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Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes
I Melamed1, G P Downey, K Aktories
1Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Insights
B cell activation involves actin polymerization, a process crucial for cell proliferation. This cytoskeletal change is mediated by guanosine triphosphate (GTP)-binding proteins and protein kinase C (PKC).
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Mechanisms of actin assembly in B lymphocytes are not fully understood.
- The role of cytoskeletal changes in B cell activation requires further investigation.
Purpose of the Study:
- Investigate molecular signals initiating actin polymerization in B cells.
- Elucidate the involvement of guanosine triphosphate (GTP)-binding proteins and protein kinase C (PKC).
- Relate early cytoskeletal events to later B cell activation, including proliferation.
Main Methods:
- Stimulation of B cells via antigen receptor cross-linking (Staphylococcus aureus Cowan I, anti-IgM) or PKC activation (phorbol ester).
- Assessment of filamentous actin content and actin assembly.
- Inhibition studies using pertussis toxin, botulinum C2 toxin, and cytochalasin D.
- Direct stimulation with GTP gamma S in permeabilized cells.
Main Results:
- Antigen receptor cross-linking and PKC stimulation increased filamentous actin content in a time- and concentration-dependent manner.
- PKC inhibition reduced actin assembly, suggesting signaling occurs downstream of PKC.
- Pertussis toxin inhibited receptor-mediated actin assembly, while GTP gamma S induced it, indicating GTP-binding protein involvement.
- Disruption of actin polymerization inhibited B cell proliferation.
Conclusions:
- Human B cell activation involves actin polymerization mediated by signaling pathways coupled to GTP-binding proteins.
- Cytoskeletal changes, specifically actin polymerization, play a role in B cell activation and proliferation.
Abstract:
The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.