697
for the variations in microtubule polymerisation are unknown, nucleotides (some of which bind to the tubules), calcium, microtubuleassociated proteins, and organising centres seem to play regulatory roles. Intact sulphydryl groups are also essential and microtubule function can be hampered by oxidised glutathione,4the concentration of which may be affected by the redox state of the cell.
sponsible
Malfunctioning Microtubules THE organs of complex animals have been evolved to perform specific functions, all of which must be executed, to a greater or lesser degree, by the single cells which together constitute the tissues of higher forms of life. In the single cells many of these processes are performed by specialised intracellular structures, the organelles, which often bear a striking resemblance to the organs of complex
Nowhere is this more apparent than in the musculoskeletal system where the "muscles" of the protective phagocytes, the neutrophils and macrophages, are made up of the same microfilaments, actin and myosin, that give muscle its contractile properties. They are responsible for similar functions such as movement, and the ingestion of food and closure of the mouth of the endocytic vacuole. To be effective these muscular contractions must be supported and coordinated with other cellular functions, and this appears to be the role of other filamentous structures, the microtubules. Microtubules2.3 are straight hollow cylinders, approximately 25 nm in diameter with walls 5 nm thick. The wall of the tubule is made up of thirteen helical protofilaments lying parallel to each other and to the long axis of the tubule. The major protein component of these protofilaments is tubulin, which exists in its native state as a 115 000 dalton dimer, known as 6S tubulin; under proteindenaturing conditions it can be dissociated into two similar subunits, oc- and &bgr;-tubulin (3-4S). A state of
organisms.
dynamic equilibrium exists between the isolated protofilaments and their structured aggregated form. Although the controlling mechanisms re-
The microtubules may be attached to each other by bridges or arms. In the case of cilia and flagella these arms are called dynein arms and they seem to form transient attachments to, and thereby control the sliding movements of, adjacent microtubules over each other. The best-known function of the microtubules is in the formation of the spindle of the mitotic apparatus, which helps separate the chromosomes in the anaphase of mitosis. However, microtubules have other diverse functions including the ordered movements of intracellular organelles ; the development and maintenance of cell shape; cellular motility, through appendages such as cilia, flagella, and sperm tails, or ordered amoeboid movement; and sensory transduction.
syndrome of dysfunction of movement neutrophils has been attridystrophy" of the microfilaments,5 so is the link between distinct pathological entities and dysfunction of the microtubules of the "skeleton" of the cell being established. The Chediak-Higashi syndrome,6which is characterised by partial albinism, the presence of giant granules in granule-containing cells, and an increased susceptibility to bacterial infection, is observed in man, and also in killer whales, the beige mouse, mink, and cattle. The defective neutrophil function in this syndrome, which is attributable to a combined defect involving both poor movement of cells to the site of infection, and to delayed degranulation into the phagocytic vacuole, was observed to be very similar to the effects induced by colchicine, which is known to associate with and "poison" the microtubules by inhibiting tubulin assembly. The funcJust
as a
and phagocytosis by buted to a "muscular
tion of the microtubules in this condition
fluorescence: where the microtubules are intact the surface proteins remain ordered and their coalescence to form a "cap" is prevented. Microtubule function does seem to be defective in this condi-
4. Kuriyama, R., Sakai, H. J. Biochem. 5. Boxer, L. A., Hedley-Whyte, E. T., 1. Stossel, T. P. Fedn Proc. 1977, 36, 2181. 2. Olmsted, J. B., Borisy, G. G. A. Rev. Biochem. 1973, 3. Snyder, J.A., McIntosh, J. R. ibid. 1976, 45, 699.
1974, 76, 651. Stossel, T. P. New Engl. J. Med. 1974,
291, 1093. 42, 507.
was
investigated by direct electron microscopic observation, and by exposure of the cells to fluoresceinisothiocyanate-conjugated concanavalin A and assessment of the polarisation, or capping, of
6. 7.
Blume, R. S., Wolff, S. M. Medicine, 1972, 51, 247. Oliver, J. M. Am. J. Path. 1976, 85, 395.
698
tion,7 which explains the association of albinismdue to maldistribution of the granules in melanocytes-the "constipated" engorged giant vacuoles, and defective killing of bacteria due to "ataxic" degranulation.
insulin, 12 and thyroid hormones,13 and in the release of lipoproteins from the liver14—it would be fascinating to know whether pancreatic secretion of insulin and of the digestive enzymes is delayed or incomplete in the Chediak-Higashi syndrome,
functions such as be the reladegranulation appear regulated by tive concentrations of the cyclic nucleotides A.M.P. (inhibitory) and G.M.P. (stimulatory).8These can be manipulated by cholinergic agonists and antagonists, and by the redox state of the cell, which is dependent upon the state of reduction of such compounds as ascorbic acid and glutathione.9 It was therefore exciting, and strong supportive evidence for defective microtubular function, when mediators such as dibutyryl cyclic G.M.P. (which can enter the cell), cholinergic agonists, and ascorbate, were seen to correct the in-vitro abnormalities observed in cells from patients with the Chediak-
and whether antidiabetic drugs that stimulate the secretion of insulin influence microtubular function. Kartagener’s syndromels comprises a number of seemingly unrelated conditions-why should dextrocardia be associated with sinusitis and bronchiectasis ? The answer came with the discovery of defective dynein arms on microtubules in the tails of spermatozoa from an infertile man with immotile spermatozoa and situs inversus. 16 The microtubular structure of cilia is similar to that of the sperm tail, and the function of cilia from the respiratory tract of patients with immotile but otherwise apparently normal spermatozoa was examined and found to be defective, and to be associated with abnormal dynein arms.17 Infection of the respiratory tract in this "immotile-cilia syndrome" is due to inadequate clearance of organisms because of mucociliary dysfunction. The situs inversus that may be associated could be due to failure of the normal control that cilia exert over the positioning of organs.18 In the absence of this control, the sidedness of the organs would become a random affair and situs inversus would be expected to occur in half the patients-the observed proportion. These examples illustrate the progress in the understanding and treatment of disease that can accrue from advances in molecular and cellular biology. The test-tube may soon displace the stethoscope from the physician’s pocket.
Microtubule-mediated
to
Higashi syndrome.7,10 Malakoplakia is a rare inflammatory granuloma characterised by the accumulation of macrophages which have large intracytoplasmic inclusions containing bacteria in various stages of digestion. patients are prone to chronic bacterial infecand tion, monocytes from one of them" demonstrated defective killing of ingested bacteria in association with large intracytoplasmic granules, and poor lysosomal enzyme release after phagocytosis. These factors pointed to defective function of the microtubule system, and it was therefore very gratifying for the investigators of this case when they detected low intracellular levels of cyclic-G.M.P. which, together with all the indicators of aberrant monocyte function, returned to normal after treatment of the patient with the cholinergic agonist bethanechol. 11 This suggests that the expression of the primary pathogenic lesion in malakoplakia is similar to, but less generalised than, that of the Chediak-Higashi syndrome, and results from a reversible dysfunction of the microtubular system. Now that it has been shown that dysfunction of microtubules can result in macrophage defects via poorly coordinated and incomplete degranulation of cytoplasmic granules into the phagocytic vacuole, the examination of this system and its regulation must be extended to other cells which These
have specialised cytoplasmic granules-notably, the endocrine and exocrine glands. The microtubule system may be important in the secretion of
8. Zurier, R. B., Weissmann, G., Hoffstein, S., Kammerman, S., Hsiung Tai, H. J. clin. Invest. 1974, 53, 297. 9. Mittal, C. K., Murad, F. Proc. natn. Acad. Sci., U.S.A. 1977, 74, 4360. 10. Boxer, L. A., Watanabe, A. M., Rister, M. Besch, H. R., Allen, J., Baehner,
R. L. New Engl. J. Med. 1976, 295, 1041. 11. Abdou, N. I., NaPombejara, C., Sagawa, A., Ragland, C., Stechschulte, D. J., Nilsson, U., Gourley, W., Watanabe, I., Lindsey, N. J., Allen, M. S. ibid. 1977, 297, 1413. 12. Lacy, P. E., Howell, S. L., Young, D. A., Fink, C. J. Nature, 1968, 219, 1177.
13.
Williams, J. A., Wolff, J. Proc. natn. Acad. Sci., U.S.A. 1970, 67, 1901.
Peace at the Last? IT is more than forty years since Lord HORDEE observed that dying in pain and being killed do not exhaust the possibilities available to the dying patient. Yet these two options often seem the only two open to many patients dying of cancer. PARKES compared hospital-centred and home-centred terminal care by means of post-bereavement visits to the surviving spouse, and found that about 20% of the hospital patients died with their severe and mostly continuous pain unrelieved.2 A similar percentage experienced pain of comparable intensity
14. Stein, O., Sanger, L., Stein, Y. J. cell. Biol. 1974, 62, 90. 15. Kartagener, M. Beitr. Klin. Tuberk. 1933, 83, 489. 16. Pedersen, H., Rebbe, H. Biol. Reprod. 1975, 12, 541. 17. Eliasson, R., Mossberg, B., Camner, P., Afzelius, B. A. New
1977, 297, 1. 18. Afzelius, B. A. Science, 1976, 193, 317. 1. Horder, Lord. Speech in the House of Lords, December, 2. Parkes, C. M. JlR. Coll. gen. Practnrs, 1978, 28, 19.
Engl. J. Med.
1936.
for the variations in microtubule polymerisation are unknown, nucleotides (some of which bind to the tubules), calcium, microtubuleassociated proteins, and organising centres seem to play regulatory roles. Intact sulphydryl groups are also essential and microtubule function can be hampered by oxidised glutathione,4the concentration of which may be affected by the redox state of the cell.
sponsible
Malfunctioning Microtubules THE organs of complex animals have been evolved to perform specific functions, all of which must be executed, to a greater or lesser degree, by the single cells which together constitute the tissues of higher forms of life. In the single cells many of these processes are performed by specialised intracellular structures, the organelles, which often bear a striking resemblance to the organs of complex
Nowhere is this more apparent than in the musculoskeletal system where the "muscles" of the protective phagocytes, the neutrophils and macrophages, are made up of the same microfilaments, actin and myosin, that give muscle its contractile properties. They are responsible for similar functions such as movement, and the ingestion of food and closure of the mouth of the endocytic vacuole. To be effective these muscular contractions must be supported and coordinated with other cellular functions, and this appears to be the role of other filamentous structures, the microtubules. Microtubules2.3 are straight hollow cylinders, approximately 25 nm in diameter with walls 5 nm thick. The wall of the tubule is made up of thirteen helical protofilaments lying parallel to each other and to the long axis of the tubule. The major protein component of these protofilaments is tubulin, which exists in its native state as a 115 000 dalton dimer, known as 6S tubulin; under proteindenaturing conditions it can be dissociated into two similar subunits, oc- and &bgr;-tubulin (3-4S). A state of
organisms.
dynamic equilibrium exists between the isolated protofilaments and their structured aggregated form. Although the controlling mechanisms re-
The microtubules may be attached to each other by bridges or arms. In the case of cilia and flagella these arms are called dynein arms and they seem to form transient attachments to, and thereby control the sliding movements of, adjacent microtubules over each other. The best-known function of the microtubules is in the formation of the spindle of the mitotic apparatus, which helps separate the chromosomes in the anaphase of mitosis. However, microtubules have other diverse functions including the ordered movements of intracellular organelles ; the development and maintenance of cell shape; cellular motility, through appendages such as cilia, flagella, and sperm tails, or ordered amoeboid movement; and sensory transduction.
syndrome of dysfunction of movement neutrophils has been attridystrophy" of the microfilaments,5 so is the link between distinct pathological entities and dysfunction of the microtubules of the "skeleton" of the cell being established. The Chediak-Higashi syndrome,6which is characterised by partial albinism, the presence of giant granules in granule-containing cells, and an increased susceptibility to bacterial infection, is observed in man, and also in killer whales, the beige mouse, mink, and cattle. The defective neutrophil function in this syndrome, which is attributable to a combined defect involving both poor movement of cells to the site of infection, and to delayed degranulation into the phagocytic vacuole, was observed to be very similar to the effects induced by colchicine, which is known to associate with and "poison" the microtubules by inhibiting tubulin assembly. The funcJust
as a
and phagocytosis by buted to a "muscular
tion of the microtubules in this condition
fluorescence: where the microtubules are intact the surface proteins remain ordered and their coalescence to form a "cap" is prevented. Microtubule function does seem to be defective in this condi-
4. Kuriyama, R., Sakai, H. J. Biochem. 5. Boxer, L. A., Hedley-Whyte, E. T., 1. Stossel, T. P. Fedn Proc. 1977, 36, 2181. 2. Olmsted, J. B., Borisy, G. G. A. Rev. Biochem. 1973, 3. Snyder, J.A., McIntosh, J. R. ibid. 1976, 45, 699.
1974, 76, 651. Stossel, T. P. New Engl. J. Med. 1974,
291, 1093. 42, 507.
was
investigated by direct electron microscopic observation, and by exposure of the cells to fluoresceinisothiocyanate-conjugated concanavalin A and assessment of the polarisation, or capping, of
6. 7.
Blume, R. S., Wolff, S. M. Medicine, 1972, 51, 247. Oliver, J. M. Am. J. Path. 1976, 85, 395.
698
tion,7 which explains the association of albinismdue to maldistribution of the granules in melanocytes-the "constipated" engorged giant vacuoles, and defective killing of bacteria due to "ataxic" degranulation.
insulin, 12 and thyroid hormones,13 and in the release of lipoproteins from the liver14—it would be fascinating to know whether pancreatic secretion of insulin and of the digestive enzymes is delayed or incomplete in the Chediak-Higashi syndrome,
functions such as be the reladegranulation appear regulated by tive concentrations of the cyclic nucleotides A.M.P. (inhibitory) and G.M.P. (stimulatory).8These can be manipulated by cholinergic agonists and antagonists, and by the redox state of the cell, which is dependent upon the state of reduction of such compounds as ascorbic acid and glutathione.9 It was therefore exciting, and strong supportive evidence for defective microtubular function, when mediators such as dibutyryl cyclic G.M.P. (which can enter the cell), cholinergic agonists, and ascorbate, were seen to correct the in-vitro abnormalities observed in cells from patients with the Chediak-
and whether antidiabetic drugs that stimulate the secretion of insulin influence microtubular function. Kartagener’s syndromels comprises a number of seemingly unrelated conditions-why should dextrocardia be associated with sinusitis and bronchiectasis ? The answer came with the discovery of defective dynein arms on microtubules in the tails of spermatozoa from an infertile man with immotile spermatozoa and situs inversus. 16 The microtubular structure of cilia is similar to that of the sperm tail, and the function of cilia from the respiratory tract of patients with immotile but otherwise apparently normal spermatozoa was examined and found to be defective, and to be associated with abnormal dynein arms.17 Infection of the respiratory tract in this "immotile-cilia syndrome" is due to inadequate clearance of organisms because of mucociliary dysfunction. The situs inversus that may be associated could be due to failure of the normal control that cilia exert over the positioning of organs.18 In the absence of this control, the sidedness of the organs would become a random affair and situs inversus would be expected to occur in half the patients-the observed proportion. These examples illustrate the progress in the understanding and treatment of disease that can accrue from advances in molecular and cellular biology. The test-tube may soon displace the stethoscope from the physician’s pocket.
Microtubule-mediated
to
Higashi syndrome.7,10 Malakoplakia is a rare inflammatory granuloma characterised by the accumulation of macrophages which have large intracytoplasmic inclusions containing bacteria in various stages of digestion. patients are prone to chronic bacterial infecand tion, monocytes from one of them" demonstrated defective killing of ingested bacteria in association with large intracytoplasmic granules, and poor lysosomal enzyme release after phagocytosis. These factors pointed to defective function of the microtubule system, and it was therefore very gratifying for the investigators of this case when they detected low intracellular levels of cyclic-G.M.P. which, together with all the indicators of aberrant monocyte function, returned to normal after treatment of the patient with the cholinergic agonist bethanechol. 11 This suggests that the expression of the primary pathogenic lesion in malakoplakia is similar to, but less generalised than, that of the Chediak-Higashi syndrome, and results from a reversible dysfunction of the microtubular system. Now that it has been shown that dysfunction of microtubules can result in macrophage defects via poorly coordinated and incomplete degranulation of cytoplasmic granules into the phagocytic vacuole, the examination of this system and its regulation must be extended to other cells which These
have specialised cytoplasmic granules-notably, the endocrine and exocrine glands. The microtubule system may be important in the secretion of
8. Zurier, R. B., Weissmann, G., Hoffstein, S., Kammerman, S., Hsiung Tai, H. J. clin. Invest. 1974, 53, 297. 9. Mittal, C. K., Murad, F. Proc. natn. Acad. Sci., U.S.A. 1977, 74, 4360. 10. Boxer, L. A., Watanabe, A. M., Rister, M. Besch, H. R., Allen, J., Baehner,
R. L. New Engl. J. Med. 1976, 295, 1041. 11. Abdou, N. I., NaPombejara, C., Sagawa, A., Ragland, C., Stechschulte, D. J., Nilsson, U., Gourley, W., Watanabe, I., Lindsey, N. J., Allen, M. S. ibid. 1977, 297, 1413. 12. Lacy, P. E., Howell, S. L., Young, D. A., Fink, C. J. Nature, 1968, 219, 1177.
13.
Williams, J. A., Wolff, J. Proc. natn. Acad. Sci., U.S.A. 1970, 67, 1901.
Peace at the Last? IT is more than forty years since Lord HORDEE observed that dying in pain and being killed do not exhaust the possibilities available to the dying patient. Yet these two options often seem the only two open to many patients dying of cancer. PARKES compared hospital-centred and home-centred terminal care by means of post-bereavement visits to the surviving spouse, and found that about 20% of the hospital patients died with their severe and mostly continuous pain unrelieved.2 A similar percentage experienced pain of comparable intensity
14. Stein, O., Sanger, L., Stein, Y. J. cell. Biol. 1974, 62, 90. 15. Kartagener, M. Beitr. Klin. Tuberk. 1933, 83, 489. 16. Pedersen, H., Rebbe, H. Biol. Reprod. 1975, 12, 541. 17. Eliasson, R., Mossberg, B., Camner, P., Afzelius, B. A. New
1977, 297, 1. 18. Afzelius, B. A. Science, 1976, 193, 317. 1. Horder, Lord. Speech in the House of Lords, December, 2. Parkes, C. M. JlR. Coll. gen. Practnrs, 1978, 28, 19.
Engl. J. Med.
1936.
