Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

Abstract

The leukocyte common antigen, CD45, is a critical immune regulator whose activity is modulated by cytoskeletal interactions. Components of the spectrin-ankyrin cytoskeleton have been implicated in the trafficking and signaling of CD45. We have examined the lateral mobility of CD45 in resting and activated T lymphocytes using single-particle tracking and found that the receptor has decreased mobility caused by increased cytoskeletal contacts in activated cells. Experiments with cells that have disrupted ␤⌱ spectrin interactions show decreased cytoskeletal contacts in resting cells and attenuation of receptor immobilization in activated cells. Applying two types of population analyses to single-particle tracking trajectories, we find good agreement between the diffusion coefficients obtained using either a mean squared displacement analysis or a hidden Markov model analysis. Hidden Markov model analysis also reveals the rate of association and dissociation of CD45-cytoskeleton contacts, demonstrating the importance of this analysis for measuring cytoskeleton binding events in live cells. Our findings are consistent with a model in which multiple cytoskeletal contacts, including those with spectrin and ankyrin, participate in the regulation of CD45 lateral mobility. These interactions are a major factor in CD45 immobilization in activated cells. Furthermore, cellular activation leads to CD45 immobilization by reduction of the CD45-cytoskeleton dissociation rate. Short peptides that mimic spectrin repeat domains alter the association rate of CD45 to the cytoskeleton and cause an apparent decrease in dissociation rates. We propose a model for CD45-cytoskeleton interactions and conclude that the spectrin-ankyrin-actin network is an essential determinant of immunoreceptor mobility.The lymphocyte receptor-like protein tyrosine phosphatase, CD45, 2 is a transmembrane protein that acts as a critical component of T cell activation (1). The phosphatase activity of the CD45 cytoplasmic domain contributes to T cell activation through the removal of an inhibitory phosphate group on a membrane-associated Src family kinase, such as Tyr 505 of p56 lck (Lck) (2). Other CD45 substrates have been established in addition to Lck, including Fyn, SKAP-55, JAK, ZAP-70, and CK2 (1). The involvement of CD45 as a regulator of critical signaling pathways and autoimmune diseases highlights the importance of understanding its function.In addition to phosphoprotein substrates, CD45 interacts with a variety of molecular partners that modulate its activity. Its heavily glycosylated extracellular domain interacts with Thy1 (3) and galectins (4), and it self-associates to form dimers. Dimer formation is a process linked to both regulation of its phosphatase activity and sialylation of its ectodomain (1, 5, 6). The cytoplasmic domain of CD45 contains one catalytically active protein-tyrosine phosphatase domain (Domain 1) and one inactive domain (Domain 2) and binds to key cellular regulators of immune function such as CD45AP (7), CD2 (8), CD11a (9),...

Guanylate Binding Protein 1–Mediated Interaction of T Cell Antigen Receptor Signaling with the Cytoskeleton

“…3D) that interfere with the cytoskeleton and early TCR signaling: stomatin-like protein 2 (STOML2) (32,33), myosin regulatory L chain MRCL3 variant (MRCL3) (34), and the bII-spectrin. bII-Spectrin was described to be involved in CD3 and CD45 surface display and IL-2 production (35,36). We found that CD45 and CD3 surface expression was enhanced in GBP-1-silenced Jurkat T cells, whereas the levels of several other surface receptors such as CD11a, CD18, and CD43 were not altered (Fig.…”

“…It was described that the bII-spectrin cytoskeleton influences IL-2 production in T cells by mediating CD3 and CD45 surface expression (35,36). We show in this article that GBP-1 interferes with CD3 and CD45 surface display (Fig.…”

“…It is known that GBP-1 binds (besides homodimerization and heterodimerization with GBP family members) (41,42) to the cytoskeletal proteins b-IIITubulin and PIM1 (14). Five among the 10 potential GBP-1 binding partners identified by us by mass spectrometry (multisynthetase complex auxiliary component p43, plastin-2, STOML2, MRCL3, and bIIspectrin) are described to be involved in the remodeling of the (actin-) cytoskeleton (33,36,43,44) and 4 of these 5 (plastin-2, STOML2, MRCL3, and bII-spectrin) to influence T cell activation (30,(32)(33)(34)(35)(36)(44)(45)(46). Based on the fact that IL-2 expression is abrogated in plastin-2 knockout mice, we suspected plastin-2 to be involved in the GBP-1-mediated IL-2 regulation.…”

Membrane Protein Dynamics and Functional Implications in Mammalian Cells

“…SPT measurements showed that CD45 mobility decreased upon cell activation, and this decrease in mobility was attenuated by disruption of the actin cytoskeleton. Peptide fragments of β1 spectrin were used to show that CD45 utilized spectrin to maintain cytoskeletal linkages with actin and with ankyrin (Cairo et al, 2010). These spectrin–ankyrin and spectrin–actin links stabilized CD45 in the membrane.…”

Spectrin: Structure, function and disease

“…A vesicular spectrin-ankyrin adaptor protein trafficking system may mediate the capture and transport of many membrane proteins while cooperating with vesicular target molecules to transport cargo proteins. Recently, Cairo et al [25] used bioinformatics methods to establish a model of the CD45-cytoskeleton interaction and elucidated that the spectrin-ankyrin-actin network significantly controls immunoreceptor mobility.…”