg of ampicillin (amp)/ml.

2.Pick three or four colonies and grow in

2 ml of LB-amp until slightly cloudy (OD 600 0.3–0.4, usually 4 h). Then 1 ml of each culture is removed and transferred to a fresh tube and 1 µl of 1 M IPTG is added. The original tube, with 1 ml remaining, is retained

for use as an uninduced control. The IPTG induced culture is returned to a shaking incubator for 4 h. Following induction, 500 µl of both induced and uninduced bacteria are harvested by low-speed centrifugation (5000 rpm for 2 min) and the supernatants discarded. Then 100 µl of TE (10 mM Tris pH 8, 1 mM EDTA) is added and the bacteria resuspended by vigorous vortexing. Then 35 µl of 4× SDS-PAGE gel loading dye is added and the solution gently mixed; 15 µl of this bacterial lysate is sufficient to run on a typical SDSPAGE minigel. If the solution is too viscous to pipette, a 1 ml syringe fitted with a 25-gauge needle can be used to shear the bacterial genomic DNA. Coomassie blue staining and destaining of the gel will reveal which colony (or colonies) provided the best expression. From the original uninduced culture, the best expressing colony should be streaked out onto a fresh LB-amp plate.

3.Day 2: select a single isolated colony and grow in 11 ml of LB-amp. This culture is grown for 6–8 h, until very cloudy, and two glycerol stock tubes are prepared by mixing 500 µl aliquots of

bacteria with an equal volume of sterile 50% glycerol and stored at −80 ◦C. The

remainder of the culture ( 10 ml) is stored at 4 ◦C overnight.

4.Day 3: use the entire 10 ml culture from the refrigerator to inoculate 500 ml of

LB-amp supplemented with 10 ml of 20% glucose. When the culture reaches an A600 of 0.8, IPTG is added to 0.5 mM. Induction is allowed to proceed for 6 h, and the bacteria harvested by centrifugation in a swinging bucket rotor at 4000 rpm for 15 min. The use of a swinging bucket rotor produces a thin pellet of bacteria on the bottom of the 250 ml bottle, instead of a thick pellet in the corner of a fixed angle rotor. This pellet geometry aids resuspension of the bacteria; complete and thorough resuspension is essential for a clean inclusion body preparation. Following centrifugation, the bacterial pellet is frozen overnight.

B.Isolation and purification of inclusion bodies (IBs) [3, 5]

Reagents and equipment

Centrifuge and rotors (for harvesting bacterial cultures and washing of IBs, e.g. Sorvall RC5C, HS4 rotor, SS34 rotor)

Lysozyme

DNase I

RNase A

Procedure

1.Thaw bacterial pellets in centrifuge bottles by the addition of a GET buffer (50 mM glucose, 25 mM Tris pH 8, 10 mM EDTA) containing 10 mg/ml egg white lysozyme (Sigma L-6876), 10 ml per centrifuge bottle (20 ml per 500 ml culture). The bottle is vigorously vortexed to resuspend the pellet and create a homogeneous suspension. The cell suspension is transferred to a disposable 50 ml screwcap tube using a Pasteur pipette. Any remaining clumps are broken up by rapid pipetting.

2. Incubate the bacterial suspension in a 37 ◦C water bath for 15–20 min.

During this time, the suspension should change from a homogeneous brown/grey/beige colour into a more clumped suspension. This indicates completion of the lysozyme digestion and beginning of bacterial lysis.

3.Produce complete lysis by the addition of an equal volume of 20 mM Tris pH 7.5, 0.2 M NaCl, 1 mM EDTA 1% deoxycholic acid, 1%NP-40. The bacterial solution and the lysis buffer should be gently rocked back and forth

several times over several minutes to mix the solutions; the solution should rapidly turn viscous (more of a gelatinous blob than a suspension). This indicates lysis and release of bacterial genomic DNA.

4.Add magnesium chloride (1 M stock), DNase I (Sigma D-5025, stock solu-

tion 10 mg/ml) and RNase A (Sigma

R4875),	10 mg/ml stock	solution)
to final	concentrations of	10 mM

(MgCl2) and 10 µg/ml (RNase and DNase). The bacterial lysate is again mixed by rocking back and forth, then incubated at 37 ◦C. Over the course of 15–30 min, digestion of the bacterial DNA by DNase I will convert the solution from a viscous gel into a watery yellowish solution with an off-white precipitate at the bottom of the tube. Mixing of the tube will form a homogeneous solution, but the IBs will again sediment to the bottom.

5.When digestion is complete, as evidenced by a watery, non-viscous con-

sistency, transfer the solution ( 20 ml) to a round bottom centrifuge tube and centrifuge in an SS34 rotor at 6500 rpm (10 000g) for 10 min. IBs form a large white/tan coloured pellet; the supernatant is discarded. (The pellet should be firm, and the supernatant can be poured off without danger of losing the pellet.) Each tube,

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333

corresponding to 250 ml of original culture, is sequentially washed with 10 ml each of no salt, low salt, high salt, and no salt buffers.

6.Add 10 ml of 0.5% TritonX-100 (TX100), 1 mM EDTA to the tube and resuspend the IBs by pipetting. IBs are then pelleted by centrifugation as before.

7. Add 10 ml of a low salt solution

(10 mM Tris pH 8, 5 mM EDTA,

0.15 KCl, 0.5% TX-100) to the pellet and resuspend the pellet by pipetting. Careful resuspension and disruption of clumps are essential to prepare inclusions free of major contaminants. Collect IBs by centrifugation.

8.Wash IBs with 10 ml of a high salt solution (10 mM Tris pH 8, 5 mM EDTA, 1.5 M KCl, 0.5% TX-100) and collect by centrifugation.

9.Wash IBs with 10 ml 0.5% TX-100, 1 mM EDTA and collect by centrifugation.

10.Resuspend each tube of IBs in 4 ml (8 ml total volume for 500 ml culture) of 20 mM Tris pH 8, 1 mM EDTA, 8 M urea. Pipette the solution up and down; the IBs should dissolve and yield a slightly yellow solution that is not viscous. If viscous, the DNase digestion was incomplete. Small brown clumps with a translucent halo around them are clumps of bacteria that were not resuspended.

C.Purification and preparation of spin labelled vimentin

Reagents and equipment

FPLC, e.g. Pharmacia

Source S column

Spin label compound (O-87500, (1-oxyl- 2,2,5,5,-tetramethyl- 3-pyrroline-3-

334 IN VITRO TECHNIQUES

methyl) methanethiosulfonate [MTSL], Toronto Research Chemicals, Toronto, Canada)

Superose column

TCEP (tris-(2-carboxylethyl) phosphine, Molecular Probes, Eugene, OR)

Procedure

1.Dissolve IBs (from 500 ml of bacterial culture) in 8 ml of 8 M urea (20 mM Tris pH 8, 1 mM EDTA, 8 M urea) and filter through a 0.2 micron filter (Pall Serum Acrodisc, Fisher Scientific).

2.Chromatograph 4 ml of inclusion body solution on a Hi Load 16/60 Superdex 200 column. The column is run with

20% buffer B, giving conditions of 20 mM Tris, pH 8, 1 mM EDTA, 0.2 M NaCl. Electrophorese column fractions on an SDS-PAG and visualize the proteins by Coomassie blue staining. Pool peak fractions.

3.Desalt the pooled vimentin peak by chromatography over a High-Prep26/10 desalting column.

4.Concentrate the desalted vimentin by chromatography over a Source 15 S column. The peak, typically 2.0 ml, is used for spin labelling.

5. Add	TCEP (100 mM stock in H2O)
to a	final concentration of 100 µM
and	incubate at room temperature for

30 min.

6.Spin label the reduced vimentin by addition of spin label to a final concentration of 500 µM; continue incubation for 1 h.

7.After spin labelling, add 8 ml of buffer A (8 M urea, 20 mM tris, 1 mM EDTA, pH 8.0) and chromatograph the spin labelled protein over the Source 15S

as before. Collect the purified peak and store at −80 ◦C.

Figure 6.36 Representative spin-labelled vimentin samples. Samples of Source S fractions 21 and 22 of spin-labelled vimentin mutants cys342, cys345, cys346 and cys349 are shown following electrophoresis on SDS-PAGE and staining with Coomassie Blue. In each pair of lanes, fraction 21 is first, followed by fraction 22; Lanes 1, 2: vimentin cys342; lanes 3,4, vimentin cys345; lanes 5,6, vimentin cys346; lanes 7,8, vimentin cys349. Markers in lane M are Benchmark protein standards from Invitrogen. Vimentin migrates between the 50 and 60 kD bands

Figure 6.36 shows the results of the above purification/labelling scheme, for four separate vimentin mutants.

D.Assembly of intermediate filaments

Equipment

Dialysis tubing and clips, e.g. Spectra/Por 6 regenerated cellulose, 10 000 molecular weight cut-off dialysis tubing (Fisher Scientific)

Procedure

1.Assemble purified vimentin in 8 M urea into filaments by dialysis against buffers without urea. Single-step dialysis can be performed using 20 mM Tris pH 7.5 and either 160 mM KCl or NaCl [6, 7]. Dialysis is performed at room temperature, overnight. For EPR studies, protein concentrations >1 mg/ml are used. For observation of filaments by electron microscopy, protein concentrations

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335

Figure 6.37 Intermediate filaments assembled from spin-labelled vimentin 342C. Spin-labelled vimentin was assembled by dialysis against 20 mM Tris pH 7.5, 160 mM NaCl, overnight followed by negative staining and visualization by EM

of 0.2–0.5 mg/ml are used. Figure 6.37 shows an example of filaments assembled from vimentin cys342.

2.If EPR spectra are to be recorded at multiple steps during vimentin assembly, dialysis can be performed in a stepwise fashion [8]. Starting conditions are

20 mM Tris pH 8.0, 1 mM EDTA, 8 M urea (buffer A + 8 M urea). Vimentin

dimers can be produced by dialysis against buffer A + 4 M urea. Vimentin tetramers can be produced by dialysis against 5 mM Tris pH 8 [9]. Filaments can be assembled from any of these

intermediate steps by dialysis against assembly buffer, 20 mM Tris pH 7.5, 160 mM NaCl. Perform dialysis for 6–8 h, followed by overnight dialysis, for filament formation.

EPR

EPR measurements can be carried out in a JEOL X-band spectrometer fitted with a loop-gap resonator (Molecular Specialties, Inc., Milwaukee, WI), or equivalent [10, 11]. An aliquot of purified, spinlabelled protein is placed in a sealed quartz capillary (0.84 mm OD, Ruska Instrument

8 M urea

4 M urea

IF

Figure 6.38 Normalized EPR spectra from spin-labelled vimentin cys342. Spectra were collected from monomers (8 M urea), dimers (4 M urea) and filaments (20 mM Tris pH 7.5, 160 mM NaCl)

Corp., Houston, TX) and inserted into the resonator. Spectra of samples at room temperature (20–22 ◦C) were obtained by a single 60 s scan over 100 G at a microwave power of 2 mW, a receiver gain of 250–400 and a modulation amplitude optimized to the natural line width of the individual spectrum. For intact filaments, spin label concentration is typically in the range of 50–70 µM. Intermediate filament samples tend to aggregate upon further concentration, so instrument sensitivity should be optimized to obtain good signal to noise, especially for specimens displaying broad line widths. However, protofilaments of IF oligomers formed in low ionic strength, can be concentrated using centrifugal devices providing excellent signal to noise ratio with even less sensitive instrumentation. For spectra obtained at −100 ◦C, the microwave power is reduced (100 µW) to avoid saturation. A representative example of EPR data is shown in Figure 6.38.

336 IN VITRO TECHNIQUES

Acknowledgements

This research was supported by a UC Davis Health System Research Grant (JFH), a US Army Medical Research Acquisition Activity (grant number DAMD17-02-1-0664, JFH), National Institutes of Health Grant R01EY08747 (PGF) and core grant P30EY-12576 (JFH and PGF) and The March of Dimes Birth Defect Foundation (JV).

References

1.Strelkov, S., Herrmann, H., Parry, D., Steinert, P. and Aebi, U. (2000) J. Invest. Dermatol., 114, 779.

2.Strelkov, S. V., Herrmann, H., Geisler, N., Wedig, T., Zimbelmann, R., Aebi, U. and

Burkhard, P. (2002) EMBO (European Molecular Biology Organization) J., 21, 1255–1266.

3.Hess, J. F., Voss, J. C. and FitzGerald, P. G. (2002) J. Biol. Chem., 277, 35 516–35 522.

4.Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: a Laboratory Manual, 3rd edn (J. Sambrook, ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY.

5.Nagai, K. and Thøgersen, H. C. (1987) Method. Enzymol., 153, 461–481.

6.Herrmann, H., Hofmann, I. and Franke, W. W. (1992) J. Mol. Biol., 223, 637–650.

7.Eckelt, A., Herrmann, H. and Franke, W. W. (1992) Euro. J. Cell Biol., 58, 319–330.

8.Carter, J. M., Hutcheson, A. M. and Quinlan, R. A. (1995) Exp. Eye Res., 60, 181–192.

9.Rogers, K. R., Herrmann, H. and Franke, W. W. (1996) J. Struct. Biol., 117, 55–69.

10.Froncisz, W. and Hyde, J. S. (1982) J. Magn. Reson., 47, 515–521.

11.Hubbell, W. L., Froncisz, W. and Hyde, J. S. (1987) Rev. Sci. Instrum., 58, 1879–1886.

PROTOCOL 6.35

Neurofilament assembly

Shin-ichi Hisanaga and Takahiro Sasaki

Introduction

Neurofilament (NF) is the neuron-specific intermediate filament (IF) [1, 2]. NF is the most abundant cytoskeleton in axon, essential for radial growth of axons. NF is composed of three subunits, NF-L (61 kDa), NF-M (95 kDa) and NF-H (110 kDa). Like other IF proteins, each subunit consists of three domains; the N-terminal head, central α-helical rod and C-terminal tail domain. The head domain is thought to be the region regulating the filament assem- bly–disassembly by phosphorylation. The rod domain is involved in the filament formation via coiled-coil interaction. The tail domain, which of NF-M and NF-H are highly phosphorylated and much longer than NF-L and other IF proteins, extrudes from the filament core and interacts with each other and with other cellular components [3, 4]. Therefore, NF-L is the basic subunit for filament formation, and NF-M and NF-H are suggested to be incorporated at the surface of filaments.

The assembly of NF has been extensively studied, but how these subunits are arranged in NF has not been discovered yet. Furthermore, abnormal metabolism of NF proteins has been reported recently with several neurodegenerative diseases. Accumulation of NF in the cell body and proximal axons of motor neurons is a well-known pathology of amyotrophic lateral sclerosis (ALS) [5]. The deletion and insertional mutations of the NF-H tail

domain are discovered in sporadic ALS patients and are suggested to be a risk factor. Several point mutations in the NF-L gene are recently reported to be associated with Charcot-Marie-Tooth disease (CMT), the most common form of hereditary motor and sensory neuropathy [6, 7]. NF-L with CMT mutants (P8R and Q333P) is suggested to be an inability to form filaments properly and disturbs axonal transport in neurons [8, 9]. The point mutation of NF- M is also found in familial Parkinson’s disease [10]. It will be important to determine the effects of these mutations on filament assembly.

The NF assembly has usually been assessed in vitro with purified proteins and in cultured cells by transfection. The NF-L purified in a denaturing solution containing 6 8 M urea can assemble into long 10 nm filaments upon dialysis against a physiological solution (i.e. 0.15 M NaCl and several mM MgCl2 at pH around 7) at 37 ◦C. NF-M and NF-H need NF-L to form 10 nm filaments, although they themselves form small oligomeric aggregates. The in vitro reassembly system is used to examine the assembly conditions and processes, and capable of investigating the detailed filament structures by electron microscopy. It is also possible to examine disassembly of filaments by phosphorylation with several protein kinases including PKA, PKC, CaMKII and Rho kinase [11]. The expression of NF proteins in SW-13

338 IN VITRO TECHNIQUES

cl.2/Vim− cells which lack the cytoplasmic IF proteins is also often used to estimate the filament assembly in cellular conditions. This assay can be performed without purification of NF proteins. This method revealed that rodent NF proteins are obligate heteropolymers; NF-L required coexpression of either or both NF-M and NF-H for filament formation [12, 13]. We will describe here the assembly system methods in vitro with NF proteins purified from mammalian spinal cords and from E. coli- expressed NF-L and in SW-13 cl.2/Vim− cells with transfection of cDNAs encoding NF protein.

Reagents

A. For assembly in vitro
PEM buffer (100 mM	Pipes, pH 6.8,
1 mM EGTA, 2 mM	MgCl2, 1 mM

DTT, 0.4 mM Pefabloc SC (Merck) 1 and 10 µg/ml leupeptin)

PEMU buffer (PEM buffer containing 6 M urea)

PEMN buffer (PEM buffer containing 0.15 M NaCl)

Bovine or porcine spinal cords

Rat or mouse NF-L cDNA cloned into an

E.coli-expression vector

B. For assembly in SW-13 cl.2/Vim− cells

cDNAs of NF subunits cloned into human cytomegalovirus promoteror rous sarcoma virus promoter-driven expression vectors

Transfection reagent, Fugene 6 (Roche) or Lipofectamine 2000 (Invitrogen)

All standard laboratory reagents should be of high purity and good quality deionized water used

Equipment

A. For assembly in vitro

Polytron homogenizer for spinal cords 2 or sonicator for E. coli

DEAE cellulose (DE52, Whatman) column and Mono Q column (Amersham Biosciences)

Centricon-10 3 (Millipore)

Centrifuge

Materials for preparation of negatively stained EM specimens

Transmission electron microscope

B. For assembly in SW-13 cl.2/Vim− cells

Facilities for cell culturing

Antibody to NF proteins

Materials for preparation of immunofluorescent staining

Fluorescence microscope or laser scanning microscope

Procedure

I. NF assembly in vitro

A. Preparation of crude NF proteins

(a)NF proteins from spinal cords

1.Bring bovine or porcine spinal cords

from a local slaughterhouse to the lab on ice as quickly as possible. 4

2.Remove meninges from the spinal cords and measure the weight.

3.Homogenize the spinal cords in an equal volume (w/v) of PEM buffer (100 mM Pipes, pH 6.8, 1 mM EGTA, 2 mM MgCl2, 1 mM DTT, 0.4 mM Pefabloc SC (Merck) and 10 µg/ml leupeptin) with a polytron homogenizer.

4.Centrifuge the homogenate at 10 000g for 15 min at 4 ◦C.

5.Take the supernatant and recentrifuge it at 100 000g for 60 min at 4 ◦C.

6.Discard the supernatant and dissolve the pellet in PEM buffer containing 6 M urea (PEMU).

7.Centrifuge the suspension at 100 000g for 30 min at 4 ◦C.

8.Remove the supernatant as the crude NF proteins.

(b)NF-L from E. coli 5

The expression of NF-L protein in E. coli is performed by a standard protocol using a T7 promoter-driven system. E. coli strain ‘BL21(DE3) pLysS’ carrying pET23-rat NF-L is induced to express NF-L protein by 0.5 mM IPTG for 3 h at 37 ◦C.

1. Collect	E. coli by	centrifugation at
8 000g	and wash	once with 0.85%
NaCl.

2.Suspend cells with 5 ml of PEM buffer per 100 ml of culture.

3.Sonicate the suspension to disrupt cells with cooling.

4. Centrifugate the homogenate at 100 000g for 30 min at 4 ◦C.

5.Dissolve the pellet with 5 ml of PEMU per 100 ml of culture. 6

6.Stand for 1 h at room temperature.

7.Centrifugate the suspension at 100 000g for 30 min at 4 ◦C.

8.Recover the supernatant as the crude NF-L preparation.

B. Purification of NF proteins

1.Apply the crude NF proteins or crude NF-L to a DEAE-cellulose column equibrated with PEMU.

2.Wash the column with 3 5 column volumes of PEMU.

PROTOCOL 6.35

339

3.Elute the NF proteins with 5 column

volumes of buffer with a linear gradient of NaCl from 0 to 0. 25 M. 7

4.Collect the NF-L- or NF-M-rich frac-

tions	and apply	it to a Mono Q
anion	exchanger	column equibrated
with PEMU.

5.Wash the column with 5 column volumes of PEMU.

6.Elute the NF proteins with 10 column volumes of PEMU with a linear gradient of NaCl from 0 to 0.5 M.

7.Collect NF-L and NF-M separately, and concentrate to 2 3 mg/ml by Cetricon-10.

C. NF assembly

The	reconstitution		of NF		is performed
by	dialysis of	the	NF	protein solution
(2 3 mg/ml)		against		the	PEM buffer

containing 0.15 M NaCl (PEMN) at 37 ◦C for more than 3 h. 8 For the assembly of filaments composed of NF-L/M or NF- L/H heteropolymers, their ratio should be 2 : 1 for both NF-L/NF-M and NF-L/NF- H. Assembly of filaments can be checked by pelleting them by centrifugation at 100 000g for 30 min, followed by SDSPAGE. For EM observation, dilute the reassembled filaments to 0.1 mg/ml and process them for negative staining.

II. NF assembly in SW-13/cl.2 vim− cells

SW-13/cl.2 vim− cells, established by Dr R Evert (Univ. of Colorado Health Sciences Center, CO), were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37 ◦C in 5% CO2. SW-13/cl.2 vim− cells can be transfected with calcium phosphate precipitation or lipofection-based reagents, although the latter method is easier. We

340 IN VITRO TECHNIQUES

successfully transfected plasmids using Fugene 6 (Roche) or Lipofectamine 2000 (Invitrogen) according to the manufacture’s instructions. The human cytomegalovirus promoter or rous sarcoma virus promoter can be used to express the NF proteins in the cells. Expressed NF is visualized by the general immunofluorescence technique. Alternatively, selffluorescence of enhanced green fluorescent protein (EGFP) fused to the NF protein is also available. EGFP-linked to the N- terminal end, but not C-terminal end, of NF subunits can assemble into filaments. Observation of NFs can be performed by a standard fluorescence microscope, but observation by a laser scanning microscope reveals more detailed structures.

Examples of representative data are shown in Figures 6.39 and 6.40.

H

M

L

Notes

1 We use Pefabloc SC as a Serineprotease inhibitor in place of PMSF, because Pefabloc SC is water-soluble and is not inactivated in aqualous solutions.

1

2

3

4

Figure 6.39 SDS-PAGE of NF proteins purified from bovine spinal cords and from E. coli. Lanes 1–3 are NF-L, NF-M and NF-H from bovine spinal cords, respectively and lane 4 is NF-L from

E. coli

(a)

(b)

10 m

Figure 6.40 (a) A negative staining electron micrograph of NFs reassembled from porcine NF-L and

(b) a laser scanning fluorescence micrograph of rat NF-L and EGFP-tagged rat NFH co-expressed in SW-13 cl.2/Vim− cells