Abstract
The formation of an immune synapse (IS) enables B cells to capture membrane-tethered antigens, where cortical actin cytoskeleton remodeling regulates cell spreading and depletion of F-actin at the centrosome promotes the recruitment of lysosomes to facilitate antigen extraction. How B cells regulate both pools of actin, remains poorly understood. We report here that decreased F-actin at the centrosome and IS relies on the distribution of the proteasome, regulated by Ecm29. Silencing Ecm29 decreases the proteasome pool associated to the centrosome of B cells and shifts its accumulation to the cell cortex and IS. Accordingly, Ecm29-silenced B cells display increased F-actin at the centrosome, impaired centrosome and lysosome repositioning to the IS and defective antigen extraction and presentation. Ecm29-silenced B cells, which accumulate higher levels of proteasome at the cell cortex, display decreased actin retrograde flow in lamellipodia and enhanced spreading responses. Our findings support a model where B the asymmetric distribution of the proteasome, mediated by Ecm29, coordinates actin dynamics at the centrosome and the IS, promoting lysosome recruitment and cell spreading.
Highlights
The interaction of the B cell receptor (BCR) with membrane-tethered antigens initiates the formation of an immune synapse (IS), characterized by rapid cortical actin cytoskeleton rearrangements and the formation of BCR-microclusters containing signaling molecules that elicit B cell activation (Yuseff et al, 2013; Kwak et al, 2019)
We found that upon activation with antigencoated beads, Ecm29 progressively accumulated at the synaptic interface, which was quantified as the fluorescence ratio between the bead and the whole cell (Figures 1A,B), to the proteasome (Ibañez-Vega et al, 2019a)
The accumulation of Ecm29 at the centrosome of B cells was verified by immunoblot of centrosome-rich fractions, where we found that Ecm29 cofractionated with γ-tubulin, a centrosome marker (Supplementary Figure 1A)
Summary
The interaction of the B cell receptor (BCR) with membrane-tethered antigens (mAg) initiates the formation of an immune synapse (IS), characterized by rapid cortical actin cytoskeleton rearrangements and the formation of BCR-microclusters containing signaling molecules that elicit B cell activation (Yuseff et al, 2013; Kwak et al, 2019). The lateral diffusion of the BCR is restricted by the cortical actin network, which becomes disassembled upon antigen engagement, enabling BCR lateral diffusion and subsequent clustering to promote downstream signaling (Mattila et al, 2016; Tolar, 2017; Freeman et al, 2018). During this phase, B cells exert a rapid spreading response by forming lamellipodia to expand the contact area with the antigenpresenting surface, thereby increasing the number of BCRs coupled to mAg (Fleire et al, 2006). Exocytic events that occur at the synaptic membrane rely on cortical actin cytoskeleton remodeling, where local actin depletion at the synaptic membrane is required for secretion of lytic granules in NK and cytotoxic T cells (Ritter et al, 2015; Gil-Krzewska et al, 2017)
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