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T Slaughter

Publications and source records attributed to T Slaughter.

9 recordsLinked to original sources
PubMed1998

Acute inactivation of tau has no effect on dynamics of microtubules in growing axons of cultured sympathetic neurons.

Tau is a developmentally regulated microtubule (MT)-associated protein in neurons that has been implicated in neuronal morphogenesis. On the basis of test tube studies, tau has been proposed to function in axon growth by stabilizing MTs and thereby promoting MT assembly. We have tested this hypothesis by examining the effects of acute inactivation of tau on axonal MTs. Tau was inactivated by microinjecting purified antibodies against recombinant tau into neurons before they extended axons. The injected antibodies quantitatively precipitated tau into aggregates in the soma. With these conditions the neurons elaborate normal-appearing axons, and MTs extend throughout the axons and into the growth cones, but the axons and their MTs are depleted of tau. The immunodepletion of tau had no detectable effect on several parameters of the dynamics of axonal MTs. Depletion of tau also was not accompanied by a reorganization of other major MT-associated proteins or actin filaments in these neurons. Thus, neurons effectively depleted of tau can extend axons that resemble those of control cells, and the axons contain normal-appearing MT arrays with normal dynamic behavior. These observations are exactly the opposite of those expected on the basis of the hypothesis that the stability of axonal MTs is a direct function of their content of tau, indicating that tau in growing axons of cultured sympathetic neurons is not specialized to promote microtubule assembly and stability.

Adrenergic Fibers↗
PubMed1997

Microtubule transport from the cell body into the axons of growing neurons.

The present studies test the hypothesis that microtubules (MTs) are transported from the cell body into the axons of growing neurons. Dissociated sympathetic neurons were cultured using conditions that allow us to control the initiation of axon outgrowth. Neurons were injected with biotin-labeled tubulin (Bt-tub) and then stimulated to extend axons. The newly formed axons were then examined using immunofluorescence procedures for MTs with or without Bt-tub. Because the Bt-tub is fully assembly competent, all MTs that assemble after injection will contain Bt-tub. However, MTs that exist in the neuron at the time of injection and persist during the subsequent incubation will not contain Bt-tub. Because the neurons were injected before extending axons, MTs without Bt-tub are initially localized to the cell body. We specifically determined whether these MTs appeared in the newly formed axon. Such a result can only be explained by the transport of these MTs from their initial location in the cell body into the axon. The newly formed axons of many neurons contained MTs both with and without Bt-tub. MTs without Bt-tub were detected all along the axon and in some neurons represented a substantial portion of the total polymer in the proximal and middle regions of the axon. These results show that MTs are transported from the cell body into growing axons and that this transport is robust, delivering MTs to all regions of the newly formed axon.

Animals↗
PubMed1996

Tau is enriched on dynamic microtubules in the distal region of growing axons.

It is widely held that tau determines the stability of microtubules in growing axons, although direct evidence supporting this hypothesis is lacking. Previous studies have shown that the microtubule polymer in the distal axon and growth cone is the most dynamic of growing axons; it turns over more rapidly and is more sensitive to microtubule depolymerizing drugs than the polymer situated proximally. We reasoned that if the stability of axonal microtubules is directly related to their content of tau, then the polymer in the distal axon should have less tau than the polymer in the proximal axon. We tested this proposition by measuring the relative tau content of microtubule along growing axons of cultured sympathetic neurons immunostained for tau and tubulin. Our results show that the tau content of microtubules varies along the axon, but in the opposite way predicted. Specifically, the relative tau content of microtubules increases progressively along the axon to reach a peak near the growth cone that is severalfold greater than that observed proximally. Thus, tau is most enriched on the most dynamic polymer of the axon. We also show that the gradient in tau content of microtubules does not generate corresponding gradients in the extent of tubulin assembly or in the sensitivity of axonal microtubules to nocodazole. On the basis of these findings, we propose that tau in growing axons has functions other than promoting microtubule assembly and stability and the key sites for these functions are the distal axon and growth cone.

Adrenergic Fibers↗
PubMed1994

Microtubule-associated protein 1b (MAP1b) is concentrated in the distal region of growing axons.

Microtubule-associated protein 1b (MAP1b) is expressed at especially high levels in neurons actively extending axons, and although it appears to be required for axon growth, the nature of its role is unknown. We reasoned that a detailed description of the localization of MAP1b in growing axons would help define how MAP1b participates in axon growth. Therefore, we have stained cultured sympathetic neurons with various antibodies against MAP1b, and then used digital image processing and analysis procedures to quantify MAP1b distribution, phosphorylation and association with microtubules (MTs) in actively elongating axons. MAP1b is present on MTs all along the axon. Quantitative analyses showed that MAP1b has a nonuniform distribution along growing axons. It is present at relatively low and constant levels along the axon shaft until approximately 130 microns from the axon tip, where the amount of MAP1b begins to increase sharply and reaches a peak close to the growth cone. The peak amount of MAP1b in the distal axon is an order of magnitude greater than the average amount in the axon shaft. The enrichment of MAP1b in the distal axon was observed for total MAP1b and assembled MAP1b, and was even more pronounced for phosphorylated MAP1b. This distribution pattern remains after correcting the relative amount of MAP1b along the axon for variations in axonal volume. Thus, the concentration of MAP1b in the distal axon exceeds by severalfold that in the rest of the axon. The amount of assembled MAP1b relative to the amount of MT polymer also varies along the axon, and is greatest distally near the growth cone. This pattern of MAP1b localization in axons focuses attention on the distal axon and growth cone as the principal sites of MAP1b function in axon growth. We discuss the possibility that MAP1b regulates MT dynamics in the distal axon so that it is properly coordinated with growth cone events involved in axon extension.

Antibodies, Monoclonal↗
PubMed1993

Composite microtubules of the axon: quantitative analysis of tyrosinated and acetylated tubulin along individual axonal microtubules.

We have shown previously, using immunoelectron microscopy, that axonal microtubules (MTs) are composite, consisting of distinct domains that differ in their content of tyrosinated alpha-tubulin (tyr-tubulin). Here, we extend these studies using a novel preparation that permits visualization of individual axonal MTs over distances of several tens of micrometers using conventional immunofluorescence procedures. Neurons are cultured on a substratum of poly-lysine and laminin and then extracted with a MT stabilizing solution containing Triton X-100 and NaCl. These extraction conditions cause a loosening of the axonal MT array so that individual MTs separate from each other for variable distances along their length. We call this phenomenon fraying. Within the axon shaft, individual MTs can often be traced for several tens of micrometers, but fraying is most extensive in the distal 100-200 microns of the axon, where individual MTs can frequently be traced for distances of 50 to 100 microns or more to their plus ends. In some cases MTs separate completely from the axon, permitting visualization of both of their ends. Double-staining of frayed preparations with various combinations of antibodies against tyr-tubulin, acetylated alpha-tubulin (Ac-tubulin) or beta-tubulin, clearly revealed the composite nature of axonal MTs. Composite MTs consisted of two distinct domains, one that was relatively rich in tyr-tubulin and poor in Ac-tubulin, and the other that was relatively poor in tyr-tubulin and rich in Ac-tubulin. The transition between these domains was relatively abrupt, with the tyr-tubulin-rich domain extending from the transition to the plus-end of the MT. Quantitative analyses of fluorescence intensity along individual MTs using digital image processing revealed that the relative amount of tyr-tubulin increased by approximately 800% across the transition, whereas the relative amount of Ac-tubulin decreased by approximately 60%. Within the tyr-tubulin-rich domains, the relative amount of tyr-tubulin was generally not constant, but increased from the transition to the plus-end of the MT in a nonlinear manner. We propose that the specific pattern of variation in the extent of post-translational modification along an individual MT represents a snapshot of that polymer's growth history.

Acetylation↗
PubMed1992

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons.

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.

Animals↗
PubMed1991

Microtubule dynamics in axons and dendrites.

We have investigated the stability, alpha-tubulin composition, and polarity orientation of microtubules (MTs) in the axons and dendrites of cultured sympathetic neurons. MT stability was evaluated in terms of sensitivity to nocodazole, a potent anti-MT drug. Nocodazole sensitivity was assayed by quantifying the loss of MT polymer as a function of time in 2 micrograms/ml of the drug. MTs in the axon and the dendrite exhibit striking similarities in their drug sensitivity. In both types of neurites, the kinetics of MT loss are biphasic, and are consistent with the existence of two types of MT polymer that depolymerize with half-times of MT polymer that depolymerize with half-times of approximately 3.5 min and approximately 130 min. We define the more rapidly depolymerizing polymer as drug-labile and the more slowly depolymerizing polymer as drug-stable. The proportion of MT polymer that is drug-stable is greater in axons (58%) than in dendrites (25%). On the basis of current understanding of the mechanism of action of nocodazole, we suggest that the drug-labile and drug-stable polymer observed in both axons and dendrites correspond to two distinct types of polymer that differ in their relative rates of turnover in vivo. In a previous study, we established that in the axon, these drug-stable and drug-labile types of MT polymer exist in the form of distinct domains on individual MTs, with the labile domain situated at the plus end of the stable domain (Baas and Black, J Cell Biol 111:495-509, 1990). Because of the great difference in drug sensitivity between the drug-labile and drug-stable MT polymer, we were able to dissect them apart by appropriate treatments with nocodazole. This permitted us to evaluate the drug-labile and drug-stable polymer in terms of polarity orientation and relative content of alpha-tubulin variants generated by posttranslational detyrosination or acetylation. In both the axon and the dendrite, the modified as well as unmodified alpha-tubulins are present in both drug-labile and drug-stable polymer, but at different levels. Specifically, the modified forms of alpha-tubulin are enriched in the drug-stable MT polymer compared to the drug-labile MT polymer. In studies on MT polarity orientation, we demonstrate that in axons, MTs are uniformly plus-end-distal, whereas in dendrites, MTs are non uniform in their polarity orientation, with roughly equal levels of the MTs having each orientation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗