Abstract
To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including RhoB. Here we describe an activation assay of RhoB in leukocytes migrating on ICAM-1Fc coated dishes using commercially available Rhoteikin coated agarose beads. Although this is a specific protocol for LFA-1 induced RhoB activation, it can also be used for RhoB activation induced by other soluble and insoluble factors.
Keywords: Leukocytes, LFA-1, RhoB, Small GTPases, Rhoteikin, Pull-down
Background
Leukocytes are frequently migrating to clear infections and/or to become activated in the lymph nodes. For a cell to move forward, it needs to be able to attach and reuse its integrins. These steps involve various small GTPases that control the intracellular transport of proteins. Small GTPases switch from active to inactive form by hydrolysis of GTP into GDP, which is controlled by specific GTPase-activating proteins (GAPs) and Guanine nucleotide exchange factors (GEFs) (Cavalli et al., 2001). RhoB is one of these important small GTPases that regulates endosomal trafficking of receptors in different cell types including leukocytes (Fernandez-Borja et al., 2005). We have recently found that the major integrin used by T lymphocytes, Lymphocyte function-associated antigen 1 (LFA-1) is recycled by a Rab11-dependent pathway that is controlled by RhoB activity (Samuelsson et al., 2017). Here, we present a protocol to measure RhoB activation based on the pulldown of active RhoB with commercially available Rhotekin-coated agarose beads. The Rhotekin-RBD protein specifically recognizes and binds to the active, GTP-bound form of the Rho protein. Furthermore, we describe integrin-dependent RhoB activation using coverslips coated with the integrin ligand ICAM-1Fc.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
In this protocol, we used human primary T lymphoblasts. Naturally, other cells can be used as well. See Samuelsson et al. (2017) for the primary T lymphoblast purification procedure.
Recipes
Acknowledgments
We would like to thank Dr. Nancy Hogg (The Francis Crick Institute, UK) for the kind gift of the LFA-1 antibody. This protocol was adopted from Samuelsson et al. (2017). This work was supported by Swedish Research Council awards K2010-80P-21592-01-4 and K2010-80X-215917-01-4, Foundation Olle Engvist Byggmästare, I & A Lundberg Research Foundation, Royal Swedish Academy of Science, Åke Wiberg Foundation, Jeanssons Foundation, Kocks Foundation, P & U Schybergs Foundation, Gyllenstiernska Krapperup Foundation, Gustav V 80 Jubilee Fund, Österlund Foundation, Nanna Svartz, Crafoord Awards Anna-Greta Crafoord Foundation and Royal Physiographic Society of Lund.
Competing interests
The authors declare no competing interest.
References
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