Proteasome Dependent Actin Remodeling Facilitates Antigen Extraction at the Immune Synapse of B Cells

Abstract

Engagement of the B cell receptor (BCR) with surface-tethered antigens leads to the formation of an immune synapse (IS), where cell signaling and antigen uptake are tightly coordinated. Centrosome re-orientation to the immune synapse has emerged as a critical regulatory step to guide the local recruitment and secretion of lysosomes, which can facilitate the extraction of immobilized antigens. This process is coupled to actin remodeling at the centrosome and at the immune synapse, which is crucial to promote cell polarity. How B cells balance both pools of actin cytoskeleton to achieve a polarized phenotype during the formation of an immune synapse is not fully understood. Here, we reveal that B cells rely on proteasome activity to achieve this task. The proteasome is a multi-catalytic protease that degrades cytosolic and nuclear proteins and its dysfunction is associated with diseases, such as cancer and autoimmunity. Our results show that resting B cells contain an active proteasome pool at the centrosome, which is required for efficient actin clearance at this level. As a result of proteasome inhibition, activated B cells do not deplete actin at the centrosome and are unable to separate the centrosome from the nucleus and thus display impaired polarity. Consequently, lysosome recruitment to the immune synapse, antigen extraction and presentation are severely compromised in B cells with diminished proteasome activity. Additionally, we found that proteasome inhibition leads to impaired actin remodeling at the immune synapse, where B cells display defective spreading responses and distribution of key signaling molecules at the synaptic membrane. Overall, our results reveal a new role for the proteasome in regulating the immune synapse of B cells, where the intracellular compartmentalization of proteasome activity controls cytoskeleton remodeling between the centrosome and synapse, with functional repercussions in antigen extraction and presentation.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes

“…The differences observed between the values of centrosomal area Factin of control (0.93, C3; 0.99, C9) vs PKCδ-interfered clones (1.18, P5; 1.37, P6) (Fig. 2C)were similar to those caused by proteasome inhibition in B lymphocytes (1.00 in control vs 1.25 in proteasome inhibitor-treated cells), that inhibited MTOC polarization[29]. Thus, it is conceivable that the centrosomal area F-actin changes we observed, alone or in combination with the alterations in cortical F-actin at the IS, may in part underlie the deficiency in MTOC polarization in PKCδ-interfered clones(Fig.…”

“…lymphocytes stimulated with BCR-ligand-coated beads [28]. In the same B lymphocyte model, F-actin depletion around the MTOC, F-actin reorganization at the IS, and MTOC polarization depend on proteasome activity [29]. to metaphase (400-2000 nm) [54].…”

“…The existence in T lymphocytes of at least two PKCδ-controlled, coordinated regulatory mechanisms acting on two distinct F-actin networks, both controlling MTOC polarization and secretion, may establish subtle regulatory checkpoints to finely control IS-triggered polarized secretion, as previously suggested[23]. Polarized secretion guarantees the antigen specificity of the final response in many cell lineages of the immune system, including innate NK cells[66,67], CTL[5,38], B lymphocytes[29] [62], primary CD4+ T cells[68] and Jurkat cells[38]. The association of the absence of PKCδ with the generation of B and T lymphoproliferative disorders both in human and mice[69] [70], and the participation of PKCδ in the homeostasis of blood progenitors[71],support the important role of PKCδ in maintaining cell homeostasis.…”

“…Regarding other molecules controlling MTOC polarization, paxillin (an actin-regulating adaptor protein) [24] is necessary for MTOC polarization to the IS in CTLs [25] and its phosphorylation is required for lytic granule secretion from CTL [26]. Along these lines, recent studies in B lymphocytes forming IS indicated that the MTOC is a F-actin organizing center [27], and F-actin depletion around the MTOC crucially facilitates MTOC polarization towards the IS in B-cell receptor (BCR)-stimulated B lymphocytes [28] [29]. In summary, taking together these observations, it is clear that MTOC and MVB polarization induced by IS formation are regulated by HS1/WASp/Arp2/3dependent cortical and formin-dependent non-cortical actin networks, and that PKCδ may regulate the reorganization of both actin networks.…”

“…dynamic and plastic signalling platform induced by antigen receptors stimulation and triggers exquisite mechanisms leading to optimal polarized and focused secretion at the synaptic cleft, to avoid the stimulation (or death) of bystander cells[65] [9][48] [1][35], or the non-specific extraction of antigens from APC[29] [62]. The existence in T lymphocytes of at least two PKCδ-controlled, coordinated regulatory mechanisms acting on two distinct F-actin networks, both controlling MTOC polarization and secretion, may establish subtle regulatory checkpoints to finely control IS-triggered polarized secretion, as previously suggested[23].…”