The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.

The expression of peripherin, an intermediate filament protein, had been shown by biochemical methods to be localized in the neurons of the PNS. Using immunohistochemical methods, we analyzed this expression more extensively during the development of the rat and compared it with that of the low-molecular-mass neurofilament protein (NF-L), which is expressed in every neuron of the CNS and PNS. The immunoreactivity of NF-L is first apparent at the 25-somite stage (about 11 d) in the ventral horn of the spinal medulla and in the posterior part of the rhombencephalon. The immunoreactivity of peripherin appears subsequently, first colocalized with that of NF-L. Both immunoreactivities then spread out along rostral and caudal directions, but whereas the immunoreactivity of NF-L finally becomes noticeable in every part of the nervous system, that of peripherin remains localized to (1) the motoneurons of the ventral horn of the spinal medulla; (2) the autonomic ganglionic and preganglionic neurons; and (3) the sensory neurons. These results demonstrate that, in the neurons that originate from migrating neural crest cells, the immunoreactivities of peripherin and of NF-L become apparent only when they have reached their destination. The results also show that peripherin is expressed more widely than has been previously observed and that this protein occurs in neuronal populations from different lineages (neural tube, neural crest, placodes) with different functions (motoneurons, sensory and autonomic neurons). The common point of these neurons is that they all have axons lying, at least partly, at the outside of the axis constituted by the encephalon and the spinal medulla; this suggests that peripherin might play a role in the recognition of the axonal pathway through the intermediary of membrane proteins.

The multiple functions of microtubules are mediated by various structural and motor microtubule-associated proteins (MAPs). To harmonize these functions in different places of a single cell, the key problem is to regulate the interactions of these proteins with microtubules. The chemical diversity of tubulin isoforms, which constitute the microtubule wall, could represent a molecular basis for this control. Using an in vitro assay of ligand blotting, we found that the microtubule-associated protein Tau interacts differentially with the diverse posttranslationally-modified isotubulins: its binding is mainly restricted to moderately-modified alpha- and beta-tubulin isoforms. We obtained evidence that the recently-discovered polyglutamylation, which consists of the sequential, posttranslational addition of one to six glutamyl units to both alpha- and beta-tubulin subunits, regulates the binding of Tau as a function of its chain length. The relative affinity of Tau, very low for unmodified tubulin, increases progressively for isotubulins carrying from one to three glutamyl units, reaches an optimal value, and then decreases progressively when the polygutamyl chain lengthens up to six residues. Our results suggest that the unmodified C-terminus of tubulin exerts a constitutive inhibition on Tau binding, probably by locking the MAP-binding site, and that this inhibition could be first released and then restored as the polyglutamyl chain grows. As the posttranslational chain does not appear to interact directly with Tau, it is thought that the growth of this chain from one to six glutamyl units causes a progressive, conformational shift in the structure of the C-terminal domain of tubulin, thus leading to the observed modulation of affinity.

A low-molecular-weight (7000), heat-stable protein-HU-that stimulates transcription of bacteriophage X DNA by E. coli RNA polymerase was purified from E. coli extracts using affinity chromatography on DNA-cellulose. HU binds to native DNA, resulting in an apparent thickening of the DNA chains as revealed by electron microscopy. Contrary to DNA unwinding proteins, it causes no destabilization of the double helix. HU differs from previously described transcription factors (HI, D, etc.) and from the lowmolecular-weight w subunit of the RNA polymerase. By its amino-acid composition and characteristics, HU displays an interesting. resemblance to some eukaryotic histones, such as H2B and Hi. A variety of low-molecular-weight proteins from Escherichia coli that stimulate RNA synthesis in vitro have been characterized (1-8). The heat-stable protein, HI (1, 2), was shown to enhance X-lac DNA transcription by E. colt RNA polymerase (3) while causing reduction of ribosomal RNA synthesis in an E. coli DNA-dependent system (4). A heatstable protein, the D factor, was reported to increase the specificity of X-DNA transcription by the E. colt polymerase (5). Another class of small, heat-stable proteins has also been described which stimulates in vitro the replication of RNA bacteriophage (6, 7). That these small protein factors could act by locally affecting the stability of nucleic acid secondary structure, hence favoring or inhibiting the action of polymerases, has already been suggested (1, 2, 5, 8), and it has been proposed that some of these entities, such as the H1 and the D factors, could represent the prokaryotic counterpart of eukaryotic nuclear proteins (3,(5)(6)(7)(8).In. the frame of this hypothesis, we have undertaken a more systematic analysis of DNA binding proteins by means of affinity chromatography on DNA-cellulose columns. We report, here, the purification from E. coli extracts of a small, heat-stable protein-HU-that stimulates transcription of bacteriophage X-DNA and displays by its amino-acid composition and physicochemical behavior some properties characteristic of eukaryotic histones. Fig. 1 MATERIALS AND METHODS Bacterial

Interaction of rat kinesin and Drosophila nonclaret disjunctional motor domains with tubulin was studied by a blot overlay assay. Either plus-end or minus-end-directed motor domain binds at the same extent to both alpha- and beta-tubulin subunits, suggesting that kinesin binding is an intrinsic property of each tubulin subunit and that motor directionality cannot be related to a preferential interaction with a given tubulin subunit. Binding features of dimeric versus monomeric rat kinesin heads suggest that dimerization could drive conformational changes to enhance binding to tubulin. Competition experiments have indicated that kinesin interacts with tubulin at a Tau-independent binding site. Complementary experiments have shown that kinesin does not interact with the same efficiency with the different tubulin isoforms. Masking the polyglutamyl chains with a specific monoclonal antibody leads to a complete inhibition of kinesin binding. These results are consistent with a model in which polyglutamylation of tubulin regulates kinesin binding through progressive conformational changes of the whole carboxyl-terminal domain of tubulin as a function of the polyglutamyl chain length, thus modulating the affinity of tubulin for kinesin and Tau as well. These results indicate that microtubules, through tubulin polymorphism, do have the ability to control microtubule-associated protein binding.

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Previous eectrophysiological and p logical studies on central and peripheral glia revealed the presence of voltage-gated Na channels with properties that are similar but not identical to those of neuronal Na channels. Here we report the isolation and characterization of a cDNA encodilg the C-terminal portion of a putative gial Na-channel (Na-G) a subunit. The amino acid sequence deduced from this cDNA indicates that the Na-G represents a separate molecular class within the Na-channel mulgene family. By Northern blot, RNase protecio, and in situ hybridization says, we demonstrate that, in addition to brain astrogila, the Na-G mRNA is expressed in cultures of Schwann cells derived from dorsal root ganglia or from sciatic nerve. In vivo, the Na-G mRNA is detected not only in brain, dorsal root gangia, and sciatic nerve, but also in tissues outside the nervous system lung cardiac and skeletal muscle and lang. Its level varies according to the tissue and is developmentally regulated. The sequence and expression data concur in desinatng Na-G as an distinct type of Na channel, preumably with low sensitivity to tetrodotoxin.Voltage-gated Na channels are essential for voltagedependent modulation ofNa ion permeability, inherent to the generation and propagation of action potentials in nerve and muscle (1). They consist of a large (--260 kDa) transmembrane glycoprotein-the a subunit (2)-that, in brain and skeletal muscle, is associated with smaller glycosylated (-subunit polypeptides (3, 4). cDNAs encoding the a subunits of four closely related rat brain Na-channel isotypes (I, II, III, and Ila) were cloned and characterized (5-7). Two Na-channel subtypes were identified in skeletal muscle and two were identified in cardiac muscle, of which one is common to both tissues (8-11).Voltage-gated ionic channels are not restricted to excitable cells (reviewed in refs. 12 and 13) and evidence is available for their presence in both central and peripheral glia in culture (14-18) as well as in vivo (19,20). The fundamental properties ofthe glial channels are quite similar to those found in central nervous system neurons. They are, however, not identical and differ in channel kinetics (13) and sensitivity to neurotoxins (12, 21). Particularly striking is their low sensitivity to the Na-channel blocker tetrodotoxin (TTX), and to a lesser extent to saxitoxin, observed in cortical astrocytes in culture (12,15,17) and Schwann cells in vivo (19). Thus, the question that arose was whether such differences have a structural basis or are the consequence of channel environment.Probes specific for brain Na-channel isotypes were recently shown to hybridize to mRNA from cultured central neurons but not to mRNA from astroglial cells (ref. 22; unpublished data). Moreover, a "common" Na-channel probe (23) that, in brain and muscle, recognizes Na-channel mRNAs of 9 + 0.5 kilobases (kb) reveals in astroglia an mRNA species of 7.5 kb (22). In the present study, we report the partial sequence of this 7.5-kb glial mRNA, show its homology to k...

The relationship between microtubule dynamics and polyglutamylation of [3H]glutamate into a-and 3-tubulin, whereas taxol had no effect for a-tubulin and a stimulating effect for f-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of a-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of a-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (lst-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated a-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated a-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of a-and ,B-tubulin suggest that distinct enzymes are involved.