Richard Cumming

Ontogeny of microtubule-associated protein 2 in rat cerebellum: Differential expression of the doublet polypeptides

We have examined the ontogeny of the microtubule-associated protein, MAP2 in rat cerebellum using biochemical and immunocytochemical techniques. In adult animals MAP2 can be resolved as a doublet of polypeptides (referred to here as MAP2a and MAP2b). In young rats only the lower molecular weight MAP2b was present in cerebellar cytosol; MAP2a appeared by 20 days postnatally. The identification of the polypeptides as MAP2a and MAP2b in heat-stable fractions of cytosol was confirmed by peptide mapping which also demonstrated that MAP2a and MAP2b shared virtually identical peptide maps. Using a rabbit anti-MAP2 antibody that was characterised by immunoblotting of cerebellar homogenates, the localisation of MAP2 was examined in 10-day-old and adult cerebella. In 10-day-old animals, MAP2 immunoreactivity was detected in the external germinal layer, granular layer and most strikingly in Purkinje cell bodies and dendrites. Parallel fibre axons were unstained. In contrast both axons and dendrites were stained by an antibody against alpha-tubulin. In adult animals MAP2 immunoreactivity was largely confined to Purkinje cell dendrites. The absence of assembled microtubules from Purkinje cell dendrites of young animals despite the presence of MAP2 and tubulin immunoreactivity may be explained by the presence of only a single MAP2 polypeptide (MAP2b) whereas the presence of the MAP2 doublet polypeptides in the adult may facilitate polymerization. Since MAP2b does not co-assemble with microtubules efficiently in vitro it may represent a form of MAP2 unable to stimulate microtubule assembly. Therefore, the possibility exists that MAP2b may play an alternative role during dendritic growth.

Evidence for the presence of high-Mr microtubule-associated proteins and their Ca2+-dependent proteolysis in synaptosomal cytosol

Calcium‐dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M r 340 000 and 300 000. These latter polypeptides comigrated with high‐M r microtubule‐associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca2+‐dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca2+.

Immunofluorescence distribution of α tubulin, β tubulin and microtubule-associated protein 2 during in vitro maturation of cerebellar granule cell neurones

In vivo developmental immunocytochemical studies have recently shown that granule cell axons (parallel fibres) undergo progressive modification of alpha tubulin as they form permanent synapses, a change that is not observed in other microtubule-containing structures (e.g. Purkinje cell dendrites). In this study we have used primary cultures of young rat cerebellum enriched in granule cells to follow maturation of their axons under defined in vitro conditions. Our results show that alpha tubulin is not modified in culture as assessed by immunofluorescence using monoclonal antibodies that recognise different forms of alpha tubulin. We have also confirmed in vivo data showing that microtubule-associated protein 2 is absent from granule cell axons at all developmental stages in vitro. The distribution of axonal cytoskeletal proteins is compared and contrasted during in vivo and in vitro maturation, and considered in relation to differences between the two systems.

Compartmentalization of neuronal cytoskeletal proteins Review

Introduction The neuronal cytoskeJeton controls a myriad of functions in the adult and developing central nervous system including cell division, growth, motility, cellular shape, axonal and dendritic transport, neurosecretion, etc (1). The assembled fibrillar components of the neuronal cytoskeleton may be divided into microtubules (approx. 25 nm diameter)~ neurofilaments (approx. 10 nm diameter), and micro- filaments (approx. 6 nm diameter). Although microfilaments and mlcrotubules

The enigma of microtubule coils in brain synaptosomes

When synaptosomes are prepared from rat brain and incubated in Krebs solution, the presynaptic bulb develops a coil of microtubules (mts). Various considerations indicate that the coil does not have a cytoskeletal supportive function. Synaptosome coil mts show certain peculiarities, e. g. they thrive during incubation in Krebs solution (dendritic mts are depolymerized in Krebs solution) and they show no protofilament molecular substructure with tannic acid. Dendritic mts show clearly a 13 protofilament substructure when processed in the same way. Synaptosomal coil mts are sensitive to micromolar calcium and are depolymerized by treatment of the synaptosomes with veratridine or A23187. Our evidence indicates that coil mts of synaptosome and synaptic vesicle clothed mts of ‘intact’ albumin-treated synapses are different morphological and functional entities. As mentioned above, the function of coil mts remains enigmatic, while the mts seen in albumin-treated synapses could well have a role in synaptic vesicle translocation.

Differential immunocytochemical localisation of α-tubulin and β-tubulin in cerebellum using monoclonal antibodies

Abstract We have utilised monoclonal antibodies against yeast tubulin and rat brain tubulin that specifically recognise α and β subunits of rat brain tubulin when characterised by immunoblotting of brain cytosol onto nitrocellulose sheets. Light microscopic immunocytochemical studies in rat cerebellum demonstrate that while dendrites and glia are labelled with the two antibodies, there is a selective deficiency of α-tubulin compared with β-tubulin immunoreactivity in parallel fibre axons and in myelinated axons in the white matter.

Immunocytochemical evidence for tubulin in the presynaptic terminal of synaptosomes

Ultrastructural studies of intact tissue rarely show presynaptic microtubules, and immunocytochemical studies on tissue sections have previously been unable to demonstrate tubulin in the nerve terminal. In contrast, a microtubular coil can be readily detected in the presynaptic nerve terminal of synaptosomes. We have developed an immunocytochemical procedure on the synaptosome preparation and demonstrated, using monoclonal antibodies, that in the presynaptic terminal alpha and beta tubulin subunits are specifically restricted to the equatorial microtubular coil.

Taxol stabilizes synaptosomal microtubules without inhibiting acetylcholine release

Synaptosomes assemble an equatorial coil of microtubules during incubation at 37 degrees C. Stimulation of synaptosomes by veratridine or A23187 for 5 min results in disassembly of the microtubules. The role of microtubule disassembly in neurotransmitter release was investigated using the microtubule-stabilizing drug taxol. Taxol stimulates microtubule assembly in synaptosomes and prevents microtubule disassembly caused by A23187. However, taxol has no effect on the release of [3H]acetylcholine triggered by veratridine or A23187. These results suggest that microtubule turnover is not necessary for neurotransmitter release.