Microtubule-associated Protein 1A Research Articles

Estrogen-enhanced neurite growth: evidence for a selective induction of Tau and stable microtubules

Estrogen stimulates the neurite outgrowth response of medial basal hypothalamic neurons maintained in culture. We show here that one effect of estrogen is to promote an increase in tau, but not in tubulin, microtubule-associated protein 1a (MAP-1a), or MAP-2 protein levels. This response precedes and accompanies an increase in stable microtubules and in neurite length. Taken collectively, our data suggest that estrogen-enhanced neurite growth is mediated by a selective induction of microtubule-stabilizing factors, namely, the tau proteins.

Differential vulnerability of microtubule components in cerebral ischemia

Differential vulnerability of the major components of microtubules was examined in ischemic gerbil brains by a light microscopic, immunohistochemical method using monoclonal antibodies for microtubule-associated protein (MAP) 1A and MAP2, polyclonal antibody for MAP1 and 2 as well as monoclonal antibody for alpha-tubulin. Progressive cerebral ischemia during unilateral carotid occlusion for 5, 15 and 120 min and reperfusion for 3, 12 and 48 h following bilateral carotid occlusion for 10 min were studied. Ischemic lesions in the subiculum-CA1 region were visualized by all antibodies after ischemia for 5 min but the antibody for alpha-tubulin was less sensitive. The antibody for alpha-tubulin was also less sensitive than antibodies for MAPs for detection of early postischemic lesions. Differential sensitivity was also observed in the cerebral cortex and other brain regions. Microtubules in myelinated axons were more stable than those in dendrites. The observed loss of immunohistochemical reactivities for MAPs and alpha-tubulin may have been caused by activation of calcium-dependent proteolytic enzymes such as calpains. The discrepancy between MAPs and alpha-tubulin could be due to differences in affinities or topographic distributions of these proteins within microtubules.

Selective purification of microtubule-associated proteins 1 and 2 from rat brain using poly( l-aspartic acid)

A rapid and selective purification procedure for microtubule-associated protein (MAP) 1 and MAP 2 has been established. This procedure is based upon the fact that poly( l-aspartic acid) (PLAA) can specifically remove MAP 1 from microtubules polymerized by taxol (Nakamura et al., 1989, J. Biochem. 106, 93–97). MAP 1 released by PLAA was further purified by column chromatography on phosphocellulose and Bio-Gel A-15m. The purified MAP 1 contained MAPs 1A and 1B. From microtubules devoid of MAP 1, MAP 2, consisting of MAPs 2A and 2B, could also be isolated by exposure to high ionic strength solutions in the presence of taxol without heat treatment. Both MAPs 1 and 2 cosedimented with microtubules consisting of purified tubulin.

Microtubule-associated protein 1A is the fibroblast HMW MAP undergoing mitogen-stimulated serine phosphorylation

A variety of antibodies to microtubule-associated protein (MAP) have been used to demonstrate that phosphorylation of a 350 kDa microtubule-associated protein is stimulated 2–3 fold by epidermal growth factor or serum in quiescent 3T3-L1 fibroblasts and by insulin in 3T3-L1 adipocytes. Phosphorylation occurs on serine residues, and is maximal by 15–20 min. The phosphoprotein has been identified as MAP1A by specific immunoprecipitation with a well-characterized monoclonal antibody.

Microtubule-associated protein 1A (MAP 1A) is a ganglion cell marker in adult rat retina

We have used antibodies raised against a cytoskeletal protein, microtubule-associated protein 1A (MAP 1A), to stain adult rat retina. In cryostat and polyethylene glycol-embedded radial sections, the fiber layer, ganglion cell layer, and inner plexiform layer were highly immunoreactive, a finding that suggested that the ganglion cell somata, axons, and dendrites were recognized by these antibodies. Retrograde labeling of retinal cell somata from the superior colliculus and dorso-lateral geniculate nucleus to identify ganglion cells showed colocalization of the tracer and immunoreactive cells. Double labeling with nuclear stains revealed that many cells in the ganglion cell layer, which are likely displaced amacrine cells, were not recognized by these antibodies. Furthermore, transection of ganglion cell axons, a procedure that causes retrograde degeneration of many of the axotomized ganglion cells, led to a decrease in the number of anti-MAP 1A immunoreactive cells in retinal wholemounts. Thus, MAP 1A antibodies preferentially stain ganglion cell somata and dendrites but not amacrine cells. These antibodies should be useful ganglion cell markers.

Effect of hypothyroidism on the expression of three microtubule-associated proteins (1A, IB and 2) in developing rat cerebellum

The microtubule-associated proteins 1A, 1B and 2 are present at high levels in Purkinje cell dendrites of normal adult rat cerebellum but show characteristic changes in localization during cerebellar development that allow examination of the effects of hypothyroidism on the development of both Purkinje cells and granule cells. Neonatal rats were made hypothyroid by treatment with propylthiouracil from the day of birth (post-natal day 0, P0). The expression of the microtubule-associated proteins 1A, 1B and 2 in the cerebellum of hypothyroid animals was examined using immunocytochemical techniques and compared to the normal developmental pattern in control animals. The normal developmental decrease in microtubule-associated protein 1A and 1B levels in parallel fibres was delayed in the cerebellum of hypothyroid animals and these proteins persisted in parallel fibres until after P20. Microtubule-associated protein 1B but not 1A was still present in parallel fibres in less mature folia at P30 in hypothyroid rats suggesting that the expression of these two microtubule-associated proteins is regulated separately. In the molecular layer staining with anti-microtubule-associated protein 2 was enriched in Purkinje cell dendrites in normal and hypothyroid cerebella and the stained Purkinje cell dendrites in hypothyroid cerebellum demonstrated a typical deformed morphology at P15. The results show that the restricted subcellular localization of these microtubule-associated proteins is maintained in the cerebellum of hypothyroid rats but the developmental changes in their expression are delayed.

Colocalization of microtubule-associated protein 1A and microtubule-associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy.

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.

The molecular structure of microtubule-associated protein 1A (MAP1A) in vivo and in vitro. An immunoelectron microscopy and quick-freeze, deep- etch study

We studied the distribution of microtubule-associated protein 1A (MAP1A) in Purkinje cell dendrites by means of electronmicroscopic immunocytochemistry, using a monoclonal antibody (McAb) against MAP1A; this was combined with the observation of the 3-dimensional cytoskeletal ultrastructure in dendrites via the quick-freeze, deep-etch technique (QF-DE). We prepared a McAb against rat brain MAP1. This McAb recognized MAP1A on a nitrocellulose filter through use of the immunoblotting method, and stained immunofluorescently Purkinje cell perikarya, dendrites, and axons. Using the McAb, we labeled rat cerebellum extracted with Triton X-100 and simultaneously fixed with aldehyde, followed by gold-labeled rabbit anti-mouse IgG. Gold particles were attached to the filamentous, fuzzy materials, mostly those connected to microtubules (MTs), but were hardly localized on those attached to neurofilaments (NFs). The 3-dimensional cytoskeletal ultrastructure of fresh Purkinje cell dendrites was revealed by QF-DE. In Purkinje cell dendrites, MT was a predominant cytoskeletal element, whereas only a few NFs were found. Fine, elaborate cross-bridges filled up the interstices among MTs, and between MTs and other cellular components. Cross-bridges linking MTs to one another were composed mainly of a fine filamentous structure, frequently branching and anastomosing at several sites, and appeared somewhat granular. We ensured that the cross-bridges observed in saponin-extracted tissues were not a result of artifactual condensations or precipitations of soluble proteins during deep etching. The molecular structure of MAP1A was further investigated by the rotary shadowing technique. The affinity-purified MAP1A was a long, thin, filamentous, and very flexible molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides.

A monoclonal antibody is described that was raised against bovine brain microtubule-associated protein (MAP) 1B. Immunoblot analysis revealed that immunoreactivity was abolished by dephosphorylation of the antigen. The antigen/antibody reaction was also directly inhibited by sodium phosphate. In whole brain tissue, MAP 1B was the primary immunoreactive species. However, the antibody was also found to react with MAP 1A as well as with the high and middle molecular weight neurofilament polypeptides. No cross-reaction with MAP 2, which is known to be extensively phosphorylated, other MAPs, or the low molecular weight neurofilament polypeptide was observed. This evidence suggests at least some sequence homology between these different polypeptide components of the neuronal cytoskeleton and points to a common mechanism for their phosphorylation.

Widespread cellular distribution of MAP-1A (microtubule-associated protein 1A) in the mitotic spindle and on interphase microtubules.

In the accompanying paper (Bloom, G.S., T.A. Schoenfeld, and R.B. Vallee, 1983, J. Cell Biol. 98:320-330), we reported that microtubule-associated protein 1 (MAP 1) from brain comprises multiple protein species, and that the principal component, MAP 1A, can be detected in both neuronal and glial cells by immunofluorescence microscopy using a monoclonal antibody. In the present study, we sought to determine the cellular and subcellular distribution of MAP 1A in commonly used cultured cell systems. For this purpose we used immunofluorescence microscopy and immunoblot analysis with anti-MAP 1A to examine 18 types of mammalian cell cultures. MAP 1A was detected in every culture system examined. Included among these were cells of mouse, rat, Chinese hamster, Syrian hamster, Potoroo (marsupial), and human origin derived from a broad variety of tissues and organs. Anti-MAP 1A consistently labeled mitotic spindles and stained cytoplasmic fibers during interphase in most of the cultures. These fibers were identified as microtubules by co-localization with tubulin in double-labeling experiments, by their disappearance in response to colchicine or vinblastine, and by their reorganization in response to taxol. The anti-MAP 1A stained microtubules in a punctate manner, raising the possibility that MAP 1A is located along microtubules at discrete foci that might represent sites of interaction between microtubules and other organelles. Verification that MAP 1A was, indeed, the reactive material in immunofluorescence microscopy was obtained from immunoblots. Anti-MAP 1A stained a band at the position of MAP 1A in all cultures examined. These results establish that MAP 1A, a major MAP from brain, is widely distributed among cultured mammalian cells both within and outside of the nervous system.