- Email: [email protected]
Noc-king out exocrine and endocrine secretion
Update
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Research Focus
Noc-king out exocrine and endocrine secretion Se´verine Cheviet, Laurent Waselle and Romano Regazzi Department of Cell Biology and Morphology, University of Lausanne, Switzerland
determine the mode of action of these GTPases, efforts have been made to identify their potential effectors, including Noc2, which was discovered seven years ago by Seino’s group [2]. Until recently, its role in exocytosis was unclear. Most Rab3 and Rab27 effectors possess domains that give a hint about their possible mode of action (Figure 1). Thus, Rabphilin-3A, RIM and synaptotagmin-like proteins (Slp1 to 5) are endowed with protein and phospholipid interaction modules known as C2 domains [3,4]. Others, such as melanophilin and Slac2c/MyRIP, bind to unconventional myosins and actin, pointing to functional interactions with the cytoskeleton [3,4]. By contrast, the structure of Noc2 gives no clues to its possible role because, apart from the Rab-binding motif, it is devoid of domains of predictable function. This is even reflected by the origin of its name (Noc2 stands for No C2 domain). The abundance of Noc2 in endocrine tissues suggests a possible involvement in the regulation of hormone release. Indeed, in one study, overexpression of the protein in catecholamine-secreting cells led to an increase in Ca2C-triggered exocytosis [2] but, in another, the same maneuver gave the opposite results [5], leaving the function of Noc2 unresolved. In a recent paper, Seino and collaborators have now readdressed
The Rab GTPase effector Noc2 was brought into the limelight by a recent publication that demonstrated its requirements at different stages of regulated exocytosis. Noc2 knockout resulted in distinct abnormalities in endocrine and exocrine cells, ranging from the accumulation of secretory granules of increased size to impairments in the regulated release of their secretory products. Explanations for these defects are beginning to emerge and they promise to reveal some of the most jealously kept secrets of regulated exocytosis. Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment-to-moment requirements of the organism. Decades of intense investigation have permitted some of the molecular components involved in this process to be identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells [1]. To Noc2
Rab
Rabphilin-3A
Rab
RIM2
Rab
Slp1
Rab
Slp2a
Rab
Slp3a
Rab
Slp4a/Granuphilin
Rab
Slp5
Rab
Melanophilin/Slac2a
Rab
Slac2b
Rab
MyRIP/Slac2c
Rab
(302)
Rab3, Rab8 and Rab27
C2A
C2B
(681)
C2A C2A
C2B
C2B
C2B
C2A
C2B
(950)
(673)
C2B
Rab27 Rab27
Rab3, Rab8 and Rab27
(730)
Rab27
Actin (590)
Rab27 (1982)
Myosin Va or VIIa
Rab3 Rab8 Rab27
(607)
C2B
C2A Myosin Va
(1530)
(567) C2A
C2A
Rab3, Rab8 and Rab27
Actin (856)
Rab27 Rab27
TRENDS in Cell Biology
Figure 1. Structural organization of Rab3 and Rab27 effectors. The functional domains of the partners of Rab3 and Rab27 are shown. The Rab GTPases capable of interacting with each effector are indicated on the right. The number of amino acids that compose each protein is given in brackets. Note that several Rab effectors can occur in different isoforms, but only one is depicted. Rab denotes a Rab-interacting domain; C2A and C2B denote domains permitting interaction with other proteins and/or phospholipids; myosin denotes a domain that mediates interaction with unconventional myosins; and actin denotes a domain involved in actin binding.
Corresponding author: Romano Regazzi ([email protected]). Available online 11 September 2004 www.sciencedirect.com
Update
526
TRENDS in Cell Biology
this issue by generating Noc2-knockout mice [6]. Analysis of the phenotype of these mice reveals multiple and previously unsuspected facets of Noc2 function. Thus, this Rab effector seems to be involved not only in the control of hormone exocytosis, as previously anticipated, but also in several aspects of exocrine secretion, including the determination of the size and shape of secretory granules (Table 1). The aim of this research update is to discuss the major defects displayed by Noc2-knockout mice and to highlight the implications for the molecular description of regulated exocytosis in endocrine and exocrine cells. Involvement of Noc2 in the regulation of endocrine secretion In view of the abundance of Noc2 in pancreatic b-cells, it was reasonable to predict that Noc2K/K mice might display defects in insulin exocytosis. Indeed, in agreement with recent data obtained in insulin-secreting cells in which Noc2 expression was abolished by RNA interference [7], isolated pancreatic islets from Noc2K/K mice release less insulin than normal in response to secretagogues. This phenomenon is observed regardless of whether secretion is triggered by glucose, the main physiological stimulus of insulin release, or by depolarizing the plasma membrane with high KC, a stimulus that triggers exocytosis bypassing the complex signaling events evoked by glucose. This indicates that the failure in insulin secretion is not caused by defects in second messenger generation but is a result of impairments in the secretory machinery. Insulin release is not only regulated by nutrients such as glucose but is also influenced by hormones and neurotransmitters [8]. Indeed, epinephrine causes a potent inhibition of insulin release. In pancreatic b-cells, epinephrine binds to a2-adrenoceptors that are coupled to the heterotrimeric GTP-binding proteins Gi and Go (Figure 2). Activation of the receptor leads to the dissociation of the a subunit of Gi and Go and, by a still unknown mechanism, to a direct inhibition of the secretory machinery [9]. Interestingly, Seino and collaborators found that Noc2K/K islets are more sensitive to the inhibitory action of a2-adrenoceptor agonists and that blockade of the Gi and Go pathway with pertussis toxin restores insulin Table 1. Phenotypic defects of Noc2K/K mice Cell type Pancreatic b-cells Pancreatic acini Salivary glands Gastric glands Smallintestinal glands Brunner glands Mucous cells (stomach) Goblet cells (duodenum) a
Granule morphology Unaffected
Granule accumulation No
Larger size
Yes
Stimulated exocytosisa Diminished; restored by PTX treatment Abolished
Larger size
Yes
n.d.
Larger size Larger size
Yes Yes
n.d. n.d.
Larger size
Yes
n.d.
Unaffected
No
n.d.
Unaffected
No
n.d.
Abbreviations: n.d., not determined; PTX, pertussis toxin.
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Vol.14 No.10 October 2004
Epinephrine
β
γ
α2-Adrenoceptor
Gαi/o PTX
–
Exocytosis
+
Rab3 Rab27
Noc2
TRENDS in Cell Biology
Figure 2. Receptor-mediated inhibition of exocytosis in pancreatic b-cells. Epinephrine binds to a2-adrenoceptors that are coupled to the GTP-binding proteins Gi and Go. Each of these GTP-binding proteins consists of three subunits: a, b and g. After activation of the receptor, the a subunits (ai or ao) dissociate from the receptor complex and inhibit a distal step of insulin exocytosis. The molecular targets of ai or ao are still unknown. In wild-type mice, Noc2 recruited on secretory granules by Rab GTPases counteracts the inhibitory action of ai and ao, and promotes insulin exocytosis. In Noc2K/K mice, the inhibition of secretion caused by epinephrine is exaggerated. Insulin secretion can be restored by treating the cells with pertussis toxin (PTX), which blocks Gi and Go signaling.
secretion. These in vitro findings are supported by observations in vivo. In fact, Noc2K/K mice display normal blood glucose levels; however, if they are subjected to stressful conditions that trigger adrenoceptor responses, the amount of insulin released is inappropriate and the animals become hyperglycemic. Therefore, the main function of Noc2 in pancreatic b-cells might be to counteract the inhibitory action of Gi and Go on exocytosis. This important and unexpected observation opens the way for investigations that will not only help in defining the role of Rabs and their effectors in exocytosis but might also contribute to clarifying the still-obscure aspects of adrenoceptor signaling in b-cells. Role of Noc2 in exocrine cells Compared with pancreatic islets and adrenal glands, exocrine tissues express only low levels of Noc2. However, in spite of this, Noc2K/K mice display striking defects in the morphology and function of a variety of exocrine glands. Indeed, all exocrine cells analyzed, including those in the pancreas, salivary, intestinal and gastric glands, are enlarged owing to accumulation of secretory granules. The excess of secretory granules is probably the result of defects in the release mechanism. In fact, pancreatic acini isolated from Noc2K/K mice are unable to secrete amylase in response to physiological stimuli such as cholecystokinin.
Update
TRENDS in Cell Biology
At present, it is not known whether the impairment in secretion is also related to an enhanced sensitivity to Gi and Go signaling that could also operate in the exocrine pancreas [10]. In addition to defects in exocytosis, most exocrine cells of Noc2K/K mice contain secretory granules of abnormally large size and irregular shape. This is reminiscent of studies in Rab3D-knockout mice in which an increase in granular size in the exocrine pancreas and parotid glands was reported [11]. Riedel et al. suggested that the morphological alterations observed in the absence of Rab3D could result from the budding of larger granule precursors from the trans Golgi network or from an increase in granule–granule fusion events [11]. The demonstration by Seino’s group that Noc2 is a potential partner of Rab3D [6] makes it tempting to speculate that a similar mechanism might be responsible for the enlarged size of secretory granules in Noc2K/K mice. Interestingly, secretory granule size is normal in endocrine cells, surface mucous cells of the stomach and goblet cells in the intestinal epithelium. In these cases, the mechanism determining the morphology of secretory granules might be different and might not involve Noc2. Alternatively, the cells might possess a compensatory mechanism that palliates the absence of Noc2. Why does the absence of Noc2 result in distinct cell abnormalities? Although Noc2 was initially thought to be a specific Rab3 effector [2,5], recently even stronger interactions with Rab27 and Rab8 have been reported [7,12,13]. The four isoforms of Rab3 and the two isoforms of Rab27 have specific tissue distributions [14–16]. Thus, according to the relative abundance of these proteins, the partner(s) of Noc2 might vary from cell to cell, possibly explaining the complexity of the phenotype of the knockout mice. The Seino group found that, in contrast to wild-type Noc2, a mutant lacking Rab3 binding was unable to rescue insulin exocytosis from Noc2K/K islets [6], and concluded that Rab3 binding is needed for Noc2 function. However, it is important to point out that this mutant has also been found to be unable to interact with Rab27 [7,13] and Rab8 (S. Cheviet et al., unpublished). Therefore, the data of the Seino group do not exclude a possible requirement for Rab27 and Rab8 binding for Noc2 function. Further experiments are needed to pinpoint the role of each Rab in the phenotypic traits of Noc2K/K mice. Such studies will be helped by the identification of mutations that prevent the association of Rab27 without altering Rab3 binding [7,13]. Evaluation of the capacity of these mutants to rescue the function of cells lacking Noc2 will help to distinguish the involvement of individual GTPases in granule formation and exocytosis. Other properties of Noc2 could also contribute to explaining the defects in Noc2-knockout mice. Regulated exocytosis is controlled by a set of Rabs and their respective partners. In pancreatic b-cells, only a fraction of Rab GTPases is in the active conformation and is capable of interacting with the effectors [17]. For this reason, the effectors present in the cells are probably in competition for their Rab partners. Noc2 seems to be devoid of domains susceptible to regulating secretion www.sciencedirect.com
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(a)
(b) E1
E3
E2
E1
E2
R
R
EE1 R
R
R E1
R
Granule R
Granule
Noc2 E3 R
Noc2
Noc2+/+ mice
Noc2–/– mice TRENDS in Cell Biology
Figure 3. Model indicating how Noc2 might modulate the interaction of Rab3 and Rab27 with their effectors. R denotes Rab3 or Rab27 in the active conformation; E1, E2 and E3 are potential partners of the Rab proteins that are associated with secretory granules. (a) In wild-type animals, Noc2 prevents the association of E3 and enables the recruitment of only a fraction of E1. (b) In the absence of Noc2, a larger fraction of E1 can be recruited on the granules, and E3 can interact with the Rab proteins. This would cause abnormalities in the secretory process.
directly, but by binding to different Rabs it can potentially modulate the availability of the GTPases for other interactions (Figure 3). If this is indeed the role of Noc2, its absence would alter the equilibrium between Rab proteins and their effectors. This is expected to cause distinct abnormalities in exocrine and endocrine cells possessing a specific set of Rab–effector complexes. Concluding remarks For several years, Noc2 was one of the most neglected Rab effectors. This situation has suddenly changed and recent advances have assigned a crucial role for this protein in regulated exocytosis, both in endocrine and exocrine cells. Further experiments are needed to define precisely the tasks of Noc2 in the late steps of the secretory pathway, from budding of granule precursors out of the Golgi network to their regulated fusion with the plasma membrane. These studies will help to clarify not only the function of Noc2 but also that of Rab3 and Rab27, a problem that has long puzzled cell biologists. Acknowledgements We thank Peter Clarke, Vale´rie Plaisance and Ce´cile Lebrand for critical reading of the manuscript. Our group is supported by Grant 3200B0–101746 from the Swiss National Science Foundation.
References 1 Burgoyne, R.D. and Morgan, A. (2003) Secretory granule exocytosis. Physiol. Rev. 83, 581–632 2 Kotake, K. et al. (1997) Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells. J. Biol. Chem. 272, 29407–29410 3 Fukuda, M. (2002) Slp and Slac2, novel families of Rab27 effectors that control Rab27-dependent membrane traffic. Recent Res. Dev. Neurochem. 5, 297–309 4 Izumi, T. et al. (2003) The roles of Rab27 and its effectors in the regulated secretory pathways. Cell Struct. Funct. 28, 465–474 5 Haynes, L.P. et al. (2001) A direct inhibitory role for the Rab3-specific effector, Noc2, in Ca2C-regulated exocytosis in neuroendocrine cells. J. Biol. Chem. 276, 9726–9732
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6 Matsumoto, M. et al. (2004) Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells. Proc. Natl. Acad. Sci. U. S. A. 101, 8313–8318 7 Cheviet, S. et al. (2004) The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis. Mol. Endocrinol. 18, 117–126 8 Sharp, G.W. (1996) Mechanisms of inhibition of insulin release. Am. J. Physiol. Cell Physiol. 271, C1781–C1799 9 Lang, J. et al. (1995) Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits. EMBO J. 14, 3635–3644 10 Tsuchida, T. et al. (1999) Inhibition of stimulated amylase secretion by adrenomedullin in rat pancreatic acini. Endocrinology 140, 865–870 11 Riedel, D. et al. (2002) Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules. Mol. Cell. Biol. 22, 6487–6497 12 Fukuda, M. (2003) Distinct Rab binding specificity of Rim1, Rim2, rabphilin, and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2. J. Biol. Chem. 278, 15373–15380
13 Fukuda, M. et al. (2004) Rabphilin and Noc2 are recruited to densecore vesicles through specific interaction with Rab27A in PC12 cells. J. Biol. Chem. 279, 13065–13075 14 Schluter, O.M. et al. (2002) Localization versus function of Rab3 proteins. Evidence for a common regulatory role in controlling fusion. J. Biol. Chem. 277, 40919–40929 15 Barral, D.C. et al. (2002) Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110, 247–257 16 Tolmachova, T. et al. (2004) A general role for Rab27a in secretory cells. Mol. Biol. Cell 15, 332–344 17 Coppola, T. et al. (2002) The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells. Biochem. J. 362, 273–279
0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.08.001
The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa Anthony Keeley1 and Dominique Soldati1,2 1 Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, UK, SW7 2AZ 2 Department of Genetics and Microbiology, Faculty of Medicine University of Geneva CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
The apicomplexans are obligate intracellular protozoan parasites that rely on gliding motility for their migration across biological barriers and for host-cell invasion and egress. This unusual form of substrate-dependent motility is powered by the ‘glideosome’, a macromolecular complex consisting of adhesive proteins that are released apically and translocated to the posterior pole of the parasite by the action of an actomyosin system anchored in the inner membrane complex of the parasite. Recent studies have revealed new insights into the composition and biogenesis of Toxoplasma gondii myosin-A motor complex and have identified an exciting set of small molecules that can interfere with different aspects of glideosome function. As the causative agent for the deadliest form of malaria in humans, Plasmodium falciparum is the most notorious member of the Apicomplexa. This phylum also includes opportunistic pathogens such as Toxoplasma gondii and Cryptosporidia, and animal pathogens such as Eimeria, which is responsible for coccidiosis in chickens. In the absence of pseudopodia, cilia or flagella, the invasive apicomplexan parasites use an unusual mode of substratedependent motility to gain entry into their host cells – a Corresponding author: Dominique Soldati ([email protected]). Available online 11 September 2004 www.sciencedirect.com
mechanism that is distinct from phagocytosis and that avoids lysosomal destruction. Gliding motility is also necessary for migration to the appropriate location in the host organism to initiate the next stage of life-cycle differentiation. In Plasmodium, the ookinetes cross the mosquito midgut epithelium [1], whereas the sporozoites migrate through the haemolymph to the mosquito salivary glands [2] and cross the sinusoidal cell layer to reach hepatocytes in the human liver [3]. Parasites that can disseminate to areas of immune privilege – for example, by crossing the placenta or blood brain barrier – are more life-threatening, and recent evidence suggests that the transmigration of T. gondii is linked to motility and is associated with acute virulence [4]. After entry into host cells, many apicomplexan parasites create a unique vacuole, termed the parasitophorous vacuole, by invagination of the host-cell plasma membrane and by the delivery of membranous material from the RHOPTRIES (see Glossary). The newly formed parasitophorous vacuole is devoid of host-cell integral membrane proteins. In particular, the absence of host soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins is probably responsible for the resistance of this vacuole to lysosomal fusion and acidification, which assures intracellular parasite survival. Formation of this unique compartment occurs at the
TRENDS in Cell Biology
Vol.14 No.10 October 2004
Research Focus
Noc-king out exocrine and endocrine secretion Se´verine Cheviet, Laurent Waselle and Romano Regazzi Department of Cell Biology and Morphology, University of Lausanne, Switzerland
determine the mode of action of these GTPases, efforts have been made to identify their potential effectors, including Noc2, which was discovered seven years ago by Seino’s group [2]. Until recently, its role in exocytosis was unclear. Most Rab3 and Rab27 effectors possess domains that give a hint about their possible mode of action (Figure 1). Thus, Rabphilin-3A, RIM and synaptotagmin-like proteins (Slp1 to 5) are endowed with protein and phospholipid interaction modules known as C2 domains [3,4]. Others, such as melanophilin and Slac2c/MyRIP, bind to unconventional myosins and actin, pointing to functional interactions with the cytoskeleton [3,4]. By contrast, the structure of Noc2 gives no clues to its possible role because, apart from the Rab-binding motif, it is devoid of domains of predictable function. This is even reflected by the origin of its name (Noc2 stands for No C2 domain). The abundance of Noc2 in endocrine tissues suggests a possible involvement in the regulation of hormone release. Indeed, in one study, overexpression of the protein in catecholamine-secreting cells led to an increase in Ca2C-triggered exocytosis [2] but, in another, the same maneuver gave the opposite results [5], leaving the function of Noc2 unresolved. In a recent paper, Seino and collaborators have now readdressed
The Rab GTPase effector Noc2 was brought into the limelight by a recent publication that demonstrated its requirements at different stages of regulated exocytosis. Noc2 knockout resulted in distinct abnormalities in endocrine and exocrine cells, ranging from the accumulation of secretory granules of increased size to impairments in the regulated release of their secretory products. Explanations for these defects are beginning to emerge and they promise to reveal some of the most jealously kept secrets of regulated exocytosis. Almost every cell in our organism releases proteins and other biological compounds using a fundamental cellular process known as constitutive exocytosis. In exocrine and endocrine glands, the cells are endowed with an additional and more refined release mechanism directly tuned by extracellular signals. This process, referred to as regulated exocytosis, ensures the timely delivery of molecules such as peptide hormones and digestive enzymes to match the moment-to-moment requirements of the organism. Decades of intense investigation have permitted some of the molecular components involved in this process to be identified, including Rab3 and Rab27, two GTPases that regulate the final steps of secretion in many cells [1]. To Noc2
Rab
Rabphilin-3A
Rab
RIM2
Rab
Slp1
Rab
Slp2a
Rab
Slp3a
Rab
Slp4a/Granuphilin
Rab
Slp5
Rab
Melanophilin/Slac2a
Rab
Slac2b
Rab
MyRIP/Slac2c
Rab
(302)
Rab3, Rab8 and Rab27
C2A
C2B
(681)
C2A C2A
C2B
C2B
C2B
C2A
C2B
(950)
(673)
C2B
Rab27 Rab27
Rab3, Rab8 and Rab27
(730)
Rab27
Actin (590)
Rab27 (1982)
Myosin Va or VIIa
Rab3 Rab8 Rab27
(607)
C2B
C2A Myosin Va
(1530)
(567) C2A
C2A
Rab3, Rab8 and Rab27
Actin (856)
Rab27 Rab27
TRENDS in Cell Biology
Figure 1. Structural organization of Rab3 and Rab27 effectors. The functional domains of the partners of Rab3 and Rab27 are shown. The Rab GTPases capable of interacting with each effector are indicated on the right. The number of amino acids that compose each protein is given in brackets. Note that several Rab effectors can occur in different isoforms, but only one is depicted. Rab denotes a Rab-interacting domain; C2A and C2B denote domains permitting interaction with other proteins and/or phospholipids; myosin denotes a domain that mediates interaction with unconventional myosins; and actin denotes a domain involved in actin binding.
Corresponding author: Romano Regazzi ([email protected]). Available online 11 September 2004 www.sciencedirect.com
Update
526
TRENDS in Cell Biology
this issue by generating Noc2-knockout mice [6]. Analysis of the phenotype of these mice reveals multiple and previously unsuspected facets of Noc2 function. Thus, this Rab effector seems to be involved not only in the control of hormone exocytosis, as previously anticipated, but also in several aspects of exocrine secretion, including the determination of the size and shape of secretory granules (Table 1). The aim of this research update is to discuss the major defects displayed by Noc2-knockout mice and to highlight the implications for the molecular description of regulated exocytosis in endocrine and exocrine cells. Involvement of Noc2 in the regulation of endocrine secretion In view of the abundance of Noc2 in pancreatic b-cells, it was reasonable to predict that Noc2K/K mice might display defects in insulin exocytosis. Indeed, in agreement with recent data obtained in insulin-secreting cells in which Noc2 expression was abolished by RNA interference [7], isolated pancreatic islets from Noc2K/K mice release less insulin than normal in response to secretagogues. This phenomenon is observed regardless of whether secretion is triggered by glucose, the main physiological stimulus of insulin release, or by depolarizing the plasma membrane with high KC, a stimulus that triggers exocytosis bypassing the complex signaling events evoked by glucose. This indicates that the failure in insulin secretion is not caused by defects in second messenger generation but is a result of impairments in the secretory machinery. Insulin release is not only regulated by nutrients such as glucose but is also influenced by hormones and neurotransmitters [8]. Indeed, epinephrine causes a potent inhibition of insulin release. In pancreatic b-cells, epinephrine binds to a2-adrenoceptors that are coupled to the heterotrimeric GTP-binding proteins Gi and Go (Figure 2). Activation of the receptor leads to the dissociation of the a subunit of Gi and Go and, by a still unknown mechanism, to a direct inhibition of the secretory machinery [9]. Interestingly, Seino and collaborators found that Noc2K/K islets are more sensitive to the inhibitory action of a2-adrenoceptor agonists and that blockade of the Gi and Go pathway with pertussis toxin restores insulin Table 1. Phenotypic defects of Noc2K/K mice Cell type Pancreatic b-cells Pancreatic acini Salivary glands Gastric glands Smallintestinal glands Brunner glands Mucous cells (stomach) Goblet cells (duodenum) a
Granule morphology Unaffected
Granule accumulation No
Larger size
Yes
Stimulated exocytosisa Diminished; restored by PTX treatment Abolished
Larger size
Yes
n.d.
Larger size Larger size
Yes Yes
n.d. n.d.
Larger size
Yes
n.d.
Unaffected
No
n.d.
Unaffected
No
n.d.
Abbreviations: n.d., not determined; PTX, pertussis toxin.
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Vol.14 No.10 October 2004
Epinephrine
β
γ
α2-Adrenoceptor
Gαi/o PTX
–
Exocytosis
+
Rab3 Rab27
Noc2
TRENDS in Cell Biology
Figure 2. Receptor-mediated inhibition of exocytosis in pancreatic b-cells. Epinephrine binds to a2-adrenoceptors that are coupled to the GTP-binding proteins Gi and Go. Each of these GTP-binding proteins consists of three subunits: a, b and g. After activation of the receptor, the a subunits (ai or ao) dissociate from the receptor complex and inhibit a distal step of insulin exocytosis. The molecular targets of ai or ao are still unknown. In wild-type mice, Noc2 recruited on secretory granules by Rab GTPases counteracts the inhibitory action of ai and ao, and promotes insulin exocytosis. In Noc2K/K mice, the inhibition of secretion caused by epinephrine is exaggerated. Insulin secretion can be restored by treating the cells with pertussis toxin (PTX), which blocks Gi and Go signaling.
secretion. These in vitro findings are supported by observations in vivo. In fact, Noc2K/K mice display normal blood glucose levels; however, if they are subjected to stressful conditions that trigger adrenoceptor responses, the amount of insulin released is inappropriate and the animals become hyperglycemic. Therefore, the main function of Noc2 in pancreatic b-cells might be to counteract the inhibitory action of Gi and Go on exocytosis. This important and unexpected observation opens the way for investigations that will not only help in defining the role of Rabs and their effectors in exocytosis but might also contribute to clarifying the still-obscure aspects of adrenoceptor signaling in b-cells. Role of Noc2 in exocrine cells Compared with pancreatic islets and adrenal glands, exocrine tissues express only low levels of Noc2. However, in spite of this, Noc2K/K mice display striking defects in the morphology and function of a variety of exocrine glands. Indeed, all exocrine cells analyzed, including those in the pancreas, salivary, intestinal and gastric glands, are enlarged owing to accumulation of secretory granules. The excess of secretory granules is probably the result of defects in the release mechanism. In fact, pancreatic acini isolated from Noc2K/K mice are unable to secrete amylase in response to physiological stimuli such as cholecystokinin.
Update
TRENDS in Cell Biology
At present, it is not known whether the impairment in secretion is also related to an enhanced sensitivity to Gi and Go signaling that could also operate in the exocrine pancreas [10]. In addition to defects in exocytosis, most exocrine cells of Noc2K/K mice contain secretory granules of abnormally large size and irregular shape. This is reminiscent of studies in Rab3D-knockout mice in which an increase in granular size in the exocrine pancreas and parotid glands was reported [11]. Riedel et al. suggested that the morphological alterations observed in the absence of Rab3D could result from the budding of larger granule precursors from the trans Golgi network or from an increase in granule–granule fusion events [11]. The demonstration by Seino’s group that Noc2 is a potential partner of Rab3D [6] makes it tempting to speculate that a similar mechanism might be responsible for the enlarged size of secretory granules in Noc2K/K mice. Interestingly, secretory granule size is normal in endocrine cells, surface mucous cells of the stomach and goblet cells in the intestinal epithelium. In these cases, the mechanism determining the morphology of secretory granules might be different and might not involve Noc2. Alternatively, the cells might possess a compensatory mechanism that palliates the absence of Noc2. Why does the absence of Noc2 result in distinct cell abnormalities? Although Noc2 was initially thought to be a specific Rab3 effector [2,5], recently even stronger interactions with Rab27 and Rab8 have been reported [7,12,13]. The four isoforms of Rab3 and the two isoforms of Rab27 have specific tissue distributions [14–16]. Thus, according to the relative abundance of these proteins, the partner(s) of Noc2 might vary from cell to cell, possibly explaining the complexity of the phenotype of the knockout mice. The Seino group found that, in contrast to wild-type Noc2, a mutant lacking Rab3 binding was unable to rescue insulin exocytosis from Noc2K/K islets [6], and concluded that Rab3 binding is needed for Noc2 function. However, it is important to point out that this mutant has also been found to be unable to interact with Rab27 [7,13] and Rab8 (S. Cheviet et al., unpublished). Therefore, the data of the Seino group do not exclude a possible requirement for Rab27 and Rab8 binding for Noc2 function. Further experiments are needed to pinpoint the role of each Rab in the phenotypic traits of Noc2K/K mice. Such studies will be helped by the identification of mutations that prevent the association of Rab27 without altering Rab3 binding [7,13]. Evaluation of the capacity of these mutants to rescue the function of cells lacking Noc2 will help to distinguish the involvement of individual GTPases in granule formation and exocytosis. Other properties of Noc2 could also contribute to explaining the defects in Noc2-knockout mice. Regulated exocytosis is controlled by a set of Rabs and their respective partners. In pancreatic b-cells, only a fraction of Rab GTPases is in the active conformation and is capable of interacting with the effectors [17]. For this reason, the effectors present in the cells are probably in competition for their Rab partners. Noc2 seems to be devoid of domains susceptible to regulating secretion www.sciencedirect.com
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(a)
(b) E1
E3
E2
E1
E2
R
R
EE1 R
R
R E1
R
Granule R
Granule
Noc2 E3 R
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Figure 3. Model indicating how Noc2 might modulate the interaction of Rab3 and Rab27 with their effectors. R denotes Rab3 or Rab27 in the active conformation; E1, E2 and E3 are potential partners of the Rab proteins that are associated with secretory granules. (a) In wild-type animals, Noc2 prevents the association of E3 and enables the recruitment of only a fraction of E1. (b) In the absence of Noc2, a larger fraction of E1 can be recruited on the granules, and E3 can interact with the Rab proteins. This would cause abnormalities in the secretory process.
directly, but by binding to different Rabs it can potentially modulate the availability of the GTPases for other interactions (Figure 3). If this is indeed the role of Noc2, its absence would alter the equilibrium between Rab proteins and their effectors. This is expected to cause distinct abnormalities in exocrine and endocrine cells possessing a specific set of Rab–effector complexes. Concluding remarks For several years, Noc2 was one of the most neglected Rab effectors. This situation has suddenly changed and recent advances have assigned a crucial role for this protein in regulated exocytosis, both in endocrine and exocrine cells. Further experiments are needed to define precisely the tasks of Noc2 in the late steps of the secretory pathway, from budding of granule precursors out of the Golgi network to their regulated fusion with the plasma membrane. These studies will help to clarify not only the function of Noc2 but also that of Rab3 and Rab27, a problem that has long puzzled cell biologists. Acknowledgements We thank Peter Clarke, Vale´rie Plaisance and Ce´cile Lebrand for critical reading of the manuscript. Our group is supported by Grant 3200B0–101746 from the Swiss National Science Foundation.
References 1 Burgoyne, R.D. and Morgan, A. (2003) Secretory granule exocytosis. Physiol. Rev. 83, 581–632 2 Kotake, K. et al. (1997) Noc2, a putative zinc finger protein involved in exocytosis in endocrine cells. J. Biol. Chem. 272, 29407–29410 3 Fukuda, M. (2002) Slp and Slac2, novel families of Rab27 effectors that control Rab27-dependent membrane traffic. Recent Res. Dev. Neurochem. 5, 297–309 4 Izumi, T. et al. (2003) The roles of Rab27 and its effectors in the regulated secretory pathways. Cell Struct. Funct. 28, 465–474 5 Haynes, L.P. et al. (2001) A direct inhibitory role for the Rab3-specific effector, Noc2, in Ca2C-regulated exocytosis in neuroendocrine cells. J. Biol. Chem. 276, 9726–9732
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6 Matsumoto, M. et al. (2004) Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells. Proc. Natl. Acad. Sci. U. S. A. 101, 8313–8318 7 Cheviet, S. et al. (2004) The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis. Mol. Endocrinol. 18, 117–126 8 Sharp, G.W. (1996) Mechanisms of inhibition of insulin release. Am. J. Physiol. Cell Physiol. 271, C1781–C1799 9 Lang, J. et al. (1995) Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits. EMBO J. 14, 3635–3644 10 Tsuchida, T. et al. (1999) Inhibition of stimulated amylase secretion by adrenomedullin in rat pancreatic acini. Endocrinology 140, 865–870 11 Riedel, D. et al. (2002) Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules. Mol. Cell. Biol. 22, 6487–6497 12 Fukuda, M. (2003) Distinct Rab binding specificity of Rim1, Rim2, rabphilin, and Noc2. Identification of a critical determinant of Rab3A/Rab27A recognition by Rim2. J. Biol. Chem. 278, 15373–15380
13 Fukuda, M. et al. (2004) Rabphilin and Noc2 are recruited to densecore vesicles through specific interaction with Rab27A in PC12 cells. J. Biol. Chem. 279, 13065–13075 14 Schluter, O.M. et al. (2002) Localization versus function of Rab3 proteins. Evidence for a common regulatory role in controlling fusion. J. Biol. Chem. 277, 40919–40929 15 Barral, D.C. et al. (2002) Functional redundancy of Rab27 proteins and the pathogenesis of Griscelli syndrome. J. Clin. Invest. 110, 247–257 16 Tolmachova, T. et al. (2004) A general role for Rab27a in secretory cells. Mol. Biol. Cell 15, 332–344 17 Coppola, T. et al. (2002) The death domain of Rab3 guanine nucleotide exchange protein in GDP/GTP exchange activity in living cells. Biochem. J. 362, 273–279
0962-8924/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2004.08.001
The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa Anthony Keeley1 and Dominique Soldati1,2 1 Department of Biological Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, UK, SW7 2AZ 2 Department of Genetics and Microbiology, Faculty of Medicine University of Geneva CMU, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland
The apicomplexans are obligate intracellular protozoan parasites that rely on gliding motility for their migration across biological barriers and for host-cell invasion and egress. This unusual form of substrate-dependent motility is powered by the ‘glideosome’, a macromolecular complex consisting of adhesive proteins that are released apically and translocated to the posterior pole of the parasite by the action of an actomyosin system anchored in the inner membrane complex of the parasite. Recent studies have revealed new insights into the composition and biogenesis of Toxoplasma gondii myosin-A motor complex and have identified an exciting set of small molecules that can interfere with different aspects of glideosome function. As the causative agent for the deadliest form of malaria in humans, Plasmodium falciparum is the most notorious member of the Apicomplexa. This phylum also includes opportunistic pathogens such as Toxoplasma gondii and Cryptosporidia, and animal pathogens such as Eimeria, which is responsible for coccidiosis in chickens. In the absence of pseudopodia, cilia or flagella, the invasive apicomplexan parasites use an unusual mode of substratedependent motility to gain entry into their host cells – a Corresponding author: Dominique Soldati ([email protected]). Available online 11 September 2004 www.sciencedirect.com
mechanism that is distinct from phagocytosis and that avoids lysosomal destruction. Gliding motility is also necessary for migration to the appropriate location in the host organism to initiate the next stage of life-cycle differentiation. In Plasmodium, the ookinetes cross the mosquito midgut epithelium [1], whereas the sporozoites migrate through the haemolymph to the mosquito salivary glands [2] and cross the sinusoidal cell layer to reach hepatocytes in the human liver [3]. Parasites that can disseminate to areas of immune privilege – for example, by crossing the placenta or blood brain barrier – are more life-threatening, and recent evidence suggests that the transmigration of T. gondii is linked to motility and is associated with acute virulence [4]. After entry into host cells, many apicomplexan parasites create a unique vacuole, termed the parasitophorous vacuole, by invagination of the host-cell plasma membrane and by the delivery of membranous material from the RHOPTRIES (see Glossary). The newly formed parasitophorous vacuole is devoid of host-cell integral membrane proteins. In particular, the absence of host soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins is probably responsible for the resistance of this vacuole to lysosomal fusion and acidification, which assures intracellular parasite survival. Formation of this unique compartment occurs at the