d1 cytoskeletons during differentiation

142

Developmental Brain Research. 5(I (Its89) t42-i4¢~

Elsevier BRESD 60332

Appearance and localization of pho horylated variants of the high molecular neurofilament protein in NB2a/dl cytoskeletons during differentiation Thomas B. Shea 1'2, Mary Lou Beermann I and Ralph A. Nixon 1'3 1Ralph Lowell Laboratories, Mailman Research Center, McLean Hospital, Belmont MA 02178 (U.S.A.) and Departments of 2Biological Chemistry and Molecular Pharmacology and 3psychiatry, and 3Program in Neuroscience, Harvard Medical School, Boston, MA 02115 (U.S.A.)

(Accepted 5 July 1989) Key words: Neurofilament; Phosphorylation; Neurite outgrowth; Axonal maturation; Neuronal culture; Neuronal differentiation;

Neuroblastoma; Cytoskeleton

We used immunoblot and immunocytochemical methodologies to characterize the appearance and intracellular localization of the high molecular weight neurofilament subunit (NF-H) within the Triton-insoluble cytoskeleton during the first 5 days of differentiation of mouse NB2a/dl neuroblastoma cells. Hypophosphorylated and partially phosphorylated forms of NF-H were detected in cells before and throughout differentiation. By contrast, some extensively phosphorylated forms of NF-H were first detected on the third day of differentiation and at least one additional 200 kDa isoform was visualized in cytoskeletons only after five days of differentiation. Extensively phosphorylated forms of NF-H were restricted to axonal neurites; by contrast, hypophosphorylated and partially phosphorylated forms of NF-H were present throughout undifferentiated and differentiated cells. The high molecular weight neurofilament subunit (NF-H) undergoes extensive phosphorylation, which profoundly influences its electrophoretic mobility on SDS-gels 2'3'11'17A9'27 and generates microheterogeneous isoforms 6'16'21'23. Since the phosphates are added predominantly to the end-domains of the molecule which protrude from the neurofilament backbone, it has been suggested that phosphorylation may mediate the interaction of neurofilaments with each other and with other cytoskeletal constituents 12'ts'23. This line of reasoning is strengthened by observations that individual phosphate groups are added and removed as neurofilaments enter and advance along a x o n s 14"24'25"27. Various investigators have reported that the appearance of NF-H is delayed relative to that of NF-M and NF-L 2'26'28'3°'31'36. Others, however, have concluded that it is the appearance of extensively phosphorylated forms of NF-H that is delayed rather than the subunit itself 1° and that the earlier findings reflect the fact that anti-NF-H antibodies used in

earlier studies recognize only extensively phosphorylated NF-H subunits 5'6'14'2°'31'35. These latter studies and others 23 have suggested that the appearance of extensively phosphorylated NF-H may be related to the development of axonal cytoskeletal stability. In the present study, we have characterized the sequential appearance and subcellular localization of NF-H and post-translationally modified forms of this subunit generated by phosphorylation within the Triton-insoluble cytoskeleton of mouse NB2a/dl neuroblastoma cells during differentiation and neurite outgrowth. Cells were cultured as described previously 33. To induce the outgrowth of axonal neurites 32,33, the medium was replaced with medium containing 1 mM dibutyryl adenosine 3",5"-cyclic monophosphate (dbcAMP; Sigma) 24 h after plating and the cells were cultured for an additional 1-5 days; these cells are defined as day 1 cells through day 5 cells, respectively, to indicate the length of dbcAMP treatment. Cells not treated with dbcAMP are referred to as day 0 cells.

Correspondence: T.B. Shea, Ralph Lowell Laboratories, McLean Hospital, Belmont, MA 02178, U.S.A.

0165-3806/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

143 Nitrocellulose replicas of Triton-insoluble cytoskeletons following SDS-gei electrophoresis were prepared as described previously33. The transferred proteins were visualized by sequential reaction of the nitrocellulose with antisera against NF-H (below) followed by secondary antibody conjugated with horseradish peroxidase and diaminobenzidine in the presence of H20 2. Triton-insoluble cytoskeletons of cultures grown in chamber slides (Lab-Tek, Naperville, MD) were fixed and processed for immunocytochemistry as described previously33. Rabbit polyclonal antisera H2 (raised against electrophoretically purified NF-H 4) was a generous gift of Dr. R.E. Majocha (McLean Hospital, Belmont, MA). Monoclonal antibodies directed against non-phosphorylated (SMI-32) and phosphorylated (SMI-31) neurofilament epitopes 35 were obtained from Sternberger-Meyer Immunochemicais (Jarretsville, MD). Monoclonal antibody RT97 ~ was a generous gift of Dr. Ursula C. Drager (Harvard Medical School, Boston, MA). SMI-32, H2 and SMI-31 were diluted 1:1000, and RT97 was diluted 1:500. We characterized by immunoblot analysis NF-H forms present in cytoskeletons of undifferentiated (day 0) NB2a/dl cells, which have only putative neurites, and cytoskeletons from cells after 3 days of differentiation, which have elaborated axonal neurites 32'33. Hypophosphorylated NF-H forms were visualized in day 0 cytoskeletons by SMI-32 (diffuse bands migrating at approximately 160 kDa and 175 kDa; Fig. la) and additional forms migrating at 175-180 kDa were stained by H2 (Fig. lb); however, extensively phosphorylated NF-H forms were not detected in day 0 cytoskeletons (Fig. lc,d). Significant changes were noted in cytoskeleton- associated levels of phosphorylated NF-H during differentiation. In day 3 cytoskeletons, hypophosphorylated NF-H subunits (approximately 160-175 kDa) were observed with SM1-32 at similar levels (Fig. le) as had been detected at day 0. By contrast, in day 3 cytoskeletons, H2 (Fig. lf) and SMI-31 (Fig. lg) labeled a range of more highly phosphorylated NF-H subunits migrating from 175-200 kDa. Additional phosphorylation-dependent NF-H epitopes were first visualized by RT97 at day 5 (Fig. li). Having demonstrated the specificities of these antibodies for NF-H subunits and their phosphorylated variants, we examined the incorporation into

the cytoskeleton and subcellular localization of NF-H forms during neuritogenesis. This is accomplished by performing immunocytochemical analyses of Triton-extracted cytoskeletons from NB2a/dl cells harvested at daily intervals from 0 to 5 days after inducing differentiation with dbcAMP. The antibodies used to examine the appearance and localization of NF-H subunits and variants generated by phosphorylation each yielded unique staining patterns. SMI-32 (Fig. 2, row 1) immunoreactivity was present in putative neuritic cytoskeletons of day 0 cells (Fig. 2a), and underwent a moderate increase in day 1 neuritic cytoskeletons (Fig. 2b); no further increase in neuritic immunoreactivity was observed (Fig. 2c-f). By contrast, perikaryal immunoreactivity increased in day 2 cytoskeletons, and apparently continued to increase during continued dbcAMP treatment (Fig. 2d-f). A similar distribution was observed in immunocytochemical analyses of the middle and low molecular weight neurofilament subunits during differentiation (not shown). Only marginal staining of perikaryai or neuritic cytoskeletons was observed with H2 (Fig. 2, row 2) until day 3 after the addition of dbcAMP, at which time immunoreactivity in perikarya and neurites increased; no further change was detected by day 5 after dbcAMP treatment (Fig, 3a-f). Both SMI-31 and RT97 (below) strongly labeled nuclei. This labeling pattern is thought to reflect the binding of neurofilament subunits by lamins 13 and/or cross-reactivity of these antibodies with histones 37, both of which share antigenic determinants with

a

b

cd

e

fgh

i

Fig. 1. Immunoblot analyses of the appearance of phosphorylated NF-H variants during NB2a/dl differentiation. Tritoninsoluble cytoskeletons from day 0 (a-d), day 3 (e-h) and day 5 (i) cells were immunostained with the following antibodies: SMI-32 (a,e); H2 (b,f); SMI-31 (c,g); RT97 (d,h,i). The migratory positions of 200 kDa, 97 kDa and 68 kDa molecular weight markers are indicated by arrowheads on the left of the figure.

144 neurofllament subunits. The extent of nuclear reactivity with these antibodies apparently decreased during differentiation of NB2a/dl cells (Fig. 4a-f;

Fig. 5a-f), which may reflect alterations in lamin expression during differentiation of these cells. Immunoreactivity with SMI-31 (Fig. 2, row 3) was

C

Fig. 2. Immunocytochemical analyses of the appearance of phosphorylated NF-H variants during NB2a/dl differentiation. Panels a-f represent Triton-extracted cytoskeletons from day 0-5 cells, respectively, which were immunostained with the following antibodies: SMI-32 (row 1); H2 (row 2); SMI-31 (row 3); RT97 (row 4).

145 marginal in p e r i k a r y a and neurites of day 0 - 2 cytoskeletons (Fig. 2 a - c ) . I m m u n o r e a c t i v i t y with SMI-31 first b e c a m e p r o m i n e n t in some neurites by day 3 (Fig. 2d, arrows), and in the m a j o r i t y of neurites by day 4 (Fig. 2e). By comparison, immunostaining with RT97 (except in nuclei) was not d e t e c t e d until 4 days after the addition of d b c A M P (Fig. 2 a - e , row 4) and at this time the staining was restricted to focal regions along the neurite (Fig. 2e, arrows). Staining of the entire neurite with RT97 was first o b s e r v e d in day 5 cytoskeletons, although in s o m e neurites RT97 immunoreactivity was more intense distally than proximally (Fig. 2f, row 4, arrows). Regions of the p e r i k a r y o n near the base of axonal neurites were occasionally labeled with SMI31 in day 4 and 5 cytoskeletons (Fig. 2e,f, rows 3 and 4, arrows); however, staining of the perikaryal cytoskeleton with RT97 was not observed at any time. These results confirm that extensively phosp h o r y l a t e d N F - H subunits are segregated within axonal neurites 33, and also suggest that the majority of h y p o p h o s p h o r y l a t e d N F - H isoforms are localized within the p e r i k a r y o n . These findings in N B 2 a / d l cells suggest, as has been d e m o n s t r a t e d by others (above) that the relative delay in a p p e a r a n c e of extensively phos-

p h o r y l a t e d N F - H isoforms during differentiation is due to d e l a y e d post-translational modification rather than d e l a y e d expression of the N F - H p o l y p e p t i d e . The additional delay in a p p e a r a n c e until day 5 of certain extensively p h o s p h o r y l a t e d epitopes suggests, as has been o b s e r v e d in vivo and in p r i m a r y culture 5-7"9"~j'j42°'3~'35, the presence of multiple

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classes of p h o s p h o r y l a t i o n - d e p e n d e n t N F - H epitopes in N B 2 a / d l cells. These results may therefore reflect the late a p p e a r a n c e and/or activation of distinct neurofilament kinases, as has been suggested for neurons in primary culture 11. Resistance of N B 2 a / d l axonal neurites to retraction following exposure to coichicine is first detected in some neurites at day 3, and has d e v e l o p e d in all neurites by day 73~. The t e m p o r a l correlation between the d e v e l o p m e n t of axonal stability and the a p p e a r a n c e within axons of N F - H subunits which have undergone the post-translational modifications associated with axonal m a t u r a t i o n in vivo suggests that these modified forms may influence neuritic stability in N B 2 a / d l cells. This research was s u p p o r t e d by the Medical Foundation, Inc./Charles A. King Trust, AG05604, and BNS-8719823.

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