- Email: [email protected]
Secretory lysosomes — a special mechanism of regulated secretion in haemopoietic cells
FORUM
Cells of the immune system defend the body against infection. To do this, the cells often need to respond rapidly to an external signal by secreting proteins that will destroy, or effect the destruction of, infectious agents. The regulated secretory pathway plays, therefore, a crucial role in the ability of such cells to function correctly. Cytotoxic T cells (CTLs), natural killer cells (NKs), basophils, neutrophils, eosinophils, mast cells, macrophages, platelets and osteoclasts all derive from the haemopoietic lineage. Several lines of evidence suggest that cells of this lineage use specialized sorting and secretory mechanisms to enable the lysosome to function as a regulated secretory organelle. This contrasts with most secretory cell types in which the secretory granules and lysosomes are entirely separate organelles. In these ‘conventional’ secretory cells, the lysosomes function to degrade proteins within the cell, whereas proteins destined for regulated secretion are targeted to, and stored in, the secretory granule prior to their release (Fig. lb). In these cells, the lysosome is not a secretory organelle. However, in secretory cells belonging to the immune system, just a single type of organelle contains both lysosomal hydrolases and specialized proteins destined for regulated secretion (Fig. lc). The ability of this ‘secretory lysosome’ to release its contents is crucial for the proper function of the immune cells listed in Table 1. The secretory process can either be exocytic - as in the case of CTLs, which release potent lytic proteins towards virus-infected cells - or internal, whereby the lysosome is ‘secreted’ into the phagosomes that have engulfed bacteria or parasites. How is it that lysosomes of cells derived from the haemopoietic lineage can function as secretory compartments? Clearly, the lysosomes of these cells are able to package specialized proteins for secretion and fuse with the plasma membrane, whereas, in other cell types, these are not functions required of lysosomes (Fig. I). The fact that cells that possess secretory lysosomes are all derived from a single lineage raises the possibility that cells of this lineage possessesspecialized sorting and/or secretory mechanisms that allow secretion from this organellel. What is the evidence for this? This article outlines the differences in the sorting and secretory processes of haemopoietic and conventional secretory cells, and suggests that there are special mechanisms for sorting and secretion in haemopoietic cells. Sorting
In CTLs and NKs, the secretory granules are termed lytic granules and contain lysosomal hydrolases as well as the soluble proteins perforin and granzymes, which are involved in target cell lysis after secretion (reviewed in Ref. 2). The sorting signals that direct these proteins to the lytic granules are quite different from those that direct secretory proteins to conventional secretory granules. The granzymes are sorted to the lytic granules by the same mechanisms that target lysosomal hydrolases to the lysosomes - namely, by the mannose 6-phosphate receptor (MPR)3,4.Both granzymes A and B acquire a mannose 6-phosphate on their N-linked glycans during biosynthesis, which trends in CELL BIOLOGY (Vol. 6) September 1996
Secretory lysosomes - a special mechanism L of regulated secretion in haemopoietic cells The secretorygranules of cells derived from the haemopoietic lineage are ‘secretorylysosomes’containing both lysosomal hydrolasesand secretoryproteins. Studies on cytotoxic T lymphocytes (CTLs) have elucidated several of the mechanisms that regulateprotein sorting to, and secretionfrom, this unusual secretoryorganelle. In particular, recent findings from a CTL mutant have led to the hypothesis that CTLs, and other cells of the haemopoietic lineage, use specialized sorting and secretory mechanisms in which the lysosomefimctions as a regulated secretorygranule.
can be recognized and sorted by the MPR. However, in cells from a patient with mucolipidosis (I-cell disease), in which the phosphotransferase adding the mannose 6-phosphate is defective, the majority of the granzymes are not sorted correctly and are secreted constitutively from these cells*. Nevertheless, granzymes are found in the granules, albeit at lower concentrations, indicating that an MPR-independent pathway can also target granzymes to the lytic granules. The lytic protein perforin is also sorted to the granules by an MPR-independent mechanism since this protein is not modified by addition of mannose-6-phosphate during its biogenesis. An MPR-independent pathway has also been identified in sorting lysosomal hydrolases in B lymphocyte@. Whether the MPRindependent pathways are the same or different for these various proteins is not clear. Secretion
How are cells of the haemopoietic lineage able to secrete their lysosomal compartment? Do they express a subset of proteins involved in exocytosis distinct from those expressed by other secretory cells? Some evidence for lineage-specific expression of proteins involved in secretion may be emerging. For example, human neutrophils have been found to 0 1996 Elsevier Science Ltd
PII:SO962-8924(96)20031-5
Ciilian M. Griffiths is at the MRC Laboratory for Molecular Cell Biology, University College London, Cower St, London, UK WC1 E 6BT. E-mail: gillian. [email protected]
329
FORUM
TABLE 1 - CELLS POSSESSING ‘SECRETORY’ LYSOSOMES Cell type
Functions
Secreted products (examples)
Cytotoxic T lymphocytes and natural killer cells
Target cell killing
Perforin Cranzymes
Eosinophils
Defence against parasites
Major basic protein Neurotoxin
Neutrophils
Inflammatory
response
Chemoattractants Histaminase
Basophils
Inflammatory
response
Histamine
Platelets
Inflammatory Clotting
response
Clotting factors Acid and neutral hydrolases
Mast cells
Inflammatory
response
Histamine Serotonin
Macrophages
Phagocytosis Antigen presentation
Lysosome ‘secretes’ into phagosome
Osteoclasts
Bone resorption
Forms lysosome with bone
express syntaxin 4, VAMP-2 and the secretory carrier membrane protein SCAMP, but syntaxin 1, VAMP-l, SNAP-25, synaptophysin and cellubrevin (which are expressed in neuronal cells) are not detected in these cells’. In addition, two novel, annexin-related proteins, which cross-react with antibodies to annexins II and IV and show specific, Ca2+-dependent associations with the secretory granules, have been identified in neutrophils but are absent from fibroblast9. The best evidence that the haemopoietic secretory cells share a common mechanism distinct from that of other conventional secretory cells comes from a mutant in which only these cells appear to have lost
(4
(b)
the ability to secrete. Chediak-Higashi syndrome (CHS) is the result of a naturally occurring genetic defect in humans, mice (where it is known as the beige mutation) and also other species. The phenotype is well characterized and appears identical in different speciesg. All cell types examined from CHS are characterized by the appearance of giant lysosomesrO. Systemically, CHS results in a greatly increased susceptibility to infection, arising from an impaired immune response. What is extraordinary is that most cell types are functionally unaffected by the giant lysosomes. Fibroblasts, for example, are able to endocytose markers to this compartment and degrade them efficiently I1. However, no immune response dependent upon CTL and NK cells can be elicitedrz-14. This results from their inability to secrete the large lytic granules formed in the mutant cellsi. This secretory defect seems to occur in all of the haemopoietic cells with secretory lysosomes, and, despite the fact that all cells examined in CHS show enlarged lysosomes, only cells with secretory lysosomes appear to be functionally impaired. Both phagolysosome fusion and degranulation have been shown to be defective in CHS haemopoietic cells1sJ6. The idea that CHS is characterized by a selective malfunction of haemopoietic secretory cells is supported by the recent success of bone marrow transplantation in treating CHS patients 17.The only exception to possession of the defect seems to be the osteoclast. There is no evidence to suggest that bone resorption is defective in CHS. However, the lysosomal secretion of osteoclasts is different from that of other haemopoietic cells. Rather than being secreted, the osteoclast lysosome is simply placed on the surface, where it forms a low-pH compartment with the bone being resorbedIs. This difference might account for the fact that osteoclast function is, apparently, unperturbed in CHS. The function of non-haemopoietic secretory
Secretion
FIGURE 1 Secretory cells from the haemopoietic lineage use the lysosomal compartment as a regulated secretory organelle. This involves specialized sorting and secretion mechanisms. In non-secretory cells such as fibroblasts (a), lysosomes do not secrete their contents. Soluble lysosomal proteins are sorted to this compartment by the mannose 6-phosphate receptor (MPR). Likewise, in ‘conventional’ secretory cells such as chromaffin cells (b), the lysosomes do not secrete their contents. Soluble lysosomal proteins are sorted to the lysosome by the mannose B-phosphate receptor, whereas proteins destined for secretion are sorted to a separate organelle, the regulated secretory granule, by completely different signals. An external stimulus triggers fusion of the secretory granule with the plasma membrane and secretion of the granule contents. By contrast, secretory cells of the haemopoietic lineage (e.g. cytotoxic T lymphocytes) secrete the contents of their lysosomes (c). In these cells, both soluble lysosomal proteins and secretory proteins are sorted to the same organelle, using both MPR-dependent and MPR-independent mechanisms. An external stimulus triggers secretion of the ‘lysosomal’ compartment, causing release of the contents of this ‘secretory lysosome’.
330
trends in CELL BIOLOGY (Vol. 6) September 1996
FORUM
FIGURE 2 Cytotoxic T lymphocytes (CTLs) stained with antibodies against tubulin and granzyme A to show the tubulin cytoskeleton (green) and the lytic granules (red). In wild-type CTLs (a), there are -50 granules per cell. In CTLs from patients with the Chediak-Higashi syndrome [CHS; (b)], only a single enlarged granule is observed. Bar (a), 3 urn; bar(b) 2 urn.
cells does not appear to be affected in CHS, with the possible exception of renal tubular cells19. Since these cells also secrete lysosomal enzymes, it is possible that their secretory mechanisms are more closely related to those that allow secretion of lysosomal contents rather than those of conventional secretory granules. What, then, is the defect that results in impaired secretion? The product of the defective gene would appear to be involved in a membrane fusion or fission event that occurs as part of lysosome biogenesis. During lytic-granule biogenesis in CTLs from CHS mutants, the granules are initially small and indistinguishable from the wild-type granules. However, as the cells mature, the CHS granules decrease in number from -50 granules per cell to l-3 large granules per cell - unlike normal cells in which the initial number is maintained or, perhaps, slightly increased1 (Fig. 2). For this reason, it is tempting to speculate that lytic granules form by processes that involve membrane fusion and/or fission, and that, in CHS, a protein that is vital for these events is defective. Curiously, although the formation of the giant lysosomes does not impair lysosomal function, it does prevent secretion in those haemopoietic cells that secrete their lysosomal compartment. One possible explanation for this could be that the lysosome is simply too big to be secreted.This seemsunlikely since CHS mast cells can be stimulated to secrete by using a Ca2+ionophorezO, and permeabilized CTLs can be triggered to secrete by GTPyS (B. D. Gomperts and G. M. Griffiths, unpublished). Therefore, it seems that the CHS defective protein results in a defect in a membrane fusion/fission event that occurs in all cells but is crucial for secretion of the lysosomal compartment of haemopoietic secretory cells. The exact stage at which this defect originates is still unclear and could be at any point between membrane-sorting events and fusion with the plasma membrane. What is clear is the simple fact that, in CHS, conventional secretory cells function normally, whereas the haemopoietic secretory cells are dramatically affected, and this demonstrates a real difference in the mechanism of secretion by these two cell types. trends in CELL BIOLOGY (Vol. 6) September 1996
Secretion
from
haemopoietic
cells
In conclusion, several lines of evidence support the hypothesis that the secretory cells of the haemopoietic lineage use specialized sorting and secretory mechanisms in which the lysosome acts as a regulated secretory organelle. The lysosomes of cells of the haemopoietic lineage are unusual in their ability to be secreted, and this pathway seems to be important not only in secreting proteins from these cells but also in secreting lysosomal hydrolases into phagosomes. The same pathway may also be important in regulation of the class II compartment that is involved in antigen presentation. In dendritic cells, class II is relocated from an intracellular lysosomal compartment to the cell surface after exposure to tumour necrosis factoP. In B cell lines, class II compartments have been observed fused with the plasma membrane, resulting in the release of class-II-containing vesiclesz2. The pathway involved in lysosomal secretion in haemopoietic cells may be utilized, therefore, to achieve a variety of different ends in different cells of this lineage. The mechanisms involved in lysosomal secretion seem to differ in some important aspects from those used to exocytose the secretory granules of conventional secretory cells. With the recent cloning of two different genes found to be mutated in the beige mouse (the mouse equivalent of human CHS), important clues regarding the mechanisms that regulate lysosomal secretion from haemopoietic cells should emergez3J4. References 1 BAETZ, K., ISAAZ, 5. and GRIFFITHS,C. M. (1995)). Immunol. 154, 6122-6131 2 LOWIN, B., PEITSCH,M. C. and TSCHOPP, 1. (1995) Curr. Top. Microbial. Immunol. 198, l-20 3 BURKHARDT,1. K., HESTER,5. and ARGON, Y. (1989) Proc. Not/. Acad. Sci. U. 5. A. 86, 7128-7132 4 CRIFFITHS,G. M. and ISAAZ, 5. (1993) 1, Cell Biol. 120, 885-896 5 CLICKMAN, 1. N. and KORNFELD, S. (1993) 1. Cell Biol. 123, 99-l 08 6 GLICKMAN, 1. N., MORTON, P. A., SLOT, J. W., KORNFELD, 5. and CEUZE, H. 1. (1993) /. Cell Biol. 123, 99-l 08 7 BRUMELL,1, H. et al. (1995) 1. Immunol. 155, 5750-5759
Acknowledgements I thank Lesley Page, Bastien Gomperts and Jim Kaufman for critical reading of the manuscript and acknowledge funding by a Wellcome Trust Senior Research Fellowship (040825).
331
FORUM
New Engl. 1. Med. 286,120-l 23 17 FISCHER,A. et al. (1986) lancet 8515, 1080-l 084 18 BARON, R., NEFF, L., BROWN, W., COURTOY, I’. I., LOUVARD, D. and FARQUHAR,M. G. (1988) /. Cell Bio/. 106, 1863-I 872 19 BRANDT, E. J., ELLIOT, R. W. and SWANK, R. T. (1975)j. Cell Biol. 67, 774-788 20 POON, K. C., LIU, P. I. and SPICER,S. S. (1981) Am. /. Pathol. 104,142-l 49 21 SALLUSTO,F. and LANZAVECCHIA, A. (1994) 1. Exp. Med. 179, 1109-1118 22 RAPOSA,C. et al. (1996) 1. Exp. Med. 183, 1161-I 172 23 PEROU,C. M. et al. (1996) Nut. Genet. 13, 303-308 24 BARBOSA,M. D. F. 5. et al. (1996) Nature 382,262-265
8 SJOLIN, Cj STENDAHL, 0. and DAHLCREN, C. (1994) 5iochem.I. 300,325-330 9 BLUME, R. and WOLFF, S. M. (1972) Medicine 51,247-280 10 OLIVER,C., and ESSNER,E. (1973) 1. Histochem. Cytochem. 21, 218-228 11 BURKHARDT,J. K., WIEBEL,F. A., HESTER,5. and ARGON, Y. (1993) I. Exp. Med. 178, 1845-l 856 12 BARAK,Y. and NIR, E. (1987) Am. 1. Pediatr. Hem&o/. Oncol. 9, 4245 13 BIRON, C. A., PEDERSEN,K. F. and WELSH, R. M. (1987) 1. Immunol. 138,2050-2056 14 KLEIN, M. et al. (1980) /. Exp. Med. 151, 1049-l 058 15 PADCETT, C. A. (1967) B/ood29,906-915 16 STOSSEL,T. P., ROOT, R. K. and VAUGHN, M. (1972)
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trends in CELL BIOLOGY (Vol. 6) September 1996
Cells of the immune system defend the body against infection. To do this, the cells often need to respond rapidly to an external signal by secreting proteins that will destroy, or effect the destruction of, infectious agents. The regulated secretory pathway plays, therefore, a crucial role in the ability of such cells to function correctly. Cytotoxic T cells (CTLs), natural killer cells (NKs), basophils, neutrophils, eosinophils, mast cells, macrophages, platelets and osteoclasts all derive from the haemopoietic lineage. Several lines of evidence suggest that cells of this lineage use specialized sorting and secretory mechanisms to enable the lysosome to function as a regulated secretory organelle. This contrasts with most secretory cell types in which the secretory granules and lysosomes are entirely separate organelles. In these ‘conventional’ secretory cells, the lysosomes function to degrade proteins within the cell, whereas proteins destined for regulated secretion are targeted to, and stored in, the secretory granule prior to their release (Fig. lb). In these cells, the lysosome is not a secretory organelle. However, in secretory cells belonging to the immune system, just a single type of organelle contains both lysosomal hydrolases and specialized proteins destined for regulated secretion (Fig. lc). The ability of this ‘secretory lysosome’ to release its contents is crucial for the proper function of the immune cells listed in Table 1. The secretory process can either be exocytic - as in the case of CTLs, which release potent lytic proteins towards virus-infected cells - or internal, whereby the lysosome is ‘secreted’ into the phagosomes that have engulfed bacteria or parasites. How is it that lysosomes of cells derived from the haemopoietic lineage can function as secretory compartments? Clearly, the lysosomes of these cells are able to package specialized proteins for secretion and fuse with the plasma membrane, whereas, in other cell types, these are not functions required of lysosomes (Fig. I). The fact that cells that possess secretory lysosomes are all derived from a single lineage raises the possibility that cells of this lineage possessesspecialized sorting and/or secretory mechanisms that allow secretion from this organellel. What is the evidence for this? This article outlines the differences in the sorting and secretory processes of haemopoietic and conventional secretory cells, and suggests that there are special mechanisms for sorting and secretion in haemopoietic cells. Sorting
In CTLs and NKs, the secretory granules are termed lytic granules and contain lysosomal hydrolases as well as the soluble proteins perforin and granzymes, which are involved in target cell lysis after secretion (reviewed in Ref. 2). The sorting signals that direct these proteins to the lytic granules are quite different from those that direct secretory proteins to conventional secretory granules. The granzymes are sorted to the lytic granules by the same mechanisms that target lysosomal hydrolases to the lysosomes - namely, by the mannose 6-phosphate receptor (MPR)3,4.Both granzymes A and B acquire a mannose 6-phosphate on their N-linked glycans during biosynthesis, which trends in CELL BIOLOGY (Vol. 6) September 1996
Secretory lysosomes - a special mechanism L of regulated secretion in haemopoietic cells The secretorygranules of cells derived from the haemopoietic lineage are ‘secretorylysosomes’containing both lysosomal hydrolasesand secretoryproteins. Studies on cytotoxic T lymphocytes (CTLs) have elucidated several of the mechanisms that regulateprotein sorting to, and secretionfrom, this unusual secretoryorganelle. In particular, recent findings from a CTL mutant have led to the hypothesis that CTLs, and other cells of the haemopoietic lineage, use specialized sorting and secretory mechanisms in which the lysosomefimctions as a regulated secretorygranule.
can be recognized and sorted by the MPR. However, in cells from a patient with mucolipidosis (I-cell disease), in which the phosphotransferase adding the mannose 6-phosphate is defective, the majority of the granzymes are not sorted correctly and are secreted constitutively from these cells*. Nevertheless, granzymes are found in the granules, albeit at lower concentrations, indicating that an MPR-independent pathway can also target granzymes to the lytic granules. The lytic protein perforin is also sorted to the granules by an MPR-independent mechanism since this protein is not modified by addition of mannose-6-phosphate during its biogenesis. An MPR-independent pathway has also been identified in sorting lysosomal hydrolases in B lymphocyte@. Whether the MPRindependent pathways are the same or different for these various proteins is not clear. Secretion
How are cells of the haemopoietic lineage able to secrete their lysosomal compartment? Do they express a subset of proteins involved in exocytosis distinct from those expressed by other secretory cells? Some evidence for lineage-specific expression of proteins involved in secretion may be emerging. For example, human neutrophils have been found to 0 1996 Elsevier Science Ltd
PII:SO962-8924(96)20031-5
Ciilian M. Griffiths is at the MRC Laboratory for Molecular Cell Biology, University College London, Cower St, London, UK WC1 E 6BT. E-mail: gillian. [email protected]
329
FORUM
TABLE 1 - CELLS POSSESSING ‘SECRETORY’ LYSOSOMES Cell type
Functions
Secreted products (examples)
Cytotoxic T lymphocytes and natural killer cells
Target cell killing
Perforin Cranzymes
Eosinophils
Defence against parasites
Major basic protein Neurotoxin
Neutrophils
Inflammatory
response
Chemoattractants Histaminase
Basophils
Inflammatory
response
Histamine
Platelets
Inflammatory Clotting
response
Clotting factors Acid and neutral hydrolases
Mast cells
Inflammatory
response
Histamine Serotonin
Macrophages
Phagocytosis Antigen presentation
Lysosome ‘secretes’ into phagosome
Osteoclasts
Bone resorption
Forms lysosome with bone
express syntaxin 4, VAMP-2 and the secretory carrier membrane protein SCAMP, but syntaxin 1, VAMP-l, SNAP-25, synaptophysin and cellubrevin (which are expressed in neuronal cells) are not detected in these cells’. In addition, two novel, annexin-related proteins, which cross-react with antibodies to annexins II and IV and show specific, Ca2+-dependent associations with the secretory granules, have been identified in neutrophils but are absent from fibroblast9. The best evidence that the haemopoietic secretory cells share a common mechanism distinct from that of other conventional secretory cells comes from a mutant in which only these cells appear to have lost
(4
(b)
the ability to secrete. Chediak-Higashi syndrome (CHS) is the result of a naturally occurring genetic defect in humans, mice (where it is known as the beige mutation) and also other species. The phenotype is well characterized and appears identical in different speciesg. All cell types examined from CHS are characterized by the appearance of giant lysosomesrO. Systemically, CHS results in a greatly increased susceptibility to infection, arising from an impaired immune response. What is extraordinary is that most cell types are functionally unaffected by the giant lysosomes. Fibroblasts, for example, are able to endocytose markers to this compartment and degrade them efficiently I1. However, no immune response dependent upon CTL and NK cells can be elicitedrz-14. This results from their inability to secrete the large lytic granules formed in the mutant cellsi. This secretory defect seems to occur in all of the haemopoietic cells with secretory lysosomes, and, despite the fact that all cells examined in CHS show enlarged lysosomes, only cells with secretory lysosomes appear to be functionally impaired. Both phagolysosome fusion and degranulation have been shown to be defective in CHS haemopoietic cells1sJ6. The idea that CHS is characterized by a selective malfunction of haemopoietic secretory cells is supported by the recent success of bone marrow transplantation in treating CHS patients 17.The only exception to possession of the defect seems to be the osteoclast. There is no evidence to suggest that bone resorption is defective in CHS. However, the lysosomal secretion of osteoclasts is different from that of other haemopoietic cells. Rather than being secreted, the osteoclast lysosome is simply placed on the surface, where it forms a low-pH compartment with the bone being resorbedIs. This difference might account for the fact that osteoclast function is, apparently, unperturbed in CHS. The function of non-haemopoietic secretory
Secretion
FIGURE 1 Secretory cells from the haemopoietic lineage use the lysosomal compartment as a regulated secretory organelle. This involves specialized sorting and secretion mechanisms. In non-secretory cells such as fibroblasts (a), lysosomes do not secrete their contents. Soluble lysosomal proteins are sorted to this compartment by the mannose 6-phosphate receptor (MPR). Likewise, in ‘conventional’ secretory cells such as chromaffin cells (b), the lysosomes do not secrete their contents. Soluble lysosomal proteins are sorted to the lysosome by the mannose B-phosphate receptor, whereas proteins destined for secretion are sorted to a separate organelle, the regulated secretory granule, by completely different signals. An external stimulus triggers fusion of the secretory granule with the plasma membrane and secretion of the granule contents. By contrast, secretory cells of the haemopoietic lineage (e.g. cytotoxic T lymphocytes) secrete the contents of their lysosomes (c). In these cells, both soluble lysosomal proteins and secretory proteins are sorted to the same organelle, using both MPR-dependent and MPR-independent mechanisms. An external stimulus triggers secretion of the ‘lysosomal’ compartment, causing release of the contents of this ‘secretory lysosome’.
330
trends in CELL BIOLOGY (Vol. 6) September 1996
FORUM
FIGURE 2 Cytotoxic T lymphocytes (CTLs) stained with antibodies against tubulin and granzyme A to show the tubulin cytoskeleton (green) and the lytic granules (red). In wild-type CTLs (a), there are -50 granules per cell. In CTLs from patients with the Chediak-Higashi syndrome [CHS; (b)], only a single enlarged granule is observed. Bar (a), 3 urn; bar(b) 2 urn.
cells does not appear to be affected in CHS, with the possible exception of renal tubular cells19. Since these cells also secrete lysosomal enzymes, it is possible that their secretory mechanisms are more closely related to those that allow secretion of lysosomal contents rather than those of conventional secretory granules. What, then, is the defect that results in impaired secretion? The product of the defective gene would appear to be involved in a membrane fusion or fission event that occurs as part of lysosome biogenesis. During lytic-granule biogenesis in CTLs from CHS mutants, the granules are initially small and indistinguishable from the wild-type granules. However, as the cells mature, the CHS granules decrease in number from -50 granules per cell to l-3 large granules per cell - unlike normal cells in which the initial number is maintained or, perhaps, slightly increased1 (Fig. 2). For this reason, it is tempting to speculate that lytic granules form by processes that involve membrane fusion and/or fission, and that, in CHS, a protein that is vital for these events is defective. Curiously, although the formation of the giant lysosomes does not impair lysosomal function, it does prevent secretion in those haemopoietic cells that secrete their lysosomal compartment. One possible explanation for this could be that the lysosome is simply too big to be secreted.This seemsunlikely since CHS mast cells can be stimulated to secrete by using a Ca2+ionophorezO, and permeabilized CTLs can be triggered to secrete by GTPyS (B. D. Gomperts and G. M. Griffiths, unpublished). Therefore, it seems that the CHS defective protein results in a defect in a membrane fusion/fission event that occurs in all cells but is crucial for secretion of the lysosomal compartment of haemopoietic secretory cells. The exact stage at which this defect originates is still unclear and could be at any point between membrane-sorting events and fusion with the plasma membrane. What is clear is the simple fact that, in CHS, conventional secretory cells function normally, whereas the haemopoietic secretory cells are dramatically affected, and this demonstrates a real difference in the mechanism of secretion by these two cell types. trends in CELL BIOLOGY (Vol. 6) September 1996
Secretion
from
haemopoietic
cells
In conclusion, several lines of evidence support the hypothesis that the secretory cells of the haemopoietic lineage use specialized sorting and secretory mechanisms in which the lysosome acts as a regulated secretory organelle. The lysosomes of cells of the haemopoietic lineage are unusual in their ability to be secreted, and this pathway seems to be important not only in secreting proteins from these cells but also in secreting lysosomal hydrolases into phagosomes. The same pathway may also be important in regulation of the class II compartment that is involved in antigen presentation. In dendritic cells, class II is relocated from an intracellular lysosomal compartment to the cell surface after exposure to tumour necrosis factoP. In B cell lines, class II compartments have been observed fused with the plasma membrane, resulting in the release of class-II-containing vesiclesz2. The pathway involved in lysosomal secretion in haemopoietic cells may be utilized, therefore, to achieve a variety of different ends in different cells of this lineage. The mechanisms involved in lysosomal secretion seem to differ in some important aspects from those used to exocytose the secretory granules of conventional secretory cells. With the recent cloning of two different genes found to be mutated in the beige mouse (the mouse equivalent of human CHS), important clues regarding the mechanisms that regulate lysosomal secretion from haemopoietic cells should emergez3J4. References 1 BAETZ, K., ISAAZ, 5. and GRIFFITHS,C. M. (1995)). Immunol. 154, 6122-6131 2 LOWIN, B., PEITSCH,M. C. and TSCHOPP, 1. (1995) Curr. Top. Microbial. Immunol. 198, l-20 3 BURKHARDT,1. K., HESTER,5. and ARGON, Y. (1989) Proc. Not/. Acad. Sci. U. 5. A. 86, 7128-7132 4 CRIFFITHS,G. M. and ISAAZ, 5. (1993) 1, Cell Biol. 120, 885-896 5 CLICKMAN, 1. N. and KORNFELD, S. (1993) 1. Cell Biol. 123, 99-l 08 6 GLICKMAN, 1. N., MORTON, P. A., SLOT, J. W., KORNFELD, 5. and CEUZE, H. 1. (1993) /. Cell Biol. 123, 99-l 08 7 BRUMELL,1, H. et al. (1995) 1. Immunol. 155, 5750-5759
Acknowledgements I thank Lesley Page, Bastien Gomperts and Jim Kaufman for critical reading of the manuscript and acknowledge funding by a Wellcome Trust Senior Research Fellowship (040825).
331
FORUM
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Assembly
of spindles around “11, A current
DNA-coated
beads in Xenopus egg extra,&@ ,I
topic in research on celli! division
centrosomes
and kinetochores
of chromatin
in the assembly of a mitotic
’;h
is the role of chromatin;“i;
These 111,,1’ pictures illustrate an in vitro system that has been used to study the rol@i” 382,420-425,
19961. Chromatin
ti beads and then incubating When
in mitoti?’ sp&dle
the chromatin
assembly.
sp&dle [Heald et al., Nature
is formed
plasmid DNA l’31’ r&~ in a Xenopus egg.extract.
theabe;@
by attaching
beads are placed’ in+
mitotic
cytop[asmic
:“extract, spindles assemble al’ound the beads. Figure 1 shows example$ of such spindles, in which the micrdtubules~a[g : ,.beads,,,(3’ in red. The spindles have a,characteristic
labelled in green and f&form
pol&, and the beads are aligned at the equat?: ,,,,,,spindles contain I
biG;ar
neither centrosomes
bead
the canonical
is sufficient;?
induce”*”
self-organization’ into, a<. ‘,’ p, ,,,s bipolar spindle. ‘j,,, In interphase extracts, nuclear envelopes containing pores assemQ+ Iar6und’the beads. The pores are functional, as fluorescent bovine
These images were kindly provided by RebeccaHeald, RegisTournebize, Eric Karsentiand Anthony Hyman who are in the Mitosis Group of the Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
332
growth
These chromaltin
nor kin&chores,
cues for spindle assembly. 6Gs, chramatin
local microtubule
shape with’ tw&1!
and subsequent
serum albumin (BSA) attached to a n&ear-localization 1. .A’ Imported’. into these artificial nu‘dlei in 1 a temperature-dependent manner (see F&J. 2). As in, cells, the N&f&A
sequence
is
is i.m-:
II, I@p@edinto the ‘nuclei’ at 2O”C, whereas 1(1111, . ia:;, at 4°C it binds to the surroundi~gi;~~~ I
II
,I,
FIGURE1
envelope but is not imported. Some
images
Bipolar spindles assembled are reproduced, around chromatin beads the Heald et &. incubated in a mitotic extract. Scale: bead ,+me@r is ;,I8 qn:*;,:;: tO..,,,$.bOy@. ,”
in Figures
1 and ‘2’
with permission;
‘@G-,,,:r,;
,,]X,,J froml;,,I, F’G”?E 2 ” $1
Nuture article rgferr.e$
“’ I,,!, ”
~*~~~~~~~~~~~~~~~~~~
-I sub$rate. lij 1)‘? ,I, 1”‘“..,$q$@ ,, ‘,
”
”
,‘,’
trends in CELL BIOLOGY (Vol. 6) September 1996