Molecular Neurobiology

, Volume 55, Issue 5, pp 4320–4332 | Cite as

Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells

  • Ayelen Bulloj
  • Arvydas Maminishkis
  • Masayuki Mizui
  • Silvia C. Finnemann


Semaphorins form a family of secreted and membrane-bound molecules that were identified originally as axonal guidance factors during neuronal development. Given their wide distribution in many including mature tissues, semaphorin 4D (sema4D) and its main functional receptor plexin B1 (plxnB1) are expected to fulfill additional functions that remain to be uncovered. A main characteristic of the plexin receptor family is its ability to reorganize the cytoskeleton. PlxnB1 specifically may directly interact with Rho family GTPases to regulate F-actin driven pathways in cells in culture. Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) of photoreceptor outer segment fragments (POS) is critical for photoreceptor function and longevity. In this process, rearrangement of RPE cytoskeletal F-actin via activation of the Rho family GTPase Rac1 is essential for POS internalization. Here, we show a novel role in POS phagocytosis by RPE cells in culture and in vivo for plexin B1 and its ligand sema4D. Exogenous sema4D abolishes POS internalization (but not binding) by differentiated RPE cells in culture by decreasing the GTP load of Rac1. In the rat eye, sema4D localizes to retinal photoreceptors, while PlxnB1 is expressed by neighboring RPE cells. At the peak of diurnal retinal phagocytosis after light onset, plxnB1 phosphorylation and sema4D levels are reduced in wild-type rat retina in situ but not in mutant RCS rat retina in which the RPE lacks phagocytic activity. Finally, increased POS phagosome content after light onset is observed in the RPE in situ of mice with either plxnB1 or sema4D gene deletion. Altogether, our results demonstrate a novel physiological function for sema4D/plxnB1 signaling in RPE phagocytosis serving as attenuating brake prior to light onset whose release enables the diurnal phagocytic burst.


Semaphorin 4D Plexin B1 Photoreceptor outer segment phagocytosis Retinal pigment epithelium 



This project was supported by NIH grant EY26215 (to S.C.F) and National Eye Institute intramural funds (to A.M.). We are grateful to Drs. Eszter Paldy and Rohini Kunar (University of Heidelberg, Germany) who generously provided the dissected tissues from plxnB1 KO mice for this study.

Compliance with Ethical Standards

All human tissue research followed the tenets of the Declaration of Helsinki and the NIH institutional review board. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and reviewed and approved according to institutional requirements.

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Kolodkin AL, Matthes DJ, Goodman CS (1993) The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 75(7):1389–1399. doi: 10.1016/0092-8674(93)90625-Z CrossRefPubMedGoogle Scholar
  2. 2.
    Takahashi T, Fournier A, Nakamura F, Wang L-H, Murakami Y, Kalb RG, Fujisawa H, Strittmatter SM (1999) Plexin-neuropilin-1 complexes form functional semaphorin-3a receptors. Cell 99(1):59–69. doi: 10.1016/S0092-8674(00)80062-8 CrossRefPubMedGoogle Scholar
  3. 3.
    Tamagnone L, Artigiani S, Chen H, He Z, Ming G-L, Song H-J, Chedotal A, Winberg ML et al (1999) Plexins are a large family of receptors for transmembrane, secreted, and gpi-anchored semaphorins in vertebrates. Cell 99(1):71–80. doi: 10.1016/S0092-8674(00)80063-X CrossRefPubMedGoogle Scholar
  4. 4.
    Gherardi E, Love CA, Esnouf RM, Jones EY (2004) The sema domain. Curr Op Struct Biol 14(6):669–678. doi: 10.1016/ CrossRefGoogle Scholar
  5. 5.
    Artigiani S, Comoglio PM, Tamagnone L (1999) Plexins, semaphorins, and scatter factor receptors: a common root for cell guidance signals? IUBMB Life 48(5):477–482. doi: 10.1080/713803563 CrossRefPubMedGoogle Scholar
  6. 6.
    Oinuma I, Ishikawa Y, Katoh H, Negishi M (2004) The semaphorin 4D receptor plexin-B1 is a GTPase activating protein for r-ras. Science 305(5685):862–865. doi: 10.1126/science.1097545 CrossRefPubMedGoogle Scholar
  7. 7.
    Rohm B, Rahim B, Kleiber B, Hovatta I, Püschel AW (2000) The semaphorin 3A receptor may directly regulate the activity of small GTPases. FEBS Letters 486(1):68–72. doi: 10.1016/S0014-5793(00)02240-7 CrossRefPubMedGoogle Scholar
  8. 8.
    Tong Y, Chugha P, Hota PK, Alviani RS, Li M, Tempel W, Shen L, Park H-W et al (2007) Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-b1 effector domain. J Biol Chem 282(51):37215–37224. doi: 10.1074/jbc.M703800200 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tong Y, Hota PK, Penachioni JY, Hamaneh MB, Kim S, Alviani RS, Shen L, He H et al (2009) Structure and function of the intracellular region of the plexin-B1 transmembrane receptor. J Biol Chem 284(51):35962–35972. doi: 10.1074/jbc.M109.056275 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Swiercz JM, Worzfeld T, Offermanns S (2008) ErbB-2 and Met reciprocally regulate cellular signaling via plexin-B1. J Biol Chem 283(4):1893–1901. doi: 10.1074/jbc.M706822200 CrossRefPubMedGoogle Scholar
  11. 11.
    Vikis HG, Li W, He Z, Guan K-L (2000) The semaphorin receptor plexin-B1 specifically interacts with active Rac in a ligand-dependent manner. Proc Natl Acad Sci U S A 97(23):12457–12462. doi: 10.1073/pnas.220421797 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Driessens MH, Hu H, Nobes CD, Self A, Jordens I, Goodman CS, Hall A (2001) Plexin-B semaphorin receptors interact directly with active Rac and regulate the actin cytoskeleton by activating Rho. Curr Biol 11(5):339–344CrossRefPubMedGoogle Scholar
  13. 13.
    Basile JR, Gavard J, Gutkind JS (2007) Plexin-B1 utilizes RhoA and Rho kinase to promote the integrin-dependent activation of Akt and ERK and endothelial cell motility. J Biol Chem 282(48):34888–34895. doi: 10.1074/jbc.M705467200 CrossRefPubMedGoogle Scholar
  14. 14.
    Ruggiero L, Connor MP, Chen J, Langen R, Finnemann SC (2012) Diurnal, localized exposure of phosphatidylserine by rod outer segment tips in wild-type but not Itgb5 −/− or Mfge8 −/− mouse retina. Proc Natl Acad Sci U S A 109(21):8145–8148. doi: 10.1073/pnas.1121101109 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Nandrot EF, Anand M, Almeida D, Atabai K, Sheppard D, Finnemann SC (2007) Essential role for MFG-E8 as ligand for αvβ5 integrin in diurnal retinal phagocytosis. Proc Natl Acad Sci U S A 104(29):12005–12010. doi: 10.1073/pnas.0704756104 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Nandrot EF, Kim Y, Brodie SE, Huang X, Sheppard D, Finnemann SC (2004) Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking αvβ5 integrin. J Exp Med 200(12):1539–1545. doi: 10.1084/jem.20041447 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Feng W, Yasumura D, Matthes MT, LaVail MM, Vollrath D (2002) Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells. J Biol Chem 277(19):17016–17022CrossRefPubMedGoogle Scholar
  18. 18.
    Finnemann SC (2003) Focal adhesion kinase signaling promotes phagocytosis of integrin-bound photoreceptors. EMBO J 22(16):4143–4154. doi: 10.1093/emboj/cdg416 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Mao Y, Finnemann SC (2012) Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase. Mol Biol Cell 23(6):1104–1114. doi: 10.1091/mbc.E11-10-0840 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Bulloj A, Duan W, Finnemann SC (2013) PI 3-kinase independent role for AKT in F-actin regulation during outer segment phagocytosis by RPE cells. Exp Eye Res 113(8):9–18. doi: 10.1016/j.exer.2013.05.002 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH, Hargrave PA (1991) Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vis Res 31(1):17–31CrossRefPubMedGoogle Scholar
  22. 22.
    Maminishkis A, Chen S, Jalickee S, Banzon T, Shi G, Wang FE, Ehalt T, Hammer JA et al (2006) Confluent monolayers of cultured human fetal retinal pigment epithelium exhibit morphology and physiology of native tissue. Invest Ophthalmol Vis Sci 47(8):3612–3624. doi: 10.1167/iovs.05-1622 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Finnemann SC, Bonilha VL, Marmorstein AD, Rodriguez-Boulan E (1997) Phagocytosis of rod outer segments by retinal pigment epithelial cells requires αvβ5 integrin for binding but not for internalization. Proc Natl Acad Sci U S A 94(24):12932–12937CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Parinot C, Rieu Q, Chatagnon J, Finnemann SC, Nandrot EF (2014) Large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells. J Vis Exp JoVE 94:52100. doi: 10.3791/52100 Google Scholar
  25. 25.
    Finnemann SC, Rodriguez-Boulan E (1999) Macrophage and retinal pigment epithelium phagocytosis: apoptotic cells and photoreceptors compete for αvβ3 and αvβ5 integrins, and protein kinase C regulates αvβ5 binding and cytoskeletal linkage. J Exp Med 190(6):861–874CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Shi W, Kumanogoh A, Watanabe C, Uchida J, Wang X, Yasui T, Yukawa K, Ikawa M et al (2000) The class IV semaphorin CD100 plays nonredundant roles in the immune system: defective B and T cell activation in CD100-deficient mice. Immunity ’(5):633–642. doi: 10.1016/S1074-7613(00)00063-7 CrossRefGoogle Scholar
  27. 27.
    Deng S, Hirschberg A, Worzfeld T, Penachioni JY, Korostylev A, Swiercz JM, Vodrazka P, Mauti O et al (2007) Plexin-B2, but not plexin-B1, critically modulates neuronal migration and patterning of the developing nervous system in vivo. J Neurosci 27(23):6333–6347. doi: 10.1523/jneurosci.5381-06.2007 CrossRefPubMedGoogle Scholar
  28. 28.
    Sethna S, Finnemann SC (2013) Analysis of photoreceptor rod outer segment phagocytosis by RPE cells in situ. Methods Mol Biol 935:245–254. doi: 10.1007/978-1-62703-080-9_17
  29. 29.
    Swiercz JM, Worzfeld T, Offermanns S (2009) Semaphorin 4D signaling requires the recruitment of phospholipase C gamma into the plexin-B1 receptor complex. Mol Cell Biol 29(23):6321–6334. doi: 10.1128/MCB.00103-09 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Koulen P, Fletcher EL, Craven SE, Bredt DS, Wässle H (1998) Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina. J Neurosci 18(23):10136–10149CrossRefPubMedGoogle Scholar
  31. 31.
    Maestrini E, Tamagnone L, Longati P, Cremona O, Gulisano M, Bione S, Tamanini F, Neel BG et al (1996) A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor. Proc Natl Acad Sci U S A 93(2):674–678CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Janssen BJ, Robinson RA, Perez-Branguli F, Bell CH, Mitchell KJ, Siebold C, Jones EY (2010) Structural basis of semaphorin-plexin signalling. Nature 467(7319):1118–1122. doi: 10.1038/nature09468 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Koppel AM, Feiner L, Kobayashi H, Raper JA (1997) A 70 amino acid region within the semaphorin domain activates specific cellular response of semaphorin family members. Neuron 19(3):531–537. doi: 10.1016/S0896-6273(00)80369-4 CrossRefPubMedGoogle Scholar
  34. 34.
    Klostermann A, Lohrum M, Adams RH, Püschel AW (1998) The chemorepulsive activity of the axonal guidance signal semaphorin D requires dimerization. J Biol Chem 273(13):7326–7331. doi: 10.1074/jbc.273.13.7326 CrossRefPubMedGoogle Scholar
  35. 35.
    Zhu L, Bergmeier W, Wu J, Jiang H, Stalker TJ, Cieslak M, Fan R, Boumsell L et al (2007) Regulated surface expression and shedding support a dual role for semaphorin 4D in platelet responses to vascular injury. Proc Natl Acad Sci U S A 104(5):1621–1626. doi: 10.1073/pnas.0606344104 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mou P, Zeng Z, Li Q, Liu X, Xin X, Wannemacher KM, Ruan C, Li R et al (2013) Identification of a calmodulin-binding domain in sema4D that regulates its exodomain shedding in platelets. Blood 121(20):4221–4230. doi: 10.1182/blood-2012-11-470609 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Basile JR, Castilho RM, Williams VP, Gutkind JS (2006) Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Proc Natl Acad Sci U S A 24:9017–9022. doi: 10.1073/pnas.0508825103 CrossRefGoogle Scholar
  38. 38.
    Oinuma I, Katoh H, Negishi M (2006) Semaphorin 4D/Plexin-B1–mediated R-Ras GAP activity inhibits cell migration by regulating β(1) integrin activity. J Cell Biol 173(4):601–613. doi: 10.1083/jcb.200508204 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Barberis D, Artigiani S, Casazza A, Corso S, Giordano S, Love CA, Jones EY, Comoglio PM et al (2004) Plexin signaling hampers integrin-based adhesion, leading to Rho-kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility. FASEB J 18(3):592–594. doi: 10.1096/fj.03-0957fje CrossRefPubMedGoogle Scholar
  40. 40.
    Serini G, Valdembri D, Zanivan S, Morterra G, Burkhardt C, Caccavari F, Zammataro L, Primo L et al (2003) Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature 424(6947):391–397. doi: 10.1038/nature01784 CrossRefPubMedGoogle Scholar
  41. 41.
    Walzer T, Galibert L, Comeau MR, De Smedt T (2005) Plexin C1 engagement on mouse dendritic cells by viral semaphorin A39R induces actin cytoskeleton rearrangement and inhibits integrin-mediated adhesion and chemokine-induced migration. J Immunol 174(1):51–59CrossRefPubMedGoogle Scholar
  42. 42.
    Myster F, Palmeira L, Sorel O, Bouillenne F, DePauw E, Schwartz-Cornil I, Vanderplasschen A, Dewals BG (2015) Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for gammaherpesvirus-induced lymphoproliferation in malignant catarrhal fever. J Virol 89(7):3630–3647. doi: 10.1128/JVI.03634-14 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Yu C-C, Nandrot EF, Dun Y, Finnemann SC (2012) Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin. Free Radic Biol Med 52(3):660–670. doi: 10.1016/j.freeradbiomed.2011.11.021 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Ayelen Bulloj
    • 1
  • Arvydas Maminishkis
    • 2
  • Masayuki Mizui
    • 3
  • Silvia C. Finnemann
    • 1
  1. 1.Department of Biological Sciences Center for Cancer, Genetic Diseases, and Gene RegulationFordham UniversityBronxUSA
  2. 2.Section on Epithelial and Retinal Physiology and DiseaseNational Eye Institute, National Institutes of HealthBethesdaUSA
  3. 3.Department of NephrologyOsaka University Graduate School of MedicineOsakaJapan

Personalised recommendations