Pierre Dustin

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons - I. Action of colchicine on axonal flow and neurotubules in the paraventricular nuclei

SummaryThe axonal flow of neurosecretory elementary granules has been studied in the paraventricular neurons of the rat (PVN), with the help of three techniques: light microscopy, radioautography after labelling with 35S-L-cysteine, and electron microscopy.Colchicine treatment does not alter the uptake of 35S cysteine in the PVN but the flow of labelled neurosecretory material towards the neurohypophysis is interrupted. Interruption of the axonal flow is also evidenced by the stagnation of neurosecretory granules at the periphery of the neuronal cytoplasm and by the presence of numerous axonal swellings, heavily loaded with neurosecretory granules and often containing abnormal elongated granules, surrounded by a single membrane, oriented more or less parallely to the long axis of the axons. Other cell organelles and neurotubules are not altered. The present experiments bring further evidence of the arrest by colchicine of the axonal flow of secretory granules without apparent changes of the neurotubules.

ACTION OF COLCHICINE ON AXONAL FLOW AND PITUICYTES IN THE HYPOTHALAMOPITUITARY SYSTEM OF THE RAT

Changes in the hypothalamo-pituitary tract and the pituicytes of the rat were studied after intrathecal and intraperitoneal injections of colchicine. Radioautography with 35S-cysteine demonstrates that intrathecal colchicine prevents the migration of neurosecretory granules from the supraoptic and paraventricular nuclei to the posterior lobe of the pituitary. This results in accumulations of neurosecretory granules and in the formation in the axons of elongated structures resembling neurosecretory products, although they sometimes have a fibrillary content. Neurotubules appear to remain intact in these conditions. The stimulation of the posterior pituitary by dehydration, in particular after injection of the diuretic furosemide, leads to an increased activity of pituicytes. When colchicine is injected at the same time as furosemide, a considerable new formation of centrioles is observed in the pituicytes. These become associated with ciliary vesicles, and form numerous cilia of the 9 + 0 type. An increased number of centrioles is also seen in the endothelial cells of the posterior lobe of the pituitary. These apparently paradoxical results were briefly discussed in relation to the action of colchicine on neurotubules and axonal flow and to the limited data from the literature indicating a stimulation of cilia formation under the action of colchicine and other drugs.

Centrioles and cilia multiplication in the pituitary of the rat after furosemid and colchicine treatment

SummaryIn adult rats, dehydration produced by the injection of Furosemid (Lasix®) induces the formation of new centrioles in the endothelial cells of the posterior lobe of the hypophysis.Colchicine considerably amplifies this phenomenon and centriologenesis and cilia formation are strikingly apparent in the pituicytes. In control animals, centrioles are quite exceptional in pituicytes and endothelial cells. All steps of the assembly of centrioles from a granular matrix, and the differentiation of atypical (mainly 9+0 and 8+1) cilia are observed in pituicytes. Dehydration increases the mitotic activity of the pituicytes. The respective roles of dehydration, Furosemid and colchicine in the stimulation of centriologenesis are briefly discussed.This work was supported by a grant (1973–1974) from the Belgian National Fund for Scientific Research and by grant no. 1.120 from the Belgian National Fund for Medical Research.

Centriolo- and ciliogenesis in the rat's pituicytes under the influence of microtubule poisons in vitro

Abstract Pituicytes of young rats have been incubated in saline solutions to which were added colchicine or vincristine (10 −5 M). The formation of new centrioles leading to ciliogenesis was observed after 1 h at 37 °C. This was more active in the presence of colchicine and vincristine, confirming earlier results in vivo. Macrotubules and crystalline aggregates of tubulin were observed after vincristine.

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons of the rat

SummaryIntrathecal administration of 20 μg of vincristine sulphate in the rat induced in vivo the formation of paracrystalline inclusions mainly in axonal processes. This is associated with an impairment in the migration of neurosecretory granules as shown by their accumulation in the perikarya of the magnocellular neurons. The granules are intermixed with numerous dense bodies of various shape, sometimes with a fibrillar content, and probably of lysosomal origin. In addition to the impairment of the flow of neurosecretory granules, there is also a striking accumulation of mitochondria and synaptic vesicles, and an apparent proliferation of the smooth endoplasmic reticulum.In the posterior lobe, the axonal endings contain a large number of neurosecretory granules, intermingled with bodies of varying shapes and electron density. Occasionally, a dense membrane surrounding a group of elementary granules is observed, reacting positively for acid phosphatase. This suggests an attempted crinophagia.

Structure and Chemistry of Microtubules

MT are regular helical assemblies of protein dimers made up of two closely related subunits, tubulins α and β, of about 50,000 molecular weight, differing slightly in electrophoretic motility. While the shape and many properties of MT proceed from the assembly of these tubulins, other proteins are closely associated with them: these play a role in tubulin assembly and in many interactions of MT with other cell constituents.

General Physiology of Tubulins and Microtubules

At this point, the chemical structure and the modes of assembly in vitro of tubulins into MT and other structures have been analyzed. Before studying complex structures made of MT, like cilia, flagella, and axonemes, more must be known about the synthesis of tubulin, its regulation, the assembly of MT in the living cell and its abnormalities. The relations of MT with various cell organelles should be considered, and interesting relations with viruses and intracellular parasites will also be described.

Secretion, Exo- and Endocytosis

The first mention of the word “microtubules” was made by Slautterback [103] in a description of secretory cells. However, it was only seven years later that Lacy et al. [53] suggested that MT play a role in the secretory activity of the B-cells of the Lan-gerhans islets. They showed that several agents known to modify MT (colchicine, VLB, VCR, and deuterium oxide) inhibited the release of insulin by these cells when stimulated by glucose. Similar findings concerning the thyroid gland were made in 1970 by two groups working independently [79, 122]. As a matter of fact, interest in colchicine and MT was revived in our laboratory by the research in these two fields carried on in Brussels [69, 78]. Since then, a large number of contributions have shown relations between MT and various modes of cell secretion, and have demonstrated that the integrity of MT is necessary for the cell activities related to the release of secretory granules.

Complex Microtubule Assemblies: Axonemes, Centrioles, Basal Bodies, Cilia, and Flagella

It was shown in Chapters 2 and 3 that tubulins may assemble not only into MT but also into various polymorphic structures such as rings, sheets, macrotubules, etc… It is remarkable that on another scale, MT themselves may assemble into complex structures, performing important functions in many cells. Axonemes are the cytoskeletal axes of long cytoplasmic extensions of several types of unicellulars; centrioles and basal bodies are important for the assembly of cilia and are present at the poles of many mitoses, and cilia and flagella are ubiquitous, playing an important part in cell motility. The relations with cell movement will be summarized in Chapter 7, and several facts about cell shape, where MT assemblies build complex cytoskeletal structures, will be reviewed in Chapter 6. Here, the modes of assembly of MT will mainly be considered: it should be mentioned at first that, as in the assembly of tubulins into MT, the various MT-associated proteins, many of which have not yet been purified or identified, are fundamental links between MT when these build large structures.

Post-Script and Outlook

At the end of this survey of MT research — by necessity incomplete, as several hundred papers on MT are published each year and only a fraction of these could be quoted — the task of considering in this last chapter the problems which remain to be solved and some future fields for tubulin and MT research is by no means easy. MT are present in all eukaryotes, and since eons have been put to use by the cells in many functions — skeletal supports, guidance tracks, traction or restraining ropes, or constituents of complex organelles such as flagella, cilia and axonemes. Tubulins are highly conserved proteins, comparable in this aspect to the histones, but have evidently undergone mutations since the origin of the first “prototubulin” molecule — perhaps a billion years ago — from which the various α and β tubulins diverged.