Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Ibañez-Vega, Jorge; Fuentes, Danitza; Lagos, Jonathan; Cancino, Jorge; Yuseff, María Isabel

doi:10.3791/59621

Cited by 5 publications

(6 citation statements)

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“…The previously characterized BCR activation linked proteins were found largely exclusively in the IS samples or showed enrichment over the control samples, well validating the approach. The notable proteins networks found in the IS samples included, for instance, cytoskeletal regulators, vesicle transport mediators and proteasome complex, all with previously reported roles in B cell activation (De Wet et al, 2011;Ibañez-Vega et al, 2019;Kuokkanen et al, 2015;Tolar, 2017). Interestingly, we also found some large protein networks without previously characterized roles in the IS, like the spliceosome and ribosomal complexes.…”

Section: Discussionsupporting

confidence: 82%

Proteomic profiling of isolated immune synapses from primary mouse B cells

Cunha

Hernández-Pérez

Awoniyi

et al. 2023

Preprint

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The immune synapse (IS) is a cell-cell interaction platform critical in lymphocyte activation by specific antigens. Despite of B cells being able to also respond to soluble antigens, in particular the in vivo importance of the IS and surface-tethered antigen recognition has strongly emerged in the recent years. The IS serves as a dynamic hub for multiple cellular actions but the molecular details of these functions, especially beyond the B cell antigen receptor (BCR) signalling, remain poorly understood. Here, to address the lack in the systems level understanding of the IS, we setup methodology for comprehensive investigation of the composition of the primary mouse B cells' IS at proteome level. Utilizing functionalized magnetic beads to mimic antigen presenting cells and trigger IS formation on them, we developed a method to specifically and robustly extract the cell adhesions on the beads, namely the IS or transferrin receptor mediated adhesion as a control. Our data revealed 661 proteins exclusively present in the IS at 15 minutes after BCR engagement, 13 exclusively in the control adhesions and 365 proteins shared between the samples. We got strong coverage of the known components of the IS as well as identified a plethora of unknown proteins and functional pathways with hitherto unknown roles in B cell IS. Thus, in this work, we validated the IS isolation method as a valuable tool to study early B cell activation by surface-bound antigens as well as unveil several novel proteins and pathways suggestive of new functional aspects in the IS.

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Section: Discussionsupporting

confidence: 82%

Proteomic profiling of isolated immune synapses from primary mouse B cells

Cunha

Hernández-Pérez

Awoniyi

et al. 2023

Preprint

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“…To this end, B cells were pretreated for 24 h with TLR ligands and stained for Lamp1+ lysosomes after incubation with activating BCR ligand+ beads for 60 min. The recruitment of lysosomes was evaluated by measuring the fluorescence of LAMP1 accumulated in the bead, as previously described [ 40 ]. Our results show that B cells treated with TLR ligands recruit fewer lysosomes to the IS upon BCR stimulation ( Figure 1 C).…”

Section: Resultsmentioning

confidence: 99%

Autophagy Induced by Toll-like Receptor Ligands Regulates Antigen Extraction and Presentation by B Cells

Lagos

Sagadiev

Díaz

et al. 2022

Cells

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The engagement of B cells with surface-tethered antigens triggers the formation of an immune synapse (IS), where the local secretion of lysosomes can facilitate antigen uptake. Lysosomes intersect with other intracellular processes, such as Toll-like Receptor (TLR) signaling and autophagy coordinating immune responses. However, the crosstalk between these processes and antigen presentation remains unclear. Here, we show that TLR stimulation induces autophagy in B cells and decreases their capacity to extract and present immobilized antigens. We reveal that TLR stimulation restricts lysosome repositioning to the IS by triggering autophagy-dependent degradation of GEF-H1, a Rho GTP exchange factor required for stable lysosome recruitment at the synaptic membrane. GEF-H1 degradation is not observed in B cells that lack αV integrins and are deficient in TLR-induced autophagy. Accordingly, these cells show efficient antigen extraction in the presence of TLR stimulation, confirming the role of TLR-induced autophagy in limiting antigen extraction. Overall, our results suggest that resources associated with autophagy regulate TLR and BCR-dependent functions, which can finetune antigen uptake by B cells. This work helps to understand the mechanisms by which B cells are activated by surface-tethered antigens in contexts of subjacent inflammation before antigen recognition, such as sepsis.

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“…Image processing and analysis were performed with FIJI (ImageJ) software ( Schindelin et al, 2012 ), as we previously described ( Ibañez-Vega et al, 2019b ). The centrosome was labeled with Centrin-GFP or α-Tubulin and determined by the brightest point where microtubules converged.…”

Section: Methodsmentioning

confidence: 99%

Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse

Ibañez-Vega

Valle

Sáez

et al. 2021

Front. Cell Dev. Biol.

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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.

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Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Cited by 5 publications

References 0 publications

Proteomic profiling of isolated immune synapses from primary mouse B cells

Proteomic profiling of isolated immune synapses from primary mouse B cells

Autophagy Induced by Toll-like Receptor Ligands Regulates Antigen Extraction and Presentation by B Cells

Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse

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