Apoptotic Cell Recognition: Will the Real Phosphatidylserine Receptor(s) Please Stand up?

Current Biology Vol 18 No 2 R76

and not from a developmental defect in the neural circuitry. Furthermore, expressing orb2 just before training restored long-term memory whereas supplying it just after training did not. Therefore Orb2 is likely to function in mushroom body gamma neurons during or shortly after training, consistent with a role in the formation of long-term courtship memory. Because Orb2 is thought to be a component of the synaptic tag and/or regulates the expression of tag components [1], finding an acute role for orb2 in the gamma neurons of the mushroom bodies suggests that courtship (perhaps pheromone) memories are represented there. Using the same restoration of orb2 expression approach, it should be easy to localize courtship memories to a smaller subset of mushroom body gamma neurons. Furthermore, if it can be determined that Orb2 is indeed localized to the synapse, activated Orb2 may ultimately allow visualization of the memory-relevant individual synapses. Keleman et al. [1] did not find a functional distinction between the Orb2A and B isoforms. It is possible that they are functionally redundant but differences may be revealed when they are expressed at lower level. It will be important to determine whether the A and B isoforms function in the same neurons and in the same way. It is worth noting that Orb2 belongs to the CPEB2 protein subfamily and that mammalian CPEB2-4 proteins bind sequences distinct from the cytoplasmic polyadenylation element [20] and therefore likely stimulate translation in a different way to CPEB1/Orb. The obvious question that arises when finding a role for an RNA-binding protein (or transcription factor) in memory is the identity of the regulated transcripts. This is especially exciting for Orb2 because these mRNAs could reveal the necessary synaptic components of long-term memory and perhaps the physical nature of a synaptic tag. The results of Keleman et al. [1] are consistent with Orb2 itself being a component of the tag and provocatively, Orb2 variants that lack the prion-like amino terminus retain function sufficient for the flies to develop, but they cannot form, or sustain, long-term memory. It will therefore be important to determine whether the Orb2DQ protein localizes appropriately in neurons and whether

the Orb2 amino terminus mediates binding to other proteins and/or whether prion-like Orb2 selfaggregation is the key to long-lasting memory [6,7]. References 1. Keleman, K., Kruttner, S., Alenius, M., and Dickson, B.J. (2007). Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat. Neurosci. 10, 1587–1593. 2. Wu, L., Wells, D., Tay, J., Mendis, D., Abbott, M.A., Barnitt, A., Quinlan, E., Heynen, A., Fallon, J.R., and Richter, J.D. (1998). CPEBmediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses. Neuron 21, 1129–1139. 3. Frey, U., and Morris, R.G. (1997). Synaptic tagging and long-term potentiation. Nature 385, 533–536. 4. Martin, K.C., Casadio, A., Zhu, H., Yaping, E., Rose, J.C., Chen, M., Bailey, C.H., and Kandel, E.R. (1997). Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 91, 927–938. 5. Casadio, A., Martin, K.C., Giustetto, M., Zhu, H., Chen, M., Bartsch, D., Bailey, C.H., and Kandel, E.R. (1999). A transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis. Cell 99, 221–237. 6. Si, K., Giustetto, M., Etkin, A., Hsu, R., Janisiewicz, A.M., Miniaci, M.C., Kim, J.H., Zhu, H., and Kandel, E.R. (2003). A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in Aplysia. Cell 115, 893–904. 7. Si, K., Lindquist, S., and Kandel, E.R. (2003). A neuronal isoform of the Aplysia CPEB has prion-like properties. Cell 115, 879–891. 8. Lantz, V., Chang, J.S., Horabin, J.I., Bopp, D., and Schedl, P. (1994). The Drosophila orb RNA-binding protein is required for the formation of the egg chamber and establishment of polarity. Genes Dev. 8, 598–613. 9. Chang, J.S., Tan, L., and Schedl, P. (1999). The Drosophila CPEB homolog, orb, is required for oskar protein expression in oocytes. Dev. Biol. 215, 91–106. 10. Chang, J.S., Tan, L., Wolf, M.R., and Schedl, P. (2001). Functioning of the Drosophila orb gene in gurken mRNA localization and translation. Development 128, 3169–3177.

11. Ejima, A., Smith, B.P., Lucas, C., Levine, J.D., and Griffith, L.C. (2005). Sequential learning of pheromonal cues modulates memory consolidation in trainer-specific associative courtship conditioning. Curr. Biol. 15, 194–206. 12. Ejima, A., Smith, B.P., Lucas, C., van der Goes van Naters, W., Miller, C.J., Carlson, J.R., Levine, J.D., and Griffith, L.C. (2007). Generalization of courtship learning in Drosophila is mediated by cis-vaccenyl acetate. Curr. Biol. 17, 599–605. 13. Kurtovic, A., Widmer, A., and Dickson, B.J. (2007). A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. Nature 446, 542–546. 14. Bray, S., and Amrein, H. (2003). A putative Drosophila pheromone receptor expressed in male-specific taste neurons is required for efficient courtship. Neuron 39, 1019–1029. 15. Siegel, R.W., and Hall, J.C. (1979). Conditioned responses in courtship behavior of normal and mutant Drosophila. Proc. Natl. Acad. Sci. USA 76, 3430–3434. 16. McBride, S.M., Giuliani, G., Choi, C., Krause, P., Correale, D., Watson, K., Baker, G., and Siwicki, K.K. (1999). Mushroom body ablation impairs short-term memory and long-term memory of courtship conditioning in Drosophila melanogaster. Neuron 24, 967–977. 17. Krashes, M.J., Keene, A.C., Leung, B., Armstrong, J.D., and Waddell, S. (2007). Sequential use of mushroom body neuron subsets during Drosophila odor memory processing. Neuron 53, 103–115. 18. Joiner, M.A., and Griffith, L.C. (1999). Mapping of the anatomical circuit of CaM kinase-dependent courtship conditioning in Drosophila. Learn. Mem. 6, 177–192. 19. McGuire, S.E., Le, P.T., Osborn, A.J., Matsumoto, K., and Davis, R.L. (2003). Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302, 1765–1768. 20. Huang, Y.S., Kan, M.C., Lin, C.L., and Richter, J.D. (2006). CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J. 25, 4865–4876.

Department of Neurobiology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA. E-mail: [email protected] DOI: 10.1016/j.cub.2007.11.053

Apoptotic Cell Recognition: Will the Real Phosphatidylserine Receptor(s) Please Stand up? The recognition of phosphatidylserine (PS) on apoptotic cells within tissues drives both their engulfment and an accompanying anti-inflammatory and tissue restorative program. Insight into the recognition of this phospholipid signal by phagocytes is provided by papers describing three new, but completely different, PS receptors. Donna L. Bratton and Peter M. Henson The recognition, engulfment and recycling of apoptotic cells is of

fundamental importance in development, remodeling, tissue homeostasis, the immune system and resolution of inflammation. Apoptotic cells are recognized and phagocytosed

Dispatch R77

by so-called ‘professional’ phagocytes, such as macrophages and dendritic cells, as well as by a wide variety of other resident cell types. A number of ligands have been reported to be displayed by apoptotic cells but the appearance of phosphatidylserine (PS) in the plasma membrane outer leaflet [1] is an almost universal recognition ligand. PS on apoptotic cells is recognized in a stereospecific manner (L-, but not D-phosphoserine) and stimulates the phagocyte both to engulf the apoptotic cell and to produce anti-inflammatory mediators, especially TGFb, which in turn inhibit production of pro-inflammatory chemokines and cytokines. Furthermore, oxidation of the fatty acyl groups appears to enhance PS recognition, at least by scavenger family receptors [2]. How PS arrives at the plasma membrane outer leaflet during apoptosis is still a mystery. Various phospholipid ‘flip-floppases’ (flip for inward, and flop for outward transbilayer movement) or ‘floppases’ have been identified, but none are universally expressed or definitively implicated in apoptosis [3]. During cell activation, enhanced transbilayer flip-flop of PS is thought to lead to PS exposure, which is then limited, temporally or spatially, by an active aminophospholipid translocase (APLT) that returns PS to the inner leaflet in living cells [4]. In aging erythrocytes, PS exposure accompanies the decline in APLT function [5]. PS exposure in apoptosis is associated with dramatic loss of APLT activity [6], but also with the possible mixing of the plasma membrane with internal cell membranes [7]. Whether designated proteins or altered lipids or membrane composition [8] lead to PS exposure in apoptosis is an open question. Recognition of the apoptosing cell by the phagocyte probably involves ‘tethering’ ligands and receptors as well as ‘tickling’ signals, including those derived from exposed PS [9]. This paradigm appears conserved throughout the metazoa. A PS receptor (PSR), along with a monoclonal antibody (mAb217) that was thought to recognize it, were originally described by Fadok et al. [10]. However, subsequent studies in a number of laboratories have demonstrated that this protein is primarily found in the nucleus and probably does not serve as a surface

receptor at all. It contains a Jumonji domain (which may suggest a demethylase function) and is required for early development. Its possible participation in other elements of apoptotic cell clearance is currently unclear. However, engagement of phagocytes with mAb217 does appear to mimic recognition of PS-exposing apoptotic cells, though its ligand remains at large. Of note, while this PSR fell by the wayside, a number of PSrecognizing bridge molecules were described (e.g. Gas6 and protein S, MFG-E8 and Del-1; Figure 1), which, acting through their respective receptors (Mer tyrosine kinase and av integrins), provided a potential explanation for PS recognition [11,12]. However, simultaneous efforts by three separate laboratories, using very different strategies have now identified three new PSRs, suggesting that the paradigm of PS-recognizing bridge molecules is not the final word. Using an expression cloning approach Miyanishi et al. [13] have identified Tim4 (T-cell immunoglobulinand mucin-domain-containing molecule) as a PSR. Screening of hybridomas for antibodies that inhibit apoptotic cell phagocytosis led to the identification of the Kat5-18 antibody. A retrovirus-mediated expression cloning system and a cDNA library from mouse peritoneal macrophages was used to identify the antigen as Tim4, a protein detected in macrophages from many sites. Heterologous expression of Tim4 in fibroblasts induced 60% of the cells to phagocytose apoptotic cells. Furthermore, Kat5-18 blocked uptake of apoptotic cells in the thymus and led to the production of autoantibodies against double-stranded DNA and cardiolipin often associated with defective apoptotic cell uptake. Using lipid overlays, the immunoglobulin variable (IgV)-like domain of Tim4 (and the related family member Tim1) was shown to bind specifically to PS with a Kd of about 2 nM. In a larger context, Tim4 and Tim1 belong to the TIM family proteins, known for homotypic and heterotypic binding to each other as well as to a diverse array of ligands [14]. Miyanishi et al. [13] went on to show that membrane expression of either Tim4 or Tim1 results in the binding of PS-exposing exosomes to the cells. These findings suggest that

PS exposure detected during non-apoptotic cell stimulation might in some cases result from the binding by Tim4 or Tim1 to such exosomes, structures known to convey a variety of signals between cells. Both Tim1 and Tim4 are associated with T-cell activation and modulation of cytokine production from the T helper cell subsets Th1 and Th2. Furthermore, Tim1 has been identified as a susceptibility locus for the development of atopy and asthma [15]. As such, genetic polymorphisms of Tim1 and Tim4 and their interactions with PS-exposing apoptotic cells may shape the inflammatory milieu, contributing either to resolution of inflammation or perhaps toward atopy, asthma or autoimmunity. Ravichandran’s laboratory has previously shown that apoptotic cell engulfment involves ELMO and Dock180 acting together as a guanine nucleotide exchange factor for the small GTPase Rac. Using yeast two-hybrid screening to identify upstream ELMO-interacting proteins, they identified brain-specific angiogenesis inhibitor 1 (BAI1), a seven transmembrane protein belonging to the adhesion-type G-protein coupled receptor (GPCR) family [16] and expressed in brain, bone marrow, spleen and J774 macrophages. Expression of BAI1 in fibroblasts enhanced both binding and engulfment of apoptotic thymocytes and also induced PS liposomes. Significantly, siRNA-mediated knockdown of BAI1 blocked uptake of apoptotic cells in vitro, and injection of its extracellular domain prevented uptake in vivo. Mutational analysis showed that the a helix of the BAI1 intracellular domain was necessary and sufficient for ELMO binding. Furthermore, the trimeric complex of BAI1–ELMO–Dock180 was associated with enhanced Rac–GTP levels and the greatest increase in apoptotic cell uptake. Genetic manipulation of the BAI1 extracellular domain showed that its thrombospondin type 1 repeats (TSRs) were required for recognition of PS on apoptotic cells, and direct binding to lipid overlays showed stereospecific binding to PS. Given the data supporting a role for thrombospondin 1 as a bridging molecule that recognizes apoptotic cells, the finding of TSRs at the business end of BAI1 is perhaps Nature repeating herself, and in this instance,

Current Biology Vol 18 No 2 R78

Membrane ruffling ingestion PS Phagocytic cell PS-binding bridge molecules

MFG-E8 Del-1 αv integrin Scavenger receptors CD36 SRA-1 etc

Gas 6 protein S MerTK

BAI1

TIM4

Stabilin 2

?

ELMO CrkII Rac

Rac

?

?

?

DOCK 180 PS receptors Current Biology

Figure 1. PS receptors and possible signaling pathways. The three new PS receptors are depicted in context with some of the other PS-recognizing systems thought to be involved in apoptotic cell removal. The bridge molecules here are shown with their cognate signaling receptors and may act in part through the ELMO, DOCK180 and CrkII signaling module that activates Rac. BAI1 was identified specifically by its binding to ELMO. Tim4 has a very short cytoplasmic domain and most likely needs a partner (at this point unidentified) to signal for uptake. Stabilin-2 acts like a scavenger receptor, a number of which are already known to bind native and/or oxidized PS, though generally with additional recognition of other anionic phospholipids.

providing the PS ‘bridge’ and downstream signaling unit in one package. One wonders whether BAI1 may also interact with other tethering receptors (such as CD36 and av integrins) that are known to interact with thrombospondin 1 in the engulfment process. While there appears no obvious homolog of BAI1 in Caenorhabditis elegans (to signal through its homologs of ELMO and Dock180), other surface molecules containing TSRs might serve the same function in this and other species. The known role of BAI1 expression in the blockade of tumor neovascularization is intriguing, since, in many instances, apoptotic cell recognition is associated with angiogenesis. A conserved GPCR proteolytic cleavage site implicated in the release of the BAI1 extracellular

domain (termed vasculostatin) and the subsequent demonstration that the resulting fragment blocks endothelial cell avb5-dependent migration and proliferation [17] raise the question of whether, under some circumstances, proteolytic cleavage of BAI1 might paradoxically inhibit the recognition and uptake of apoptotic cells, an event associated with the development of autoimmunity. The third PSR recently described is stabilin-2, a multifunctional receptor binding a large array of ligands, and perhaps best known for its scavenger receptor and endocytic functions. Park et al. [18] hypothesized that stabilin-2 might participate in the removal of aged, PS-exposing red blood cells (RBCs) and apoptotic cells. Overexpression of stabilin-2 in

fibroblasts greatly enhanced both binding and engulfment of aged, but not normal, RBCs. Remarkably, tethered aged RBCs were engulfed within 2–4 seconds. Furthermore, both binding and engulfment were inhibited by an anti-stabilin-2 antibody and by knockdown of the protein using shRNA, and findings were similar when apoptotic cells were used as targets. Recognition was specific for PS in a stereospecific manner, though the responsible molecular sequences are unknown. Stabilin-2 expression was documented in human and mouse spleen, human monocyte-derived macrophages (HMDMs), alveolar macrophages, and several macrophage cell lines but not in J774.1 or Raw246.7 cells, which are often used in apoptotic

Dispatch R79

cell uptake assays. Knockdown in HMDMs inhibited phagocytosis of aged RBC or apoptotic cells. Stabilin-2, previously associated with alternative macrophage activation and with demonstrated localization on endothelial sinuses of liver, lymph nodes, spleen and bone marrow [19], may be a particularly attractive candidate for clearance of circulating aged RBC and apoptotic leukocytes. Finally, Park et al. [18] demonstrate that engagement of stabilin-2-expressing cells with either aged RBCs or the monoclonal antibody resulted in production of TGFb, a hallmark of the anti-inflammatory program associated with recognition of apoptotic cells. Intriguingly, each of these receptors seems to use different sequence structures to recognize PS and these may be different again from such recognition domains in the PS-binding bridge molecules, or the scavenger receptors, or for that matter, those of other PS-binding proteins, such as protein kinase C, annexins or other coagulation proteins. Until crystal structures are available, much remains unknown as to how PS is recognized by each of these diverse molecular structures (e.g. whether as a monomer or multimers patched in the plasma membrane or oxidized). The overall redundancy of PS recognition in response to apoptotic cells is also noteworthy and leads to critical questions about the signaling pathways employed. Of interest here, genetic analysis in C. elegans identified signaling molecules but provided very little evidence for recognition receptors, suggesting significant common usage of signal pathways with perhaps overlap and redundancy in the receptors. BAI1 was identified by its interaction with the ELMO–DOCK180 RacGEF signaling complex and both the Gas6–MerTK and MFG-E8–av integrin complexes have also been shown to at least link to this complex [11]. Tim4 does not have a significant or obvious intracellular signaling domain and may therefore require a signaling partner — i.e. serve rather like a membrane-bound bridge molecule and/or predominantly as a tethering ligand. In other words, the finding of three new PS receptors raises many new issues, not the least of which is that it seems unlikely that we have yet found them all. How do they all interact? How is the redundancy played

out in different tissues and on different cells and at different times? Finally, it is important to note that we can put to rest the concept that there is a single phosphatidylserine receptor — there are clearly many — and the originally identified PSR needs a new name.

10.

11.

12.

References 1. Fadok, V.A., Voelker, D.R., Campbell, P.A., Cohen, J.J., Bratton, D.L., and Henson, P.M. (1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216. 2. Greenberg, M.E., Sun, M., Zhang, R., Febbraio, M., Silverstein, R., and Hazen, S.L. (2006). Oxidized phosphatidylserine-CD36 interactions play an essential role in macrophage-dependent phagocytosis of apoptotic cells. J. Exp. Med. 203, 2613–2625. 3. Williamson, P., and Schlegel, R.A. (2002). Transbilayer phospholipid movement and the clearance of apoptotic cells. Biochim. Biophys. Acta. 1585, 53–63. 4. Frasch, S.C., Henson, P.M., Nagaosa, K., Fessler, M.B., Borregaard, N., and Bratton, D.L. (2004). Phospholipid flip-flop and phospholipid scramblase 1 (PLSCR1) co-localize to uropod rafts in formylated Met-Leu-Phe-stimulated neutrophils. J. Biol. Chem. 279, 17625–17633. 5. Herrmann, A., and Devaux, P.F. (1990). Alteration of the aminophospholipid translocase activity during in vivo and artificial aging of human erythrocytes. Biochim. Biophys. Acta. 1027, 41–46. 6. Verhoven, B., Schlegel, R.A., and Williamson, P. (1995). Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J. Exp. Med. 182, 1597–1601. 7. Sorice, M., Circella, A., Cristea, I.M., Garofalo, T., Di Renzo, L., Alessandri, C., Valesini, G., and Esposti, M.D. (2004). Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. Cell Death Differ. 11, 1133–1145. 8. Elliott, J.I., Sardini, A., Cooper, J.C., Alexander, D.R., Davanture, S., Chimini, G., and Higgins, C.F. (2006). Phosphatidylserine exposure in B lymphocytes: a role for lipid packing. Blood 108, 1611–1617. 9. Hoffmann, P.R., deCathelineau, A.M., Ogden, C.A., Leverrier, Y., Bratton, D.L., Daleke, D.L., Ridley, A.J., Fadok, V.A., and Henson, P.M. (2001). Phosphatidylserine (PS) induces PS receptor-mediated

13.

14.

15.

16.

17.

18.

19.

macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 155, 649–659. Fadok, V.A., Bratton, D.L., Rose, D.M., Pearson, A., Ezekewitz, R.A., and Henson, P.M. (2000). A receptor for phosphatidylserinespecific clearance of apoptotic cells. Nature 405, 85–90. Wu, Y., Tibrewal, N., and Birge, R.B. (2006). Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol. 16, 189–197. Hanayama, R., Tanaka, M., Miwa, K., and Nagata, S. (2004). Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells. J. Immunol. 172, 3876–3882. Miyanishi, M., Tada, K., Koike, M., Uchiyama, Y., Kitamura, T., and Nagata, S. (2007). Identification of Tim4 as a phosphatidylserine receptor. Nature 450, 435–439. Wilker, P.R., Sedy, J.R., Grigura, V., Murphy, T.L., and Murphy, K.M. (2007). Evidence for carbohydrate recognition and homotypic and heterotypic binding by the TIM family. Int. Immunol. 19, 763–773. McIntire J.J., Umetsu S.E., Akbari O., Potter M., Kuchroo V.K., Barsh G.S., Freeman G.J., Umetsu D.T., and DeKruyff R.H. (2001). Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat. Immunol. 2, 1109–1116. Park, D., Tosello-Trampont, A.C., Elliott, M.R., Lu, M., Haney, L.B., Ma, Z., Klibanov, A.L., Mandell, J.W., and Ravichandran, K.S. (2007). BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450, 430–434. Kaur, B., Brat, D.J., Devi, N.S., and Van Meir, E.G. (2005). Vasculostatin, a proteolytic fragment of brain angiogenesis inhibitor 1, is an antiangiogenic and antitumorigenic factor. Oncogene 24, 3632–3642. Park, S.Y., Jung, M.Y., Kim, H.J., Lee, S.J., Kim, S.Y., Lee, B.H., Kwon, T.H., Park, R.W., and Kim, I.S. (2007). Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ. 15, 192–201. Falkowski, M., Schledzewski, K., Hansen, B., and Goerdt, S. (2003). Expression of stabilin-2, a novel fasciclin-like hyaluronan receptor protein, in murine sinusoidal endothelia, avascular tissues, and at solid/liquid interfaces. Histochem. Cell Biol. 120, 361–369.

Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA. E-mail: [email protected], [email protected] DOI: 10.1016/j.cub.2007.11.024

Reproductive Traits: Evidence for Sexually Selected Sperm Sperm exhibit extraordinary morphological divergence yet precise evolutionary causes often remain elusive. A quantitative genetic study sheds light on the major role postcopulatory sexual selection could play in determining sperm size. Oliver Y. Martin1 and Marco Demont2 Sexual selection arises because individuals vary in reproductive success [1]. This variation often

exceeds that in survivorship and sexual selection is thus a potentially powerful evolutionary force [2,3]. Classically, sexual selection is viewed as comprising competition between