Directionality of Brain Microtubule Assembly In Vitro

Abstract

Chicks were injected intra-cerebrally with [3Hjleucine, and 'H-labeled microtubules were assembled in vitro in the brain supernatants. Pieces of these 'Hlabeled microtubules were then incubated with unlabeled brain tubulin subunits under conditions where the subunits assembled onto the labeled pieces. Electron microscopic autoradiography of the negatively-stained microtubules showed all of the radioactivity at one end of the tubules as they increased in length. This clearly' demonstrated that the microtubules of brain in this in vitro system were being assembled unidirectionally.For some time our laboratory has been interested in the directionality of assembly of microtubules during flagellar growth. In the initial studies, light microscopic autoradiography was used to show that material is added primarily to the distal end ("tip") of flagella during flagellar regeneration in Ochromonas and Chlamydomonas (1, 2). These early studies did not differentiate between the addition of material to the axonemal microtubules and the flagellar membranes, butWitman, using the Chlamydomnnas flagella-regenerating system (2) coupled with techniques of flagellar fractionation (3, 10) and electron microscopic autoradiography, recently has been able to show that the microtubules of the flagella are assembled primarily by distal addition of subunits (4). However, it was unknown whether microtubules which do not arise from a basal body, as do the flagellar microtubules, also exhibit a polarity of growth, or if they instead elongate by addition of new subunits to both ends. Now, with the development of methods for the in vitro assembly of brain microtubules (5), it has been possible to determine the directionality of brain microtubule (neurotubule) assembly in vitro. The basic design of these experiments was to obtain highly-radioactive pieces of brain microtubules for use as "seeds" or nucleation centers for the assembly of unlabeled brain tubulin subunits.

Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.

“…We refer to these bands collectively as high-molecular-weight components (HMW). Bands of high molecular weight have also been observed by others using a similar assembly procedure (11)(12)(13), an assembly procedure using glycerol (14), and in purified tubule preparations stabilized in hexylene glycol (15). Previous results showed that the HMW components copurified in constant stoichiometry with tubulin through repeated cycles of assembly-disassembly (9,10,14).…”

Identification of a protein related to tubulin in the postsynaptic density.

“…Previously described methods were used for electron microscopic examination of the PSD fraction (1). Tubulin was prepared from the appropriate animal species by reassembly of tubulin subunits (6).…”

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

“…On the basis of comigration, heat-stability and co-sedimentation with microtubules two of these polypeptides in synaptosomal cytosol (Mr 340 000 and 300 000) appear to be MAPs probably corresponding to MAP 1 and MAP 2, respectively [5]. High-Mr MAPs have been shown to be particularly susceptible to a Ca 2 +-dependent protease in whole brain cytosol [9].…”

“…A number of proteins known as microtubule-associated proteins (MAPs) copurify with tubulin in assembled microtubules. MAPs promote microtubule assembly and are believed to stabilise microtubules against disassembly [5][6][7][8]. High-Mr MAPs are particularly sensitive to a Ca 2 +-dependent protease present in brain cytosol [9,10] and Ca2+-dependent proteolysis of these MAPs in brain cytosol fractions leads to an irreversible inhibition of microtubule assembly [9].…”