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T Doll

Publications and source records attributed to T Doll.

10 recordsLinked to original sources
PubMed1999

The role of CT simulation in whole-brain irradiation.

PURPOSE: Evidence is growing that incorrect field-shaping is a major cause of treatment failure in whole-brain irradiation (WBI). To evaluate the potential benefits of CT simulation in WBI we compared field-shaping based on 3D CT simulation to conventional 2D simulation. METHODS: CT head scans were obtained from 20 patients. Conventional 2D planning was imitated by drawing the block contours on digitally reconstructed radiographs (DRR) by four radiotherapists. Critical parts of the target and the eye lenses were subsequently marked and planning was repeated using 3D information ("3D planning"). The results of both methods were compared by evaluation of the minimal distance from the field edge according to each site. RESULTS: In conventional planning using DRR, major geographic mismatches (< -3 mm) occurred in the subfrontal region and both eye lenses with 1% each location. Minor mismatches (-3 to 0 mm) predominantly occurred in the contralateral lens (21%), ipsilateral lens (10%), and subfrontal region (9%). Close margins (0-5 mm) were most frequently noted at the contralateral lens (49%), ipsilateral lens (35 %), and the subfrontal region (28%). When 3D planning was used, mismatches were not found. However, close margins were inevitable at the ipsilateral lens (5%), subfrontal region (30%), and contralateral lens (70%). CONCLUSIONS: CT simulation in WBI is significantly superior to conventional simulation with respect to complete coverage of the target volume and protection of the eye lenses. The narrow passage between the ocular lenses and lamina cribrosa represents a serious limitation. These patients are safely identified with CT simulation and can be referred for modified irradiation techniques.

Brain Neoplasms↗
PubMed1997

Isoform specificity in the relationship of actin to dendritic spines.

Dendritic spines contain high concentrations of actin, but neither the isoforms involved nor the mechanism of accumulation is known. In situ hybridization with specific probes established that beta- and gamma-cytoplasmic actins are selectively expressed at high levels by spine-bearing neurons. Transfecting cultured hippocampal neurons with epitope-tagged actin isoforms showed that cytoplasmic beta- and gamma-cytoplasmic actins are correctly targeted to spines, whereas alpha-cardiac muscle actin, which is normally absent from neurons, formed aggregates in dendrites. The transfected actin cDNAs contained only coding domains, suggesting that spine targeting involves amino acid sequences in the proteins, an interpretation supported by experiments with chimeric cDNAs in which C-terminal actin sequences were found to be determinative in spine targeting. By contrast to actin, microtubule components, including tubulin and MAP2, were restricted to the dendritic shaft domain. The close association of cytoplasmic actins with spines together with their general involvement in cell surface motility further supports the idea that actin motility-based changes in spine shape may contribute to synaptic plasticity.

Actins↗
PubMed1996

Transgenic expression of embryonic MAP2 in adult mouse brain: implications for neuronal polarization.

The major neuronal microtubule-associated protein MAP2 is selectively localized in dendrites, where its expression is under strong developmental regulation. To learn more about its potential effects on neuronal morphogenesis and its sorting within the neuronal cytoplasm, we have raised transgenic mice that express high levels of the embryonic form, MAP2c, in the adult brain. One transgenic line expressed higher levels of MAP2c than endogenous adult MAP2. This had no detectable effect on either the arrangement or morphology of neurons, suggesting that although MAP2c is necessary for neuronal morphogenesis it is not involved in its regulation. Like endogenous adult MAP2, transgenic MAP2c was present in dendrites but not axons, indicating that the signal responsible for its cytoplasmic sorting is contained within the 1.5 kb of its coding sequence. In situ hybridization with specific probes showed that transgenic MAP2c mRNA was limited to cell bodies. Thus, the dendritic localization of MAP2c protein cannot be the result of previous transport of its mRNA but must depend on a signal associated with the protein itself. Furthermore, because the amino acid sequence of MAP2c is present in all forms of MAP2, this signal is also contained within adult high-M(r) MAP2 protein. This raises the possibility that, rather than the conventional scheme of mRNA sorting preceding protein localization, the transport of adult MAP2 mRNA into dendrites could depend on it being part of a translation complex in which the targeting signal is on the nascent protein.

Animals↗
PubMed1996

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.

Animals↗
PubMed1994

Sequence analysis of MAP2 function in living cells.

Microtubule-associated protein 2 (MAP2) is an abundant neuron-specific protein that binds to microtubules through a domain near its carboxyl terminus that contains either three or four similar repeats of a 31 amino acid motif. When expressed in non-neuronal cells by transfection MAP2 stabilises microtubules and induces their rearrangement into long bundles that are capable of supporting process outgrowth. To investigate which elements in the MAP2 sequence are involved in these functions we have constructed a series of deletion mutants of the short embryonic form of MAP2, MAP2c, and transfected them into non-neuronal cells. This showed that the strength of binding to microtubules increased with the number of repeats present in the construct. However, the repeat domain itself was insufficient for microtubule binding, which required in addition contiguous sequences either amino-terminal or carboxyl-terminal to the repeats themselves. Particularly on the amino-terminal side of the repeats, where there is a proline-rich domain, step-wise increases in the length of neighbouring sequence produced a gradual increase in microtubule binding. The apparent strength of binding to microtubules produced by mutant MAP2 forms was further correlated with the degree of bundling they induced as well as with the ability of the resulting microtubules to support process outgrowth. These results indicate that the interaction of MAP2 with microtubules is mediated by the combined action of several weak binding sites, including each of the repeat motifs and elements in the sequences on either side of them, whose additive effect produces the strong binding of the native MAP2 molecule. The results further indicate that both the bundling and stiffening of microtubules by MAP2 are correlated with the strength of its binding to them and suggest that these properties are a direct result of microtubule stabilisation.

Animals↗
PubMed1993

The low molecular weight form of microtubule-associated protein 2 is transported into both axons and dendrites.

In the developing brain microtubule-associated protein MAP2 occurs as both a high molecular weight form, MAP2b, which is present only in dendrites, and a low molecular weight form, MAP2c, which is also present in axons. Because the MAP2c amino acid sequence is entirely contained within that of MAP2b it is not possible to raise a MAP2c-specific antibody, so that it has been impossible to determine whether MAP2c is present in dendrites along with MAP2b. To answer this question we have generated a MAP2c cDNA clone tagged with a 10 amino acid epitope from human c-myc. This additional sequence does not alter either the binding of MAP2c to microtubules or its effects on microtubules in non-neuronal cells. When expressed in cultured primary neurons by transfection, the myc tag allowed the distribution of MAP2c to be determined independently of endogenous MAP2 protein by immunostaining with an anti-myc antibody. This showed that MAP2c is present in all processes, indicating that it can enter all kinds of processes and is stable in their cytoplasm. The results further suggest that the selective association of high molecular weight MAP2 with dendrites depends on a mechanism that prevents either its entrance or survival in the axonal compartment.

Amino Acid Sequence↗
PubMed1993

An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

Amino Acid Sequence↗
PubMed1992

Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c).

Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.

Animals↗
PubMed1989

Embryonic MAP2 lacks the cross-linking sidearm sequences and dendritic targeting signal of adult MAP2.

The most prominent microtubule-associated protein of the neuronal cytoskeleton is MAP2. In the brain it exists as a pair of high-molecular weight proteins, MAP2a and MAP2b, and a smaller form, MAP2c, which is particularly abundant in the developing brain. High-molecular weight MAP2 is expressed in dendrites, where its messenger RNA is also located, but is not found in axons; it has been shown to be present in fine filaments that crosslink dendritic microtubules. This correlates with the primary structure of high-molecular weight MAP2, which consists of a short carboxy-terminal tubulin-binding domain and a long amino-terminal arm, which forms a filamentous sidearm on reconstituted microtubules. Here we report that the high- and low-molecular weight forms of MAP2 are generated by alternative splicing and share the entire C-terminal tubulin-binding domain as well as a short N-terminal sequence. In contrast to high molecular weight MAP2, embryonic brain MAP2c lacks 1,342 amino acids from the filamentous sidearm domain. Furthermore, the mRNA for low molecular weight MAP2c is not present in dendrites, indicating that the dendritic targeting signal is specific for the high-molecular weight form.

Amino Acid Sequence↗