B Cell methods of FCRL1 localization to BCR complex

B Cells are a type of lymphocyte whose primary purpose is to generate highly specific antibodies in response to a foreign antigen. The B cell receptor (BCR) signaling pathway regulates B cell activation, and Fc receptor-like proteins may contribute to this pathway’s activation or inhibition. Previous studies have demonstrated that Fc receptor-like protein 1 (FCRL1) is found on B cell plasma membranes, associating with the BCR and enhancing the activation of the B cell. Also, growth factor receptor-bound 2 (Grb2) and Grb2-related adaptor protein (GRAP) are required to activate the extracellular signal regulated kinase pathway (ERK), which is necessary for proper B cell development in hematopoietic cells and for the formation of memory B cells. GRAP may function to recruit FCRL1 to the BCR, and therefore, determination of FCRL1 localization and function of GRAP within the localization cascade is a relevant and important field to further our understanding of B cell activation.

In all B cells, one of the main activating pathways of the BCR is the immunoreceptor tyrosine-based activation motif (ITAM), which leads to the cleavage of PIP2, resulting in a DAG/IP3 pathway that opens a ligand gated Ca2+ channel and culminates in an increase of intracellular Ca2+ concentrations. This concludes with B cell activation and transcriptional changes. However, in B cells with membrane bound IgE and IgG molecules, the immunoglobulin tail tyrosine (ITT) motif recruits growth factor receptor-bound 2 (Grb2), drastically amplifying second messenger production and skipping many of the earlier steps in the ITAM, leading directly to the DAG/IP3 pathway. GRAP and Grb2 are also required to activate the extracellular signal regulated kinase (ERK) pathway through the ITAM in hematopoietic cells.

Figure 1

BCR activation leads to FCRL1 recruitment.

Project Aims

This project aims to answer two major questions, the first of which will be the main focus of this site:

Which part of the FCRL1 protein is responsible for its localization to the BCR?
Where does GRAP function in the FCRL1 localization cascade, if at all?

Over 300 primary immune defects were known as of 2018, with the most common types being those that affect B cells by either failing to properly develop or inhibiting their activation. It has been shown that FCRL1 and GRAP play important roles in B Cell maturation and activation, so by answering the questions above, we can further our understanding of B Cell activation and develop medicine to combat B Cell diseases.

Strategy

In order to determine which regions of FCRL1 are responsible for its localization to the BCR, two constructs must be produced. The first construct contains the extracellular domain and the transmembrane portion, and the second contains the cytoplasmic domain and the transmembrane portion.

The first of these constructs will be produced through a standard PCR (named H construct), while the second will be produced through a stitching PCR (named T construct). The constructs will be tagged with a FLAG tag.
Constructs will be cloned into a lentiviral vector.
- - The presence of the construct DNA insert will be confirmed with a restriction digestion.
The lentiviral vector will be integrated into the A20 mouse B cell line through transfection and transduction.
Flow Cytometry will check for FLAG expression.
Cell sorting will separate cells expressing the construct of interest from cells that are not.
Cells expressing the construct of interest will be activated, stained, and viewed under confocal microscopy.

The image shows the construct design of FCRL1. The left side of the image shows a full wild type FCRL1, while the right shows the "H construct" consisting of the extracellular domain and transmembrane portion, and the "T construct" containing the cytoplasmic domain and the transmembrane portion.

Figure 2

Relevant portions of FCRL1 will be amplified through PCR to produce the two constructs of interest. These include the extracellular head with the transmembrane region and the cytoplasmic tail with the transmembrane region.

Hypothesis and Rationale

The hypothesis for this project is as follows:

If one region of FCRL1 is responsible for its localization to the BCR, then the cytoplasmic tail is primarily responsible for this localization.

Recent findings from our lab group (Rut Tallon, graduate student) suggest that Grb2 binds at the second tyrosine motif found on the cytoplasmic tail region of FCRL1. This points toward the cytoplasmic tail as being the region responsible for its localization to the BCR. However, ligands for mouse FCRL1 have not been identified, and so we cannot rule out the possibility that molecules interact with the extracellular region of FCRL1 that play a role in its localization.

Early Results

Both PCRs to generate the constructs of interest were successful.

Construct was successfully cloned into lentiviral vector, as confirmed with gel analysis.

A transient transfection followed by a flow cytometry was done to check for FLAG expression in 293T cells. The results were successful.

A gel confirming the FCRL1 construct inserts. More information can be found in the adjacent text box.

Figure 3

Gel analysis confirming inserts. Inserts were cut with XbaI and SalI.

Lane 1: Ladder

Lane 2: Uncut vector control

Lane 3: Lentiviral vector with GFP

Lane 4: Lentiviral vector with head insert

Lane 5: Lentiviral vector with tail insert

The GFP fragment is expected to be at 754 bp. The head construct is expected to be at 798 bp, but contains an XbaI site within the construct, cutting it into two fragments of size 390 and 408. The tail fragment is expected to be at 510 bp. All of these are found where expected.

Figure 4

Flow cytometry results. From the singlets, live cell populations were selected, and then FLAG expression was confirmed in both samples.

Next Steps

Currently in the process of completing a transduction to the A20 cell line.
Cells will be sorted and viewed under confocal microscopy.

NACE Career Readiness Requirements

From the National Association for Colleges and Employers,

What Is Career Readiness?

Career readiness is a foundation from which to demonstrate requisite core competencies that broadly prepare the college educated for success in the workplace and lifelong career management.

For new college graduates, career readiness is key to ensuring successful entrance into the workforce. Career readiness is the foundation upon which a successful career is launched. Career readiness is, quite simply, the new career currency.

For higher education, career readiness provides a framework for addressing career-related goals and outcomes of curricular and extracurricular activities, regardless of the student’s field of study. For employers, career readiness plays an important role in sourcing talent, providing a means of identifying key skills and abilities across all job functions; similarly, career readiness offers employers a framework for developing talent through internship and other experiential education programs.

Of the Eight Career Readiness Competencies, I have chosen to talk about Career & Self Development, Communication, and Teamwork.

Career & Self Development

As someone who wants to continue their career in academia, research has been the perfect launching point for me to prepare for graduate school. I have been able to travel to conferences, take additional trainings, and write on my own project(s), which has helped me further prepare for what academia has to offer.

Communication

From communicating with other students in the lab to the professional setting of a conference, being able to effectively communicate is vital to the research process. In addition, being able to write abstracts and make effective posters also teaches proper communication, as well as skills to identify and present effectively to a wide range of audiences.

Teamwork

Although projects may be individualized, it is crucial to work together as a team in the lab. Especially as an undergraduate, I cannot do everything alone, and I often lean on the guidance of graduate students in the lab. Running samples together to minimize supply waste, sharing progress at lab meetings, and graduate students helping me run machines I am not allowed to use are all examples of how we plan around each other and work together effectively as a team.

Acknowledgements

Special thanks to the NIH grant R15-AI174170, the Beckman Foundation, and the Wilson lab group.

References

Vanshylla, Kanika & Bartsch, Caren & Hitzing, Christoffer & Krümpelmann, Laura & Wienands, Jürgen & Engels, Niklas. (2018). “Grb2 and GRAP connect the B cell antigen receptor to Erk MAP kinase activation in human B cells.” Scientific Reports. 8. 10.1038/s41598-018-22544-x.

DeLuca, Jenna M., et al. “FCRL1 Regulates BCR-Induced ERK Activation through GRB2 - PMC.” PubMed Central (PMC), National Center for Biotechnology Information (NCBI), 25 Oct. 2021, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8629370/.

Zhao, Xingwang, et al. “Fc receptor–like 1 intrinsically recruits C-ABL to enhance B cell activation and function.” Science Advances, vol. 5, no. 7, 5 July 2019, https://doi.org/10.1126/sciadv.aaw0315.

American Type Culture Collection “A20” ATCC, https://www.atcc.org/products/tib-208 [5] Mamidi, Huang, Honjo, Li, Tabengwa, et al. 25 September 2023, “FCRL1 Immunoregulation in B Cell Development and Malignancy.” Frontiers, https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1251127/f ull

“CRISPR Plasmids: Nicks” AddGene, https://www.addgene.org/crispr/nick/

Smith, Tukisa, et al. “ Primary B Cell Immunodeficiencies” PubMed Central, 22 October 2018, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395616/

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