Protein isoaspartyl methyltransferase protects from Bax-induced apoptosis.

Protein L-isoaspartyl methyltransferase (Pimt) is a highly conserved enzyme utilising S-adenosylmethionine (AdoMet) to methylate aspartate residues of proteins damaged by age-related isomerisation and deamidation. We have been particularly interested in this enzyme since addition of the compound CGP3466 to primary rat astroglia cell cultures resulted in an upregulation of Pimt at the mRNA level, as shown here by semi-quantitative RT-PCR. CGP3466 is a compound related to the anti-Parkinson's drug R-(-)-deprenyl, which has been shown to protect from neural apoptosis induced by trophic factor withdrawal [Tatton et al., 1994. J. Neurochem. 63, 1572]. The pro-apoptotic gene Bax is required in the cascade of events following withdrawal [Deckwerth et al., 1996. Neuron 17, 401]. We therefore investigated whether Pimt overexpression was able to affect Bax-induced apoptosis in primary mouse cortical neurons. Our results show that Pimt is indeed able to protect from Bax-induced apoptosis. Furthermore, this activity is not restricted to brain-specific cell types, since the same effect is also demonstrated in COS1 cells. In addition, mutational analysis suggests that the protective effect is dependent on the adenosine methionine-binding motif, which is well conserved in protein methyltransferases, and that a mutation destroying this motif crucially affects cytoskeletal structures of the cell.

GFP illuminates the cytoskeleton.

Until recently, cytoskeleton research has relied primarily on immunofluorescence microscopy techniques, requiring fixation and hence killing of the specimen before the analysis. The sole method for visualizing cytoskeletal dynamics in living cells has been the microinjection of purified and fluorescently labelled protein, but technical difficulties have precluded its widespread use. The recent introduction of green fluorescent protein (GFP) has enabled visualization of proteins and cytoskeletal dynamics with only minimal perturbations of the living cell and has opened new horizons for studying the cytoskeleton.

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules.

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at approximately 20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.

Cytoskeletal plasticity in cells expressing neuronal microtubule-associated proteins.

MAP2 and tau are the two most prominent neuron-specific microtubule-associated proteins. They have been implicated in the stabilization of microtubules and consequently of neurite morphology. To investigate their influence on microtubule dynamics, we have tagged both proteins with green fluorescent protein and expressed them in non-neuronal cells. Time-lapse recordings of living cells showed that MAP2 and tau did not significantly affect the rates of microtubule growth and shrinkage. Longer recordings revealed the growth and disappearance of MAP-induced microtubule bundles coinciding with changes in cell shape. This supports the idea that microtubule dynamics are influenced by the cortical cytoskeleton. The dynamics-preserving stabilization of microtubules by MAP2 and tau thus provides a molecular basis for the morphological plasticity reported to exist in established neurites.

Functional analysis of the MAP2 repeat domain.

The neuronal microtubule-associated protein MAP2 binds to microtubules via a domain near its C terminus containing a set of 3 or 4 imperfect repeats of a 31 amino acid motif. Using naturally occurring and mutated forms of the molecule containing between 1 and 4 repeats we have examined the contribution that these repeats make to MAP2 function and explored the significance of their repetition. The experiments utilised the short 3- and 4-repeat splice variants MAP2c and MAP2d that are expressed in developing neurons and in glia respectively, and mutant 1- and 2-repeat versions that were produced by using in vitro mutagenesis to remove further 31 amino acid units while leaving the rest of the molecule unaltered. The properties of these MAP2 variants were compared both with respect to their influence on microtubules in transfected non-neuronal cells and their ability to promote microtubule assembly in vitro. We found that each of the known effects of MAP2, including the bundling of microtubules and induction of process formation in living cells, are expressed by the 1-repeat form MAP2c3, which contains only the third repeat (R3). A second 1-repeat form, MAP2c4, which contains only R4, interacts more weakly with tubulin in vitro and does not bind to microtubules in transfected cells. The microtubule-related properties of MAP2 thus arise mainly from a single predominant repeat unit, R3. In vitro assembly experiments showed that the primary effect of all the repeats is to lower the critical concentration of tubulin required for microtubule assembly but that they differ greatly in potency. The results did not reveal a separate function related to the repetition of the repeat motifs, but instead suggest that its purpose is to tailor the efficiency of MAP2 to the cellular environment in which it has to function.

Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins.

We describe the construction of pBact-NGFP and pBact-CGFP, two expression vectors that incorporate green fluorescent protein (GFP) as a fluorescent tag at the N- or C terminus of the produced protein. When transfected into recipient cells, GFP-tagged proteins can be visualised in the living cells using standard fluorescence microscopy techniques. Using these expression vectors, we have produced GFP-tagged versions of the neuronal microtubule-associated proteins (MAP), MAP2c and Tau34, in a number of different cell types. Both GFP-MAP2c and GFP-Tau34 were fluorescent and retained their ability to bind to microtubules. The pBact-NGFP and pBact-CGFP expression vectors represent a fast and convenient way to produce fluorescently tagged polypeptides of selected sequences encoding whole proteins or fragments for the analysis of function and dynamic events in living cells.

Bundling of microtubules in transfected cells does not involve an autonomous dimerization site on the MAP2 molecule.

We have searched for putative dimerization sites in microtubule-associated protein 2 (MAP2) that may be involved in the bundling of microtubules. An overlapping series of fragments of the embryonic form MAP2c were created and immunologically "tagged" with an 11 amino acid sequence from human c-myc. Nonneuronal cells were transfected simultaneously with one of these myc-tagged fragments and with full-length native MAP2c. Immunolabeling with site-specific antibodies allowed the two transgene products to be located independently within the cytoplasm of a single double-transfected cell. All transfected cells contained bundled microtubules to which the full-length native MAP2 was bound. The distribution of the tagged MAP2 fragment relative to these MAP2-induced bundles was determined by the anti-myc staining. None of the fragments tested, representing all of the MAP2c sequence in overlapping pieces, were associated with MAP2-induced microtubule bundles. These results suggest that MAP2-induced bundle formation in cells does not involve an autonomous dimerization site within the MAP2 sequence.

[Glaucomatous visual fields: observation of development with the Octopus automatic perimeter (author's transl)]. / Glaukomgesichtsfelder: Verlaufsbeobachtungen mit dem automatischen Perimeter Octopus.

The analytical program Delta was used to determine long-term fluctuations and accuracy of measurement of the program 31 of the Octopus when used with glaucoma patients. Program 31 examines the 30 degree field. The test locations are arranged in a square grid with 6 degree resolution. The program Delta determines and compares 1) the disturbed area in %; 2) the total loss, the total sensitivity being around 2000 dB; 3) the total loss in dB per mean number of disturbed points. 32 eyes of 22 patients with established glaucomatous field defects were examined twice within 2 to 6 days and 2 months later, twice again within this number of days. The size of the disturbed area served for classification of our sample into 3 groups: 1st group: disturbed area 1-33%; 2nd group: disturbed area 34-66%; 3rd group: disturbed area 67-100%. Long-term fluctuations and accuracy of measurement were determined as follows: 1) Disturbed area between 0.7 +/- 8% in group 3 and 1.7 +/- 13% in group 2. 2) The total loss increases proportionately to the disturbed area and was 4.9 +/- 29.2 dB in group 1 and 31.8 +/- 82.4 dB in group 3. 3) The total loss per mean number of disturbed points was 0.5 +/- 2 dB in group 1 and 0.3 +/- 1.2 dB in group 2. This means that, if the learning effect is over, changes of more than 2 dB, especially if several adjacent points are affected, are a significant loss, the learning effect, as determined in an earlier study, may go up as high as 2dB per point.