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Rho GTPases pp 235-246 | Cite as

Methods to Study the Role of Cdc42, Rac1, and Rac2 in B-Cell Cytoskeletal Responses

  • Natalija Gerasimcik
  • Lisa S. Westerberg
  • Eva SeverinsonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1821)

Abstract

B-cell migration and adhesion are critical to form a germinal center response, the site for B-cell production of high-affinity antibodies. Here, we describe two assays that can be used to examine B-cell cytoskeletal responses needed during the germinal center response: B-cell spreading and homotypic adhesion. Spreading of B cells is dependent on Cdc42, while Rac1 and Rac2 are necessary for homotypic adhesion. These in vitro assays can be used to examine functional responses of B cells mediated by the cell cytoskeleton, for example when comparing B cells from different gene knockout animals.

Key words

B lymphocytes In vitro responses Polyclonal activation Adhesion Spreading 

Supplementary material

Video 1

Formation of aggregates in B cells cultured with anti-CD40 + IL-4. B cells were stimulated for 2 days. Bright-field images were taken every minute. Note that big aggregates are formed by fusion of smaller ones. Magnification 10× (MOV 61702 kb)

References

  1. 1.
    Guo F, Velu CS, Grimes HL, Zheng Y (2009) Rho GTPase Cdc42 is essential for B-lymphocyte development and activation. Blood 114:2909–2916CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Burbage M, Keppler SJ, Gasparrini F, Martínez-Martín N, Gaya M, Feest C, Domart M-C, Brakebusch C, Collinson L, Bruckbauer A, Batista FD (2015) Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity. J Exp Med 212:53–72CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Walmsley MJ, Ooi SKT, Reynolds LF, Smith SH, Ruf S, Mathiot A, Vanes L, Williams DA, Cancro MP, Tybulewicz VLJ (2003) Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling. Science 302:459–462CrossRefPubMedGoogle Scholar
  4. 4.
    Henderson RB, Grys K, Vehlow A, de Bettignies C, Zachacz A, Henley T, Turner M, Batista F, Tybulewicz VL (2010) A novel Rac-dependent checkpoint in B cell development controls entry into the splenic white pulp and cell survival. J Exp Med 207:837–853CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gerasimčik N, Dahlberg CI, Baptista MA, Massaad MJ, Geha RS, Westerberg LS, Severinson E (2015) The rho GTPase Cdc42 is essential for the activation and function of mature B cells. J Immunol 194:4750–4758CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gerasimčik N, He M, Dahlberg CI, Kouznetsov N, Severinson E, Westerberg LS (2017) The small rho GTPases Rac1 and Rac2 are important for T-cell independent antigen responses and for suppressing switching to IgG2b in mice. Front Immunol 8:1264CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hobeika E, Levit-Zerdoun E, Anastasopoulou V, Pohlmeyer R, Altmeier S, Alsadeq A, Dobnecker MW, Pelanda R, Reth M (2015) CD19 and BAFF-R can signal to promote B-cell survival in the absence of Syk. EMBO J 34:925–939CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Gerasimčik N, He M, Baptista MAP, Severinson E, Westerberg LS (2017) Deletion of Dock10 in B cells results in normal development but a mild deficiency upon in vivo and in vitro stimulations. Front Immunol 8:491CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Alberini CM, Bet P, Milstein C, Sitia R (1990) Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents. Nature 347:485–487CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Natalija Gerasimcik
    • 1
  • Lisa S. Westerberg
    • 2
  • Eva Severinson
    • 3
    Email author
  1. 1.Department of Dental MedicineDivision of Periodontology, Karolinska InstitutetHuddingeSweden
  2. 2.Department of MicrobiologyTumor and Cell Biology, Karolinska InstitutetStockholmSweden
  3. 3.Department of Molecular Biosciences, The Wenner Gren InstituteStockholm UniversityStockholmSweden

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