Missing-in-Metastasis/Metastasis Suppressor 1 Regulates B Cell Receptor Signaling, B Cell Metabolic Potential, and T Cell-Independent Immune Responses

Sarapulov, Alexey V; Petrov, Petar; Hernández-Pérez, Sara; Šuštar, Vid; Kuokkanen, Elina; Cords, Lena; Samuel, Rufus V. M.; Vainio, Marika; Fritzsche, Marco; Carrasco, Yolanda R.; Mattila, Pieta K.

doi:10.3389/fimmu.2020.00599

Cited by 10 publications

(5 citation statements)

References 66 publications

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“…Lysates (450.000 primary cells/25 µl) were run on 10% polyacrylamide gels and transferred to PVDF membranes (Trans-Blot Turbo Transfer System, BioRad). Membranes were blocked with 5% milk in TBST (TBS with 0.05% Tween-20) for 1 h and incubated with primary antibodies in 5% BSA in TBST O/N at 4°C ( Sarapulov et al, 2020 ). Secondary antibody incubations (1:20.000) were done for 1 h at RT in 5% milk in TBST using HRP-conjugated secondary antibodies.…”

Section: Methodsmentioning

confidence: 99%

The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentation

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Tejeda-González

Cunha

et al. 2022

Front. Cell Dev. Biol.

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In order to fulfil the special requirements of antigen-specific activation and communication with other immune cells, B lymphocytes require finely regulated endosomal vesicle trafficking. How the endosomal machinery is regulated in B cells remains largely unexplored. In our previous proximity proteomic screen, we identified the SNARE protein Vti1b as one of the strongest candidates getting accumulated to the sites of early BCR activation. In this report, we follow up on this finding and investigate the localisation and function of Vti1b in B cells. We found that GFP-fused Vti1b was concentrated at the Golgi complex, around the MTOC, as well as in the Rab7+ lysosomal vesicles in the cell periphery. Upon BCR activation with soluble antigen, Vti1b showed partial localization to the internalized antigen vesicles, especially in the periphery of the cell. Moreover, upon BCR activation using surface-bound antigen, Vti1b polarised to the immunological synapse, colocalising with the Golgi complex, and with lysosomes at actin foci. To test for a functional role of Vti1b in early B cell activation, we used primary B cells isolated from Vit1b-deficient mouse. However, we found no functional defects in BCR signalling, immunological synapse formation, or processing and presentation of the internalized antigen, suggesting that the loss of Vti1b in B cells could be compensated by its close homologue Vti1a or other SNAREs.

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Section: Methodsmentioning

confidence: 99%

The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentation

Music

Tejeda-González

Cunha

et al. 2022

Front. Cell Dev. Biol.

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“…After which cells were lysed with 2× SDS PAGE loading buffer, sonicated and subjected to SDS PAGE. The blots were processed as described in Sarapulov et al (2020) and probed with streptavidin–HRP (Life Technologies, #S-911).…”

Section: Methodsmentioning

confidence: 99%

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

Awoniyi

Cunha

Sarapulov

et al. 2023

Journal of Cell Science

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Successful B cell activation, critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodelling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through Akt and MAPK axes.

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“…Lysates were sonicated and subjected to SDS PAGE. The blots were processed as described in ( Sarapulov et al ., 2020). Briefly, the blots were probed with antibodies against phosphorylated forms of proteins, stripped and reprobed with antibodies against total corresponding proteins.…”

Section: Methodsmentioning

confidence: 99%

BCR-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

Awoniyi

Runsala

Šuštar

et al. 2020

Preprint

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B lymphocytes form a central part of the adaptive immune system, helping to clear infections by mounting antibody responses and immunological memory. B cell activation is critically controlled by a specific antigen receptor, the B cell receptor (BCR), which triggers a complex, multibranched signaling cascade initiating various cellular changes. While parts of these pathways are reasonably well characterized, we still lack a comprehensive protein-level view of the very dynamic and robust cellular response triggered by antigen engagement. Ability to track, with sufficient kinetic resolution, the protein machineries responding to BCR signaling is imperative to provide new understanding into this complex cell activation event. We address this challenge by using APEX2 proximity labeling technique, that allows capture a major fraction of proteins in a given location with 20nm range and 1min time window, and target the APEX2 enzyme to the plasma membrane lipid raft domain, where BCR efficiently translocates upon activation. Our data provides unprecedented insights into the protein composition of lipid raft environment in B cells, and the changes triggered there upon BCR cross-linking and translocation. In total, we identified 1677 proteins locating at the vicinity of lipid raft domains in cultured mouse B cells. The data includes a majority of proteins known to be involved in proximal BCR signaling. Interestingly, our differential enrichment analysis identified various proteins that underwent dynamic changes in their localization but that had no previously known linkage to early B cell activation. As expected, we also identified, for example, a wealth of proteins linked to clathrin-mediated endocytosis that were recruited to the lipid rafts upon cell activation. We believe that his data serves as a valuable record of proteins involved in BCR activation response and aid various future studies in the field.

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Missing-in-Metastasis/Metastasis Suppressor 1 Regulates B Cell Receptor Signaling, B Cell Metabolic Potential, and T Cell-Independent Immune Responses

Cited by 10 publications

References 66 publications

The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentation

The SNARE protein Vti1b is recruited to the sites of BCR activation but is redundant for antigen internalisation, processing and presentation

B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

BCR-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics

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