Neurofilament reassembly in vitro: biochemical, morphological and immuno-electron microscopic studies employing monoclonal antibodies to defined epitopes

Brain Research, 556 (1991) 181-195 t~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116820R BRES 16820

181

Research Reports

Neurofilament reassembly in vitro: biochemical, morphological and immuno-electron microscopic studies employing monoclonal antibodies to defined epitopes Brian J. Balin, Edwin A. Clark, John Q. Trojanowski and Virginia M.-Y. Lee The Department of Pathology and Laboratory Medicine [Neuropathology], The University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283 (U.S.A.) (Accepted 26 February 1991) Key words: Reassembly; Negative staining; Immuno-electron microscopy; Intermediate filament; Protofilament

The reassembly process of purified native (phosphorylated) and enzymatically dephosphorylated bovine neurofilament (NF) subunits was studied to delineate how NF triplet proteins assemble together into intermediate-sized filaments in vitro. We determined the time course for reassembly, the ultrastructural characteristics of reassembled NFs, and the topographical disposition of NF protein subdomains within reassembled NFs using quantitative biochemical techniques, negative staining and immunoelectron microscopy. Our data indicate that: (1) ~-50% of the purified NF subunit proteins assembled within 30 min from the start of reassembly into 10- to 12-rim filaments, and by 90 rain ~85-90% of the NF proteins were reassembled, (2) low concentrations (0.15-0.5 mg/ml) of purified NF proteins were able to reassemble into long filaments, (3) the rate and ability of native phosphorylated and dephosphorylated NF proteins to assemble into NFs were comparable, (4) negative staining revealed a periodicity of ~-18-22 nm and a protofilamentous substructure in reassembled NFs, (5) immunoeleetron microscopy using domain specific anti-NF monoclonal antibodies (mAbs) to all 3 NF proteins demonstrated specific labeling patterns corresponding to the spatial relationships of subdomains within reassembled NFs, and (6) negative staining and immunolabeling revealed that reassembled NFs are very similar to isolated native NFs. We conclude that purified mammalian axonal NF triplet proteins, independent of their phosphorylation state, rapidly and efficiently reassemble in vitro to generate characteristic 10-nm filaments. Furthermore, immunological analysis reveals that the rod domains of NF-H, NF-M and NF-L are buried within the reassembled NF, whereas the head domain of NF-M and the tail domains of all 3 NF proteins remain exposed following reassembly. INTRODUCTION Neurofilaments (NFs), members of the neuron-specific type IV class of intermediate filaments (IFs), are composed of 3 subunits with apparent M r values of 70,000 (NF-L), 150,000 (NF-M) and 200,000 (NF-H) when measured using SDS gels 35'42. Previous studies on mammalian NFs suggest that the 3 polypeptides are assembled into IFs ~ 1 0 nm in diameter via coiled-coil interactions between structurally conserved ' r o d ' regions (~310 amino acids) s. NF-M and N F - H have long, highly phosphorylated carboxy (COOH)-terminal tail domains 4' 22,24,25,27 that appear to project from the homologous ' r o d ' regions of assembled NFs 6'1°'17'25'37. The tail domains are thought to represent the peripheral regions of N F - H and NF-M as demonstrated by metal shadowing 17 and proteolytic digestion of the NFs 4'6'25. Additional morphological and immunological data of axonal NFs suggest that lateral 'projections' between adjacent NFs

may represent the tail domains of N F - H and NF-M 15A6. In contrast to type III IFs, such as peripherin, desmin, vimentin and glial fibrillary acidic protein ( G F A P ) , in which polymers of single polypeptide subunits comprise the 10-nm filament 9A1'13'26'41'46'48, the formation of NFs (i.e. members of type IV IFs) into 10-nm filaments may be complicated in that they are heteropolymers of 3 different polypeptides. In vitro reassembly studies have been employed for numerous purified IFs, including NFs, to understand how IF subunits assemble into 10-nm filaments 12,17,18,47,54. These studies have established that although c o m m o n features are inherent in all IFs 5°, a degree of morphologic polymorphism for the numerous IFs exists at the ultrastructural level which, in part, may be a consequence of the different methods used to visualize them. Reassembly studies using IFs other than NFs indicated that they could be reassembled very efficiently from low concentrations (e.g. 0.1-0.5 mg/ml) of starting proteins 21'26'47. In contrast, early reassembly

Correspondence: B.J. Balin. Present address: Department of Pathology and Laboratory Medicine, Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129, U.S.A.

182 studies conducted with NFs, but in which the efficacy of reassembly was not monitored closely, revealed that protein concentrations of I>1.0 mg/ml were required for reassembly34'54. In the present study, we re-examined the ability of N F proteins to reassemble into intermediatesized NFs for two reasons: first, to monitor and determine the efficiency of reassembly of purified N F proteins into 10-nm diameter filaments u n d e r optimized reassembly conditions, and second, to gain further insight into N F structure by determining the ultrastructural and immunological characteristics of reassembled type IV filaments as compared to their isolated native counterparts. Early immunological studies showed that antisera to NF-L and NF-M continuously decorated the filament rod domains, whereas antisera to NF-H labeled structures discontinuously and peripheral to the central rod 16'43'53. However, since polyclonal antisera (which recognize multiple epitopes) were used for these previous studies, the spatial relationship of subdomains of each N F subunit within intact NFs could not be determined unequivocally. To address this issue directly, we conducted a thorough immunological 'dissection' of the N F triplet proteins to determine the location of the rod and tail domains of each protein within reassembled NFs. Our extensive library of anti-NF m A b s enabled us to select antibodies that bind to different subdomains of each N F protein, and thereby conduct a detailed immunological analysis of the NFs at the ultrastructural level. Additionally, we undertook to define the optimal conditions for the reassembly of purified bovine NFs into long 10-nm wide filaments. This work showed that the substructural composition of reassembled NFs is comparable to that of native isolated NFs as demonstrated by negative staining and i m m u n o d e c o r a t i o n , and provides further insight into the topographical relationship(s) of each N F protein to one another in the 10-nm filament. MATERIALS AND METHODS

Isolation of NFs Bovine spinal cords freshly obtained from a local slaughterhouse were enriched for white matter as previously describedz° and placed overnight at 4 °C into hypotonic Tris-buffered saline containing 10 mM Tris-HCI pH 7.6, 2 mM EGTA, 1 mM DTT, and 50 mM NaC1 at pH 7.4. Subsequently, the enriched white matter from these spinal cords was made isotonic, homogenized and centrifuged briefly at 8,000 rpm as reported elsewhere4. Sucrose (2.5 M) in TBS was added to the low speed supernatant to give a final concentration of 0.85 M sucrose and the extract was centrifuged for 3-4 h at 100,000 g in a Beckman L8-50 ultracentrifuge at 4 °C. This step eliminated most of the myelin and gray flocculent material. The clarified supernatant was collected and centrifuged overnight (14-20 h) at 100,000 g at 4 °C over a 2.5 M sucrose pad to yield a clear gel-like material highly enriched for NFs (see Results) above the sucrose pad and below the residual floating myelin. This material represents our isolated native phosphorylated NF preparation.

Purification of NF proteins Native phosphorylated and enzymatically dephosphorylated NFs were prepared for purification by HPLC. For dephosphorylation of NFs, 2.0 units of alkaline phosphatase from E. coli (Sigma Chemicals, Type III-N) per milligram of neurofilament protein were used. The phosphatase-neurofilament mixture contained 1 mM ZnSO4, 100 mM NaCl and 50 mM Tris-HCi buffer at pH 8.0 and was incubated for 14-18 h at 37 °C. To remove excess enzyme, NFs were pelleted from solution by centrifugation at 100,000 g for 2 h at 4 °C. To obtain purified phosphorylated and dephosphorylated individual NF proteins, the enriched NF preparations were homogenized in buffer A (8 M urea, 0.05 M Tris-HCi, 1% fl-mercaptoethanol) at pH 7.6 and incubated at 22 °C for 30 min. After a brief centrifugation step (i.e. 5,000 rpm, 4 °C, 20 min) to remove particulate material, the solubilized NF samples from each preparation were fractionated by HPLC (Waters, Millford, MA) using a Bio-Gei TSK DEAE 5PW anion exchange column at a flow rate of 5 ml/min. The NFs were ehited with a linear NaCl gradient in buffer A. Four main peaks were observed. The earliest peak at 0 mM NaC1 concentration (i.e. the flow-through) consisted primarily of GFAP and other contaminating proteins. The second peak consisted of purified NF-H at 100-150 mM NaCl, the third peak consisted primarily of NF-M at =200-280 mM NaCI, and the fourth peak consisted primarily of NF-L at ~300-400 mM NaCl. Fractions corresponding to the observed peaks for NF-H, NF-M and NF-L were selected for reassembly.

Reassembly of NFs The identity and purity of the NF triplet proteins were monitored by SDS-PAGE with Coomassie blue staining and protein concentrations were determined by the Bradford method 3 using BSA as the standard. Preparations of phosphorylated and dephosphorylated NF triplet proteins (0.15-0.5 mg/ml) were recombined in a protein ratio of 1:1:1 or 2:1:1 (NF-L:NF-M:NF-H) and dialyzed in buffer B [4.0 M guanidine-thiocyanate in 0.1 M (2[N-morpholino]-ethanesulfonic acid) MES buffer, 0.17 M NaCI, 0.5 mM EGTA and 1.0 mM DTT (pH 6.5)] at 37 °C for 2 h. Following this dialysis, 0.5-ml aliquots of the unassembled dephosphorylated and phosphorylated NF triplet proteins were placed into individual wells of a microdialysis system (BRL, Gaithersburg, MD) to undergo reassembly in buffer C (buffer B without guanidine-thiocyanate) for up to 24 h at 37 °C. The BRL dialysis system allows for the treatment of multiple samples under identical conditions. Buffer C was circulated by a peristaltic pump at a flow rate of ~10 ml/min. Samples (0.5 ml/well) of dephosphorylated and phosphorylated NFs undergoing reassembly were taken for biochemical and ultrastructural analysis at 0, 15, 30, 45, 60 and 90 rain and, for ultrastructural analysis alone, at 12 and 24 h after the start of dialysis in buffer C. For ultrastructural analysis, electron microscopy grids were placed directly onto the samples at each time point during dialysis (see below). For biochemical analysis, the dephosphorylated and phosphorylated triplet proteins were centrifuged at 100,000 g overnight (12-16 h) at 4 °C to pellet reassembled NFs. Supematants from this centrifugation were dialyzed against 10 mM Tris-HC1pH 7.6 for a minimum of 4 h to eliminate residual guanidinethiocyanate. Pellets containing reassembled NFs were dissolved in 8 M urea. Protein assays3 and SDS-PAGE followed by laser densitometry (LKB 2400 Gel ScanXL, Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) were used to determine the amount of NF subunit proteins in both the supematants and pellets. Each experiment was repeated at least four times.

Negative staining Isolated native phosphorylated and dephosphorylated NFs that were not subjected to purification and reassembly were resuspended at ~3-5 mg/ml by homogenization in isotonic TBS. These NFs were diluted further (1:100-1:500) in TBS and adsorbed onto nickel EM grids (see below) prior to negative staining in the dark with 2% methanolic uranyl acetate (UA) for 30-60 s at 22 °C. Similarly, grids containing reassembled NFs were rinsed briefly (<1 min) in

183 and their specificities are listed in Table I.

double-distilled water prior to negative staining in the dark with 1-2% methanolic UA for 10-60 s at 22 *C. Additional grids to which isolated native NFs or reassembled NFs were adsorbed were processed for immunoelectron microscopy.

Antibodies The antibodies used here, including their specificities and epitope assignments, have been described previously4,2s'a°'31,51 with the following exceptions. (1) An antiserum was generated to a peptide representing the last 20 amino acids (aa) of human NF-L23 by immunization of rabbits by standard methods 3°. (2) mAbs which bind to a synthetic peptide corresponding to the last 20 aa" of the carboxy terminal of human NF-M3a. (3) An anti-aMSH antisera denoted here as MAT (reactive to NF-M Amino-Terminus) which has been shown previously to recognize the first 5 aa from the amino terminus of NF-M7'45'52. All of these antibodies and their subunit and domain specificities are summarized in Table I.

Immunoelectron microscopy Optimal immunolabeling of native and reassembled bovine NFs was accomplished using a modified protocol of Birrell et al. 2. Briefly, reassembled NFs were adsorbed on carbon, formvar-coated grids and blocked (30 rain) for non-specific background labeling with a mixture of 2% new-born calf serum and 1% cold-water fish gelatin in TBS. The grids were incubated with primary rat or mouse mAbs (supernatants used undilute) and/or antisera (rabbit) (dilutions ranged from 1:100 to 1:2500) for 30 min, washed for 30 min in TBS and 1% cold-water fish gelatin, and blocked a second time for 15 min in TBS containing 2% new-born calf serum and 1% cold-water fish gelatin. Following the second blocking step, the grids were incubated with secondary antibodies diluted 1:5 to 1:10 in TBS and 1% cold-water fish gelatin for 1 h; the secondary antibodies were goat antisera to rat, rabbit or mouse IgG that were conjugated to 5 or 10 nm gold (Janssen Pharmaceutica, Piscataway, NJ). For double immunolabeling experiments, the primary antibody incubations were performed simultaneously using antibodies to different species (e.g. incubation with mouse mAb followed by rabbit antisera). Subsequently, incubation with the specific secondary antibodies followed the same sequence. After the 1-h incubation, the grids were washed (3 times) in double-distilled water over 30 rain followed by negative staining in 2% methanolic U A for 2 min. Controls for each immunolabeling experiment included: (1) incubation of NFs without a primary antibody, (2) incubation of NFs with antibodies specific for other IFs (e.g. mAb 2.2B10 which recognizes GFAP) 32, and (3) substitution of mAb supernatants with spent medium from a non-secreting mouse myeloma cell line (SP2). The panel of mAbs and the polycional antibodies used in this study

Electron microscopy Glow-discharged, carbon and formvar-coated, 400 mesh nickel EM grids were used for negative staining as well as for our immunogold labeling experiments. All grids were examined on a Hitachi 600 electron microscope at 75 kV. Tobacco Mosaic Virus was used to calibrate the magnification. Magnifications ranged from 10 to 100,000x. Quantitation of NF structure and of immunogold labeling patterns was performed both manually with a measuring magnifier and computer-assisted with an SMI (Atlanta, GA) Image Analysis System. Measurements of length were from end to end of individual non-entangled f'daments, while width measurements were from the widest part of the backbone of the filaments not including projections. The periodicity of the bead-like areas of the NFs was calculated from the distance measured between constricted parts of the NF backbone as illustrated by bars demarcating these areas in Fig. lb.

TABLE I

Antibodies for immunoelectron microscopy The specificity of the antibodies to bovine NF subunits used in this study are shown here. MAbs and two antisera (i.e. rabbit anti-NF-L, MAT) are in the left column. The phosphorylation states of the epitopes recognized by the antibodies are: P[+], diminished immunoreactivity after dephosphorylation; P[-], increased immunoreactivity after dephosphorylation; P[ind], no change after dephosphorylation. The labeling pattern is summarized for reassembled phosphorylated and dephosphorylated NF-H, NF-M and NF-L in the columns on the right with 'margins' referring to immunoreactivity primarily on the sides of the filaments, and 'backbone' as immunoreactivity on the central region of the NFs. aa, amino acid; R, rod; NR, non-rod; ND, not determined; H, head domain; T, tail domain; (-), not immunoreactive; (+/-), little immunoreactivity; (+), immunoreactive.

A BY

TA51 TA50 TA56 DP1 TA34 TA54 OC85 OC50 OC33 RMO255 MAT SE3 SE6 Rabbit anti-NF-L 2.2B10

Subunit specificity

Phosphorylation state of epitope

Epitope domain

Labeling pattern of NF-H, NF-M and NF-L Phosphorylated

Dephosphorylated

NF-H> >NF-M NF-H only NF-H only NF-H>NF-M NF-M only NF-M only NF-M only NF-M only NF-M only NF-M [Last 20 aa of COOH-term] NF-M [NH2-term ]

P[ + ] P[ + ] P[ind] P[-] P[ + ] P[ind] P[ind] P[ + ] P[ind] P[ind]

T T R T T R T T T T

(+), (+), (-) (-) (+), (-) (+), (+), (+), (+),

(+/-) (+/-) (-)

ND

H

NF-L only NF-Lonly NF-L [Last 20 aa of COOH-term] GFAP

P[ind] P[ind] P[ind]

NR R T

ND

ND

(+), periodic, clumps & pairs, margins (+), uniform, margins (-) (+), heavy, uniform, margins and backbone (-)

clumps, margins clumps, margins

(+), clumps, margins uniform, margins

(+/-) (-)

heavy, uniform, margins uniform, margins heavy, uniform, margins clumps, margins

(+), heavy, uniform, margins

(-)

(+), heavy, uniform, margins (+), clumps, margins (+), periodic, clumps & pairs, margins (+), uniform, margins

(-)

(+), heavy, uniform, margins and backbone

(-)

184

Fig. 1. Isolated native bovine NFs negatively stained with 2% methanolic UA (a). The diameter of the NFs is ~10-12 nm while the lengths of the NFs are from 0.75 to >2.0~tm. Gel analysis of these IFs (a, inset) show that NF-H, NF-M and NF-L are the major protein components, although small amounts of other cytoskeletal protein contaminants such as GFAP also are present (arrowheads; also see lane 1 in Fig. 3a). A high power micrograph (b) demonstrates bead-like areas along the NFs with a periodicity of ~22 nm (segmented areas) and numerous projections (small arrows) which may represent branches from the parent NF sheared off during homogenization or NF fragments sticking onto the elongated filaments. Bars = (a) 200 nm; (b) 100 nm.

185 RESULTS

Isolation and ultrastructural analysis of native NFs Native, phosphorylated NFs from bovine spinal cord were isolated as a translucent gel-like material following the final centrifugation step in the isolation procedure. Examination of these native NFs by electron microscopy revealed an interconnected array of negatively stained 10-nm wide filaments (Fig la). Coomassie blue-stained gels of this preparation indicated that the NF triplet proteins represented >80% of the total protein with some contamination by other cytoskeletal proteins (Fig. la, inset). Typically, the length of the isolated NFs ( - 1 0 0 counted) ranged from ~0.75 to t>2.0 /~m and the diameter of the backbone of the filaments measured ~10-12 nm ( - 5 0 measurements) (Fig. la,b). Notably, the native NFs displayed a periodic (i.e. ~-21-22 nm) bead-like segmentation pattern (Fig. lb) which may correspond to the axial repeats that purportedly characterize all classes of intermediate filaments 14'36. Within some filaments, a protofilamentous substructure was evident, but it was difficult to determine the exact numbers of protofilaments present. Numerous arrays projecting from the filament rods appeared to appose or interconnect other NFs. These projections appeared to

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bifurcate into branches identical in width to the parent NF, but this phenomenon was more evident in the reassembled NFs (see below). Dephosphorylated NFs were similar ultrastructurally to their untreated native counterparts (Fig. 2). By negative staining, the dephosphorylated NFs were long and intertwined as observed with phosphorylated NFs, although some shorter filaments also could be discerned. Visualization of a bead-like periodicity and protofilamentous substructure of dephosphorylated NFs, while evident at high power (data not shown), was not always possible given the inherent variability of negative staining. An increase in electrophoretic mobility of dephosphorylated NF-H and NF-M but not NF-L as compared to the native phosphorylated NFs was demonstrable by SDS-PAGE (Fig. 2, inset); this mobility shift and mAbs to phosphate-dependent and non-phosphate-dependent epitopes of the triplet proteins were used to monitor the extent of dephosphorylation.

Reassembly of purified NFs Biochemical analysis The enriched NF preparations were solubilized in buffer A and proteins were purified by anion exchange

b

Fig. 2. Dephosphorylated bovine NFs negatively stained with 2% methanolic UA. Negative staining reveals tortuous, interconnected and branched 10-12 nm wide filaments with no obvious periodic segmentation. The filaments were comparable to those of native phosphorylated NFs, although more fragmented filaments were apparent after dephosphorylation. A 7.5% SDS-PAGE of control (lane a) and dephosphorylated (lane b) NF triplet protein demonstrates the electrophoretic mobility shift of dephosphorylated NF-M and NF-H (inset). B a r = 100 nm.

186 column chromatography for subsequent reassembly studies. Fig. 3 shows an SDS-PAGE gel containing the NF-enriched starting material (lane 1) and highly purified phosphorylated NF-H, NF-M and NF-L (lanes 2-4) and dephosphorylated NF-H, NF-M and NF-L, respectively (lanes 5-7). The rate of NF reassembly was monitored over 90 min. During this time, unassembled NF proteins remained in the supernatant, and reassembled NFs could be pelleted by centrifugation. Phosphorylated and dephosphorylated NFs were similar with regard to the time course for maximal reassembly. At 0-15 min from the start of dialysis against reassembly buffer, virtually all of the protein remained in the supernatant (Fig. 4, lanes 2, 3). By contrast, abundant, pelleted, reassembled NFs were detected at 30-90 min post-reassembly (Fig. 4, lanes 11-14). Protein assaysa of the supernatants and pellets of quadruplicate samples at an initial protein concentration of 0.5 mg/ml were analyzed to obtain a statistically significant depiction of the reassembly kinetics through 90 min of reassembly. These data are shown graphically in Fig. 5 and reveal that - 5 0 % reassembly can be obtained by 30 min, and -85-90% of the protein reassembled within 90 min after beginning dialysis in reassembly buffer. In all instances, NF-H was the only NF protein recovered in the supernatant at 90 min; this may represent an excess of NF-H added to the reassembly mixture or the difficulty NF-H has in forming homopolymers (see companion paper). Our results were obtained consistently when 0.5 mg/ml of purified NF subunit proteins were used for reassembly. However, when lower concentrations of protein were used (e.g. 0.15 mg/ml), the time for maximal reassembly (i.e. the time at which 85-90% of the NFs were reassembled) was extended to t>180 min (data not shown). Thus, the rate of reassembly is dependent in part on the protein

I---

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S_.

NF200-NF150--

NF70-- ~

~ i i

1

2

3

4

5

6

7

8

9

1 0 11 1 2 1 3 14

Fig. 4. SDS-polyacrylamide (7.5%) gel analysis of reassembly of purified bovine NF-H, NF-M and NF-L (0.5 mg/ml) into 10-nm filaments over a 90-min time course. Lane 1 contains the freshly isolated bovine NF-enriehed starting material. Twelve/tg of purified proteins were loaded onto lane 2. In subsequent companion lanes (other than lane 8 which is blank) the amount of NF subunit protein loaded was the same as in lane 2. However, these samples were divided into supernatant (S) and pellet (P) fractions and each fraction was loaded in different wells. A progressive diminution of unassembled NF subunits is observed in the supernatants from 0 to 90 min (lanes 2-7), while pelletable, rapidly assembled NFs appear within 30-90 min from the start of reassembly (lanes 11-14).

concentration of the NF subunits. The ultrastructural appearance of the reassembled NFs using 0.15 mg/ml was

lOO 90 80 z

7o 60

o

~. 5o m 40

2(? 10 0 15

30

45

60

75

90

TIME OF REASSEMBLY [MIN|

1

2

3

4

5

6

7

Fig. 3. SDS-PAGE (7.5% polyacrylamide) of isolated bovine NFs and HPLC purified NF proteins. Lane 1 contains NF-enriched starting material, lanes 2-4 contain phosphorylated NF-H, NF-M and N-F-L, and lanes 5-7 contain dephosphorylated NF-H, NF-M and NF-L, respectively.

Fig. 5. The time course of bovine NF reassembly was evaluated quantitatively following HPLC purification of the triplet protein as described in the text. Within 90 min from the start of dialysis in reassembly buffer, ~85% of the NF protein (H:M:L) was reassembled (initial concentration 0.5 mg/ml). Each point represents the mean percent of protein + S.E.M. (vertical bars) of quadruplicate samples collected at different time intervals.

187 the same as that o b t a i n e d from 0.5 mg/ml protein (see below).

Ultrastructural analysis of reassembled phosphorylated NFs Ultrastructurally, at 0-15 min from the start of dialysis

in the reassembly buffer, the p r e d o m i n a n t structures o b s e r v e d were unpelletable fragments <0.25 # m in length (data not shown). N F s >~0.5 /zm in length a p p e a r e d by 30 min, and at 3 0 - 9 0 min post-reassembly, n u m e r o u s grids r e v e a l e d a n e t w o r k of parallel or nearly parallel N F s which a p p e a r e d to interconnect neighboring

Fig. 6. Reassembled bovine NFs sometimes display an intersecting and parallel-like meshwork following negative staining with 2% methanolic UA (a). This pattern, which exhibits similarities with axonal NFs in situ, is revealed after placing glow discharged, formvar carbon-coated nickel grids on the surface of the NF solution undergoing dialysis for >30 min. Bead-like areas also may be observed along the reassembled NFs (b). These areas display a periodicity of ~19.8 _+ 1.6 nm, n = 75 (arrowheads), and may have short spike-like projections (b, inset, small arrows) which may interact with neighboring filaments. Additionally, branching of reassembled NFs is observed frequently (b, large arrowhead). The widths of the 3 branches in b were ~10-12 nm. Bars = (a,b) 200 nm; (b, inset) 100 nm.

188 filaments via branches and side arm-like projections (Fig. 6a). Some of these projections may represent the tail domains of NF-H and NF-M. These near-parallel arrays of reassembled NFs might be determined solely by the reassembly conditions and the adsorption onto EM grids, or they may reflect intrinsic properties of NFs that lead to a stable network of axonal NFs as observed in situ. Although small NF fragments could be observed through 60-90 min, the vast majority of NFs observed were long reassembled NFs that branched frequently as noted above (Fig. 6b). Like native NFs, reassembled NFs were ~10-12 nm in diameter and displayed a well-recognized bead-like periodic segmentation (=18-22 nm) (Figs. 6b and 7). Projections (Fig. 6b, inset) and protofilaments

(Fig. 7 and inset) were evident for a number of these reassembled filaments. Dialysis for 12 and 24 h yielded NFs that appeared quite similar to those observed at 90 min; however, at these later times, numerous grid squares contained NF fragments ~0.5-1.0/~m in length as well as NFs that appeared partially 'unraveled'. Unraveling of NFs may lead to the occasional observation of NFs appearing much greater in width than the typical intermediate filament.

Ultrastructure of reassembled dephosphorylated NFs Reassembled dephosphorylated NFs at short reassembly times (i.e. 15-30 min) appeared fragmented and relatively smooth by negative staining (Fig. 8a). In

Fig. 7. The substructure of reassembled phosphorylatedNFs is revealed after 90 min of reassemblyfollowingnegative staining with methanolic UA. Note that protofilaments comprising the NFs are recognizedespeciallyin filaments that appear to be 'unravelling' (a, inset, arrows). At later times (e.g. 12-24 h), numerous short or fragmented NFs also appear to 'unravel'. Additionally, the periodic segmentation (bars) of the reassembled NFs can be discerned readily. Bars = (a) 100 nm; (inset) 50 nm.

Fig. 8. Negative staining of dephosphorylated NFs reassembled at 15-30 min (a) as compared to !I0 min (b) revealed a dramatic difference in the structure of the reassembled proteins. Between 15 and 30 min, only short fragmented NFs could be discerned, whereas after 3040 min, long NFs predominated. These long NFs exhibited branching similar to that observed for reassembled phosphorylated NFs. Bars = (a) 100 nm; (b) 200 nm.

contrast, at longer post-reassembly times (i.e. 90 min), tortuous, long and relatively smooth NFs were apparent (Fig. 8b). From some NFs, branching, spike-like areas and projections could be discerned, but these characteristics were not as prominent as observed for phosphorylated NFs.

to a non-phosphorylated epitope in the MPR of NF-H. In contrast, TA56, a rod domain-specific mAb to NF-H, failed to label native and reassembed NFs (Fig. 9d). Thus, it appears that the tail domain epitopes of NF-H are exposed and the rod domain epitopes of NF-H are buried in reassembled NFs. NF-M. NFs probed with mAbs directed against phos-

Zmmunoelectron microscopy In the far right columns of Table I, a summary of the immunogold labeling patterns produced by domainspecific antibodies to NF-H, NF-M and NF-L has been provided for reassembled phosphorylated and dephosphorylated NF triplet proteins. The immunolabeling profile of each NF subunit is detailed separately below. NF-H. When mAbs which bind to phosphate-dependent epitopes in the multi-phosphorylation repeat (MPR) of the tail domain of NF-H31 (e.g. TA51, TA50) were used, both native and reassembled phosphorylated NFs, but not dephosphorylated NFs, were labeled with 5-nm gold particles localized primarily as interspersed clumps (>22 nm apart) along these filaments (Fig. 9a,b). These may represent collapsed tail domains or ‘lateral projections’ along both sides of the filament backbone. Similar labeling was seen when reassembled dephosphorylated NFs were decorated with mAbs specific for non-phosphorylated peripheral epitopes of NF-H (e.g. DPl in Fig. 9c). As expected, native and reassembled phosphorylated NFs were not labeled by DPl (data not shown), a mAb

phate-dependent epitopes (e.g. TA34, OCSO) in the tail domain of NF-M, displayed gold particles localized uniformly (-18-20 + 10.7 nm apart, n = 73) along the reassembled phosphorylated NFs (Fig. 10a). In contrast, non-uniform scant labeling was observed with the same mAbs along dephosphorylated NFs (Fig. lob). The minor staining of dephosphorylated NFs by mAbs that are phosphorylation-dependent probably reflects incomplete enzymatic dephosphorylation of the NF-M protein4. Antibodies to phosphate-independent epitopes of NF-M were found to label in two distinct patterns depending on the epitope location (see Table I). The first pattern observed following immunolabeling with Oc85 and 0C33 (data not shown) consisted of uniform labeling along the margins of the NFs similar to that observed with TA34 (see Fig. 10a). The second pattern which yielded gold particles primarily in pairs and clumps spaced along the NFs at irregular intervals was seen with a mAb which binds to the last 20 aa of the tail domain of NF-M (RM0255) (Fig. 10~) and with an antiserum (designated MAT) which binds to the first 5 aa of the

190

TA51

a

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m

TA

"

.

-

"

DP1

Fig. 9. Immunogold labeling of native and reassembled bovine NFs with mAbs specific for NF-H. TA51, which recognizes P[+] epitopes in the tail domain of bovine NF-H, labels native (a) and reassembled phosphorylated (b) NFs in interspersed clump-like patterns (arrowheads). Dephosphorylated NFs label only slightly with TA51. In contrast, DP1, which recognizes P[-] epitopes of the tail domain of NF-H, labels dephosphorylated NFs in an interspersed clump-like manner (c), whereas phosphorylated NFs do not label with this mAb. TA56, a mAb specific to the NF-H rod domain, does not label the NFs appreciably (d). Bars (a-d) = 100 nm.

head domain of NF-M 45'52 (Fig. 10d). In contrast, virtually no labeling was evident following staining of native and reassembled NFs with mAbs to the rod domain of NF-M (TA54) (data not shown). NF-L. The labeling observed with antibodies specific for phosphate-independent epitopes of NF-L displayed two different patterns for both native and reassembled NFs (see Table I). Immunogold decoration by SE3, a mAb which binds to a non-rod epitope, revealed labeling primarily on the margins along native and reassembled NFs (Fig. lla,b). On the other hand, a rabbit antiserum (designated anti-NF-L) which binds to the last 20 aa of the COOH terminus of NF-L labeled both the marginal and backbone regions of native (Fig. llc) and reassembled (Fig. l l d ) NFs. However, labeling of reassembled NFs was much heavier than that of the native NFs. Similarly, SE3 did not label reassembled NFs as heavily

as compared to the rabbit antiserum specific for the COOH terminus of NF-L. The reasons for this are not known, but they may reflect the unmasking of epitopes after purification and reassembly, the recognition of different non-rod domains (e.g. SE3 may recognize the head domain versus rabbit anti-NF-L which binds to the tail domain), and/or differences in the affinity of the antibodies for native vs reassembled NF-L. Like antiNF-H and NF-M rod-specific mAbs, a mAb specific to the rod domain of NF-L (e.g. SE6) produced virtually no labeling of any NFs (Fig. lie). Finally, double labeling of the reassembled NF triplet protein with SE3 (5 nm gold) and MAT (10 nm gold) demonstrated co-localization to the same filament of NF-L and NF-M (Fig. llf). The antiserum MAT was more widely dispersed along the filaments than SE3, but in numerous instances, both antibodies were in close apposition (arrows).

191

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m

Fig. 10. Immunogold labeling of epitopes within the head, rod and tail domains of the NF triplet with antibodies to NF-M. TA34 labels phosphate-dependent epitopes on native and reassembled phosphorylated NFs uniformly (a), whereas labeling of reassembled dephosphorylated NFs (b) is scant at best (arrowheads). MAbs to phosphate-independent epitopes of NF-M (e.g. RMO255) (c) label all NFs non-uniformly on their margins and backbone (small arrows). Similarly, MAT which recognizes the head domain of NF-M, labels native and reassembled phosphorylated (d) and dephosphorylated NFs in a non-uniform fashion. TA54, which recognizes the NF-M rod domain, does not label native or reassembled NFs appreciably (data not shown). Bars = 100 nm. Controls of the immunolabeling experiments (see Materials and Methods) were consistently negative in all incubations of reassembled NFs. With incubations of the native NF preparation, some labeling of filaments with 2.2B10 (anti-GFAP) was present (data not shown); this labeling was expected given the contamination of our isolated NF preparation with GFAP. DISCUSSION The data reported here demonstrate that purified mammalian NF triplet proteins can rapidly and efficiently reassemble in vitro to generate NFs that are nearly indistinguishable from isolated native NFs by ultrastructural and immunological criteria. MAbs specific for defined epitopes were used to localize the corresponding protein domains within native and reassembled NFs, and new information on the disposition of distinct protein

domains within head, rod and tail domains of each of the NF triplet proteins was obtained which allowed us to extend current insights into the structure of NFs. Types I (acidic keratins) and II (basic keratins) and type III (desmin and vimentin) IFs have been shown to undergo efficient reassembly at low protein concentrations from denaturing solutions (e.g. 8 M urea) into various reassembly buffers 21'26'47. As a result of our efforts to devise conditions for the efficient reassembly of purified NF triplet proteins similar to those observed for other IFs, we show that filament reassembly occurs within 30 rain, that it is influenced by the concentration of protein, and that the phosphorylation state of the proteins does not affect their reassembly. Furthermore, the intermediate dialysis step between urea and guanidine-thiocyanate prior to dialysis against buffer C is essential since only a few short NFs could be discerned following dialysis directly from urea to buffer C (unpub-

Fig. 11. Immunolabeling of NFs with mAbs to NF-L. SE3, specific for P[ind] epitopes of the non-rod domain(s) of NF-L, readily labels the marginal regions of reassembled phosphorylated NFs (a) and dephosphorylated NFs (b, arrowheads). In contrast, rabbit anti-NF-L, which is specific for the last 20 aa in the tail domain of NF-L, labels native NFs (c) and reassembled phosphorylated (d) and dephosphorylated NFs on their margins and backbones. Like mAbs specific to the rod domains of NF-H and NF-M, SE6 fails to label the NF triplet protein (e). Co-localization of antibodies to NF-L (SE3, 5-nm gold) and NF-M (MAT, lO-nm gold, arrows) reveals the disposition of subunit domains along the NF triplet proteins (f). Bars = 100 nm.

lished observations). Interestingly, we have observed that NFs can be reassembled into lo-nm filaments in low concentrations of guanidine-thiocyanate (e.g. 0.1-1.0 M), suggesting that under these conditions NF subunit

proteins probably have a high affinity for one another (data not shown). By negative staining with methanolic UA, native, axonally derived NFs, comprised of the more extensively

193 phosphorylated isoforms of NF subunits5'28"29'49, and reassembled phosphorylated NFs appear indistinguishable with regard to their periodicity, branching from parent filaments, and presence of 'side arm-like projections'. Although protofilaments of the NFs are not apparent in isolated NFs, they can be observed in reassembled NFs. A protofilamentous substructure has been observed previously for Types I, II and III IFs 1A4'21 and, more recently, for type IV NF-L proteins TM. Similarly, the periodic bead-like repeating units that we observe (i.e. 18-22 nm axial periodicity) approximate those reported earlier in studies of metal-shadowed NFs 14'17'18'36. These bead-like repeats exhibited a distinct substructure with widely separated protofilaments and/or protofibrils that appeared to coil with one another (see results Fig. 7). These findings are consistent with the view that IFs are comprised of a protofilamentous substructure 39,48. Reassembled dephosphorylated NFs underwent reassembly within a time course comparable to reassembled phosphorylated NFs (data not shown). These results extend earlier studies, suggesting that dephosphorylation of NFs has no effect on assembled NFs 4, or on the reassembly of NF subunits into intermediate-sized filaments 12'18. However, it should be noted that enzymatic dephosphorylation does not completely dephosphorylate NF subunits 4. Thus, the phospho-amino acids that are resistant to dephosphorylation may be present in NF subunits, and they may play a role in filament assembly and/or stability. The close similarity of the immunolabeling patterns observed with mAbs specific to epitopes localized to the rod and tail domains of NF-H, NF-M and NF-L in isolated native as well as in enzymatically dephosphorylated NFs and in reassembled NFs further substantiates the structural homology between reassembled and native NFs. As expected, mAbs to phosphate-dependent epitopes (such as TA51 and TA34) labeled phosphorylated NFs, but not dephosphorylated NFs, whereas DP1 only labeled dephosphorylated NFs. Because DP1 labeling was comparable to that observed for TA51 (see below), DP1 and TA51 may label the same epitopes or spatially proximate epitopes in different states of phosphorylation within the MPR of NF-H 31. Further, since mAbs to the rod domains of each NF subunit yielded scant or no labeling of native and reassembled NFs, we speculate that the epitopes within the rod domains are masked as a result of the a-helical coiled-coil interactions between their conserved rod regions8. These findings are consistent with the notion that the rod domain of each NF subunit is intimately involved in the formation of the NF backbone. In contrast, epitopes within the peripheral tail domains of each triplet protein do not appear to be

blocked or masked since antibodies to the head and tail domains readily label the NFs. Finally, double immunolabeling demonstrates that heteropolymers of NF-H, NF-M and NF-L are indeed formed following reassembly. Native and reassembled phosphorylated NFs are labeled by TA51 and TA50 in interspersed dumps; this pattern of labeling suggests that either multiple sites within the MPR of NF-H may be recognized by these mAbs and/or that the tail domain(s) of NF-H and/or NF-M is collapsed onto the filament backbone. Since it is unlikely that multiple antibody molecules can bind to a single MPR, and because the distance between the groups of particles is greater than the -22-nm periodic repeat, the most logical explanation of the appearance of interspersed dumps is that the tail domains are collapsed against the filament backbone. This scenario appears likely since the tail domains of NF-H and NF-M are quite large and would tend to fold and clump following negative staining. Antibodies that recognize epitopes closer to the rod domains may label more evenly; for example, the labeling patterns that are seen with TA34 and with anti-NF-L (Figs. 10a and lld). The location of the extreme NH 2 and COOH termini of NF-M relative to other NF subunit domains within the NF triplet protein is not yet known. We observed a discontinuous clump-like distribution of gold particles along the reassembled filaments following immunolabeling with both MAT (NH 2 terminus) and RMO255 (COOH terminus) (see Fig. 10). This labeling pattern suggests that the extreme ends of the COOH and NH2-termini of NF-M probably are exposed. However, immunolabeling of the NF-M tail domain with 2 tail domain-specific mAbs (i.e. TA34 recognizes P[+] epitopes in this region) and RMO255 (recognizes the last 20 aa of the tail domain) reveals that TA34 labels the tail domain continuously, whereas RMO255 labels this region discontinuously. This suggests that the COOHtermini of NF-M may interact with themselves and therefore mask much of the labeling with RMO255. Notably, the extreme COOH-terminal region of NF-M does contain a segment of heptad repeats capable of forming a-helical coiled-coils, and it is this motif that is conserved in a variety of species44. Indeed, recent evidence from our laboratory supports the notion that this extreme COOH-terminal heptad repeat region interacts with a second NF-M tail domain containing the heptad repeat region. The different labeling patterns observed for antibodies to the non-rod region(s) of NF-L indicate that SE3 and rabbit anti-NF-L bind to different epitopes. The antiNF-L antisera binds to the last 20 aa in the COOH terminus of NF-L, but the exact location of SE3 binding

194 has yet to be identified. It is possible that SE3 recognizes the head domain given the differences in its labeling p a t t e r n c o m p a r e d with the a n t i - N F - L antiserum. Labeling of reassembled NFs with a n t i - N F - L was abolished when co-labeling these filaments with SE3 (data not shown). The abolition of labeling suggests that the epitopes seen by these two antibodies are p r o b a b l y situated close to one a n o t h e r within the fully assembled N F protein. That rabbit a n t i - N F - L antibody labels both peripherally and on the b a c k b o n e of the filaments suggests that the C O O H terminal domain of N F - L is situated very close to the filament core and is exposed superficially following reassembly of the N F triplet protein. This p a t t e r n of labeling contrasts with that seen using antibodies to the tail domains of N F - H and N F - M (e.g. TA51 and RMO255, respectively). These differences probably reflect variations in the length of the projecting domains of each of the N F subunit proteins and in the REFERENCES 1 Aebi, U., Fowler, W.E., Rew, P. and Sun, T.-T., The fibrillar substructure of keratin filaments unraveled, J. Cell Biol., 97 (1983) 1131-1143. 2 Birrell, G.B., Hedberg, K.K. and Griffith, O.H., Pitfalls of immunogold labeling: analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy, J. Histochem. Cytochem., 35 (1987) 843-848. 3 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254. 4 Carden, M.J., Schlaepfer, W.W. and Lee, V.M.-Y., The structure, biochemical properties and immunogenicity of neurofilament peripheral regions are determined by phosphorylation, J. Biol. Chem., 260 (1985) 9805-9817. 5 Carden, M.J., Trojanowski, J.Q., Schlaepfer, W.W. and Lee, V.M.-Y., Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns, J. Neurosci., 7 (1987) 3489-3504. 6 Chin, T.K., Eagles, P.A.M. and Maggs, A., The proteolytic digestion of ox neurofilaments with trypsin and alpha-chymotrypsin, Biochem. J., 215 (1983) 239-252. 7 Drager, U.C., Edwards, K.L. and Kleinschmidt, J., Neurofilamerits contain a-melanocyte-stimulating hormone (a-MSH)-like immunoreactivity, Proc. Natl. Acad. Sci. U.S.A., 80 (1983) 6408-6413. 8 Geisler, N., Kaufmann, E., Fischer, S., Plessmann, U. and Weber, K., Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminai extensions increasing in size between triplet proteins, EMBO J., 2 (1983) 1295-1302. 9 Geisler, N., Kaufmann, E. and Weber, K., Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments, J. Mol. Biol., 182 (1985) 173-177. 10 Geisler, N. and Weber, K., Self-assembly in vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments, J. Mol. Biol., 151 (1981) 565-571. 11 Geisler, N. and Weber, K., The amino acid sequence of chicken muscle desmin provides a common structural model for intermediate filament proteins, EMBO J., 1 (1982) 1649-1656. 12 Georges, E., Lefebvre, S. and Mushynski, W.E., Dephosphorylation of neurofilaments by exogenous phosphatases has no

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