Fractionation of Brain Microtubule‐Associated Proteins

Herzog, Wilhelm; Weber, Klaus

doi:10.1111/j.1432-1033.1978.tb12716.x

Cited by 310 publications

(142 citation statements)

References 29 publications

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“…26 The assembly rate of tubulin to form polymers was monitored using a light scattering assay. 27,28 Purified tubulin was diluted in PEM buffer to a final concentration of 20 µM (according to the monomer, around 1.1 mg.ml -1 ). GST-recombinant proteins were concentrated using Vivaspin columns (Vivascience, Germany) in EB buffer (100 mM Tris-HCl, 120 mM NaCl, 10% glycerol).…”

Section: Introductionmentioning

confidence: 99%

The DCX Superfamily 1: Common and Divergent Roles for Members of the Mouse DCX Superfamily

Coquelle¹,

Levy²,

Bergmann³

et al. 2006

Cell Cycle

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The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.

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Section: Introductionmentioning

confidence: 99%

The DCX Superfamily 1: Common and Divergent Roles for Members of the Mouse DCX Superfamily

Coquelle¹,

Levy²,

Bergmann³

et al. 2006

Cell Cycle

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“…Although very little is known about its regulation in the developing nervous system, it is believed that microtubule-associated proteins (MAPs) are involved (4)(5)(6)(7)(8)(9). Three MAP species have been shown by immunohistochemistry to be differentially distributed in the brain, differing both in the types of cells in which they occur and in their distribution within the neuronal cytoplasm (10).…”

mentioning

confidence: 99%

Differential expression of distinct microtubule-associated proteins during brain development.

Riederer¹,

Matus²

1985

Proc. Natl. Acad. Sci. U.S.A.

214

169

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The levels of three different microtubuleassociated proteins (MAP1, -2, and -3) in brain were found to undergo large changes during postnatal development. MAPi was barely detectable at birth but thereafter steadily increased, reaching adult levels by postnatal day 20 (P20). Both MAP2 and MAP3 showed differential expression patterns of their component peptides. At birth, MAP2 was represented by the smaller of two Mr 280,000 peptides (MAP2b) and three antigenically related Mr 70,000 peptides. The larger of the M, 280,000 peptides (MAP2a) first appeared between P10 and P20, and the Mr 70,000 components disappeared at the same time. Of the two MAP3 peptides, the larger (MAP3a) was present in the late embryo, several days before MAP3b appeared. Between P10 and P20, both MAP3 components underwent a striking decrease in abundance (a factor of 10), which correlated with their disappearance from all neuronal compartments except neurofrlament-containing axons. These developmental changes in expression are different and characteristic for each of the three MAPs, yet in each case they are detectable in brain homogenates, indicating that they occur concurrently throughout the brain.The assembly of microtubules is an essential step in the growth of neuronal processes (1-3). Although very little is known about its regulation in the developing nervous system, it is believed that microtubule-associated proteins (MAPs) are involved (4-9). Three MAP species have been shown by immunohistochemistry to be differentially distributed in the brain, differing both in the types of cells in which they occur and in their distribution within the neuronal cytoplasm (10). MAPi is, according to our observations, associated exclusively with neurons in the adult brain and is more abundant in dendrites than in axons (11-13). MAP2 is also neuron specific and is predominantly, and probably exclusively, located in dendrites (12, 14-18). MAP3 occurs in both neurons and glia, but in neurons it is restricted to neurofilament-rich axons (10,19). In a recent immunohistochemical study, we followed the appearance of these proteins in the developing rat cerebellum and found that each exhibits a characteristic developmental pattern (26). MAPi appears in axons before dendrites, but the balance changes during the first 3 postnatal weeks by the gradual accumulation of MAPi in dendrites and by its simultaneous decrease in axons. MAP2 is exclusively associated with dendrites throughout development. MAP3 appears transitorily in nascent axons (from which it is absent in mature tissue) and later appears in glial cell bodies and processes.These changes in cellular distribution indicate that the expression of these proteins is developmentally regulated. In the present study, we have used monoclonal antibodies to trace the appearance of MAPi, -2, and -3 in rat brain from the late embryo to the end of the third postnatal week. We find that each MAP shows a distinct developmental pattern, which includes large changes in the abundance of their components that occ...

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“…Both MAP2 and tau have been shown to promote the polymerization of microtubules in vitro when added to purified tubulin [8,9]. Hitherto the role of MAPl in polymerization was not known.…”

Section: Discussionmentioning

confidence: 99%

“…A family of four low-M, proteins (tau proteins), M , ranging from 55000 to 68000, has also been shown to copurify with tubulin [6,7]. Purified MAPz or tau proteins can induce polymerization of pure tubulin at low magnesium concentration in the absence of glycerol [8,9]. However, nothing is known of the ability of MAPl to influence polymerization.…”

mentioning

confidence: 99%

Microtubule‐Associated‐Protein MAP1 is not Implicated in the Polymerization of Microtubules

Villasanté

Torre

Manso-Martínez

et al. 1980

European Journal of Biochemistry

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Addition of DNA to microtubule protein solutions results in a decrease of the amount of the high-molecular-weight microtubule-associated proteins (MAP1 and MAP2) available for polymerization. Complete removal of MAPl from microtubules requires lower concentrations of DNA compared to MAP2. Removal of MAPl from microtubules has little or no effect on microtubule polymerization (nucleation and propagation). In contrast, removal of MAP2 produces a marked decrease in microtubule polymerization.Incubation of microtubular protein with DNA results in complete disappearance of the 30-S ring-shaped oligomers at concentrations of DNA which are not markedly inhibitory of polymerization.Brain microtubules purified through cycles of polymerization-depolymerization contain, in addition to tubulin, several other proteins [l -31. These proteins have been classified according to their molecular weights, as high-M, and low-M, proteins [4]. Among the high-M, proteins two of them, named MAPl and MAP2 ( M , equal to 350000 and 300000, respectively), copurify in a constant ratio with tubulin through cycles of polymerization-depolymerization [5]. A family of four low-M, proteins (tau proteins), M , ranging from 55000 to 68000, has also been shown to copurify with tubulin [6,7]. Purified MAPz or tau proteins can induce polymerization of pure tubulin at low magnesium concentration in the absence of glycerol [8,9]. However, nothing is known of the ability of MAPl to influence polymerization. Two recent reports have independently shown the ability of purified MAPl to hydrolize ATP [lo] and the ability of ATP to enhance both nucleation and the flux of tubulin through the microtubule [ll].Here we report the ability of low concentrations of DNA to remove MAPl selectively and completely, from solutions of microtubular protein. The MAP1-free microtubular protein shows polymerization properties which are identical to those of complete microtubular protein. In contrast, the ability of microtubular protein to polymerize is strongly decreased following the removal of MAP2 by high concentrations of DNA.Abbreviations. MAP1 and MAP2, high-molecular-weight microtubule-associated proteins 1 and 2; Pipes, 1,4-piperazineethanesulfonic acid.Also interesting is the ability of low DNA concentrations to destroy the 30-S rings [12,13] in microtubular protein without having a marked effect on polymerization. MATERIALS AND METHODS Protein PreparationMicrotubule protein was obtained from porcine brain by three cycles of temperature-dependent polymerization-depolymerization using buffer A (i.e. 0.1 M Pipes, 0.5 mM MgC12, 2 mM EGTA, pH 6.7) containing 1 mM GTP. The purification protocol was that described by Shelanski et al. [14] but 4 M glycerol was only included in the first polymerization step. The resulting microtubule protein at 15 -20 mg/ml was frozen in liquid nitrogen and stored at -70 'C.To obtain microtubular protein depleted of highmolecular-weight microtubule-associated proteins (i.e. MAPl, MAP2), samples of complete microtubule protein were incubated ...

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Fractionation of Brain Microtubule‐Associated Proteins

Cited by 310 publications

References 29 publications

The DCX Superfamily 1: Common and Divergent Roles for Members of the Mouse DCX Superfamily

The DCX Superfamily 1: Common and Divergent Roles for Members of the Mouse DCX Superfamily

Differential expression of distinct microtubule-associated proteins during brain development.

Microtubule‐Associated‐Protein MAP1 is not Implicated in the Polymerization of Microtubules

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