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The expression of phosphorylated and non-phosphorylated forms of MAP5 in the amphibian CNS.
Viereck C
,
Matus A
.
???displayArticle.abstract??? MAP5 is a microtubule-associated protein that in rat and quail is more abundant in the developing than in the adult brain. Previous studies in our laboratory have shown that MAP5 can be resolved into two forms by SDS-PAGE, MAP5a and MAP5b (Mr 300,000-320,000 Da) with MAP5a representing a highly phosphorylated form of this protein. In the present study, the relationship between MAP5 expression and neuronal growth and plasticity was investigated by assessing the amount and distribution of MAP5a and MAP5b in both the developing Xenopus brain and in different regions of the adult brain where neurons of varying growth potential and plasticity are present. In the larval and metamorphic Xenopus brain, like the neonatal rat brain, MAP5 is present in the highly phosphorylated form, MAP5a, and in concentrated in neuronal processes. In the adult Xenopus brain, MAP5a remains high in the optic tectum but, like the situation in the adult rat brain, is undetectable in the telencephalon. Immunohistochemistry showed that MAP5 was concentrated in the outer layer of the tectum, where ingrowing and plastic retinal ganglion cell axons are found. The correlation between MAP5 expression and phosphorylation and growth potential suggests that this molecule plays an important role in the regulation and organization of the neuronal cytoskeleton during neurite outgrowth and plasticity.
Fig. 1. Immunoblot analysis of MAP5 phosphorylation in the adult
Xenopus brain. In control preparations incubated in the absence of
alkaline phosphatase (lane 1), the presence of alkaline phosphatase
and 10 mM 5-nitrophenylphosphate (lane 3), or in the presence of
10 mM 5-nitrophenylphosphate alone (lane 4), two forms of MAP5,
MAP5a and MAP5b, (M r 300,000-320,000 Da) can be identified.
Following alkaline phosphatase treatment (lane 2) only the lower
polypeptide, MAP5b, is detectable. A 4-6% linear gradient of
acrylamide was used. The top of the gel is indicated by an arrow.
Standards corresponding to M r 200,000, 116,000, 92,000, 66,000 and
45,000 Da are indicated on the right.
Fig. 2. Temporal expression of MAP5 forms in total Xenopus brain.
The immunoblot stained with monoclonal MAP5 antibody reveals
that both the level and relative amount of MAP5a and MAP5b in
whole brain supernatants ($1) and microtubules (MT) remain
unchanged during development. Nieuwkoop and Faber's 31 developmental
stages are indicated. A 4-8% gradient of acrylamide was
utilized. The arrow indicates the origin of the gel. Only the portion
of the immunoblot above the M,+ 116,000 Da marker is shown.
Fig. 3. Regional distribution of MAP5 forms in the adult Xenopus
brain. Immunoblots stained with monoclonal MAP5 antibody reveal
that the predominant MAP5 form expressed by the optic tectum
(OT) is MAP5a while the telencephalon (TEL) expresses only
MAP5b. A 4-8% linear gradient of acrylamide was utilized. Only
the portion of the immunoblot above the M r 200,000 Da marker is
shown.
Fig. 4. Distribution of MAP5 in the adult Xenopus retina. Adjacent
horizontal frozen sections through the optic nerve head of the adult
Xenopus retina stained with monoclonal MAP5 (A) and/3-tubulin
(B) antibodies. Anti-MAP5 staining is most intense in the optic fiber
layer as well as in processes in the outer plexiform layer.
Anti-/3-tubulin staining is more intense in the inner and outer
plexiform layers, onh, optic nerve head; off, optic fiber layer; ipl,
inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform
layer: onl, outer nuclear layer; os, outer segments; pe, pigmented
epithelium. Bar = 50 ~tm.
Fig. 5. Distribution of MAP5 and fl-tubulin in the stage 51 Xenopus
tectum as revealed by immunohistochemistry. Adjacent transverse
sections stained with monoclonal MAP5 (A) and /3-tubulin (B)
antibodies. Anti-MAP5 staining can be seen in all tectal layers and
is similar to that of anti-fl-tubulin. Bar = 50/~m.
Fig. 6. Distribution of MAPs and fl-tubulin in the superficial adult
Xenopus rectum as revealed by immunohistochemistry. Adjacent
transverse frozen serial sections stained with monoclonal MAP5
(A),/~-tubulin (B), MAP2 (C), and tau (D) antibodies. The tectal
layers are indicated. Anti-MAP5 staining is found in only a subset
of axons, those found in layer 9 (compare A with D), though it
cannot be ruled out that it is present in dendrites as well, since there
is considerable anti-MAP2 staining in layer 9. Anti-tau can
additionally be found in lower tectal layers. Bar = 50/~m.
Fig. 7. Distribution of MAPs and fl-tubulin in the adult telencephalon as revealed by immunohistochemistry. Adjacent transverse frozen serial
sections were stained with monoclonal MAP5 (A), fl-tubulin (B), MAP2 (C), and tau (D) antibodies. The control section (E) was incubated
with secondary antibody only. Anti-MAP5 staining, in contrast to the other primary antibodies used, is faint but appears to be present in axons
and dendrites. Bar = 50 um.
