How does the phosphorylation status of PLCγ1 (Tyr783) regulate phase separation in T cell signaling pathways?

1. What is the formation and regulatory mechanism of immune receptor signaling microclusters?

When immune cells are activated, receptors on the cell surface (such as TCR, BCR, CAR, etc.) and their downstream signaling molecules undergo dynamic self-assembly, forming nano- to micro-scale microclusters enriched with various signaling proteins. These microclusters are the core platforms for signal transduction, and their formation mechanisms directly affect the strength and specificity of immune responses. Recent studies have revealed that liquid-liquid phase separation (LLPS) of biomacromolecules is an important physicochemical principle driving the assembly of such subcellular structures. In T cells, the key adaptor protein LAT (Linker for Activation of T cells) can undergo phase separation after phosphorylation, thereby promoting the aggregation of signaling complexes and the activation of downstream pathways. However, how LAT phase separation is precisely regulated, particularly the roles played by other signaling molecules, remains a critical question in the field.

2. What is the non-canonical function of PLCγ1 in T cell signaling?

Phospholipase Cγ1 (PLCγ1) is a core enzyme in the TCR signaling pathway. Its canonical function is to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) after being recruited to phosphorylated LAT at the membrane, producing the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG), which initiate calcium influx and protein kinase C (PKC) activation, respectively, and are essential for full T cell activation. However, recent studies have discovered that beyond this canonical catalytic function, PLCγ1 also plays a novel role in regulating upstream signal assembly, independent of its enzymatic activity.

Research shows that PLCγ1 can significantly promote the phase separation of LAT protein and the stability of microclusters. This effect does not depend on its ability to hydrolyze PIP2 but is achieved through interactions between its protein domains. PLCγ1 contains two tandem SH2 domains and one SH3 domain, endowing it with the potential to act as a "molecular crosslinker."

3. By what molecular mechanism does PLCγ1 promote LAT phase separation?

1. Acting as a molecular crosslinker to stabilize microcluster structure: The two SH2 domains of PLCγ1 can simultaneously recognize and bind multiple phosphorylated LAT molecules. Through this "multivalent binding" effect, PLCγ1 effectively crosslinks discrete LAT molecules, promoting local concentration of LAT and thereby stabilizing the signal microcluster structure formed by LAT phase separation, preventing excessive diffusion or disintegration.

2. Protecting LAT from dephosphorylation: The protein tyrosine phosphatase CD45 on the plasma membrane can broadly dephosphorylate signaling molecules, including LAT, thereby negatively regulating TCR signaling. Studies have found that after PLCγ1 binds to phosphorylated LAT, its physical presence may create steric hindrance for CD45 or alter the conformational state of LAT, thus protecting key phosphorylated tyrosine residues on LAT from rapid removal by CD45. This maintains LAT's activated state, further promoting its sustained phase separation capability.

3. Concentration-dependent "bidirectional" regulation: Interestingly, PLCγ1's promotion of LAT phase separation exhibits precise concentration dependence. At physiologically relevant intermediate concentrations, its crosslinking effect is optimized, effectively promoting phase separation. However, when PLCγ1 concentration is too high, it instead inhibits the formation of large condensates. Computer simulation analysis provides a plausible explanation for this phenomenon: excess PLCγ1 molecules may act as "terminal nodes" in the microcluster network, occupying too many binding sites without effectively increasing internal connections in the crosslinked network, thereby hindering the growth of larger, more stable condensate structures. This reflects the sensitive regulation of phase separation systems to component concentrations.

4. What is the research value of the PLCγ1 (Tyr783) phosphorylation antibody?

PLCγ1's own activity is strictly regulated by its phosphorylation state. During T cell activation, PLCγ1 is phosphorylated at multiple tyrosine sites (e.g., Tyr771, Tyr783), and these phosphorylation events are crucial for its membrane localization and full enzymatic activity. Among these, phosphorylation at the Tyr783 site is a key marker of its activation.

In this context, specific tools such as recombinant rabbit monoclonal antibodies against PLCγ1 (Tyr783) hold significant research value:

1. Precise analysis of activation kinetics: This antibody can be used to accurately measure the timing and intensity of PLCγ1 phosphorylation at the Tyr783 site after T cell stimulation. This helps correlate the time course of PLCγ1's canonical catalytic activity activation with the functional time window of its role as a "phase separation regulator."

2. Exploring functional correlations: By comparing the spatial distribution of phosphorylated PLCγ1 (p-Tyr783) in cells with the localization of LAT microclusters/condensates, it is possible to visually verify whether phosphorylated PLCγ1 is indeed recruited to the phase-separated LAT signaling platform, thereby spatially linking its activated state with its phase separation regulatory function.

3. Revealing regulatory networks: Using this tool, researchers can study how other kinases, phosphatases, or signaling molecules indirectly regulate LAT phase separation and overall T cell signal strength by influencing the phosphorylation state of PLCγ1 at Tyr783.

4. Drug effect evaluation: When screening or evaluating drugs targeting T cell signaling pathways (such as immune checkpoint inhibitors or CAR-T optimization strategies), detecting PLCγ1 p-Tyr783 levels can serve as an important downstream readout for assessing TCR signal activation and effectiveness.

5. What implications does this discovery have for understanding other signaling pathways?

PLCγ1 is not only a key node in TCR signaling but also plays a central role in B cell receptor (BCR), Fc receptor, and various growth factor receptor (e.g., EGFR, FGFR) signaling pathways. Its dysfunction is associated with immunodeficiency, autoimmune diseases, and cancer, among other conditions.

This study's revelation of PLCγ1's non-canonical function as a phase separation regulator raises a universal new question: Does PLCγ1, in other receptor signaling pathways, similarly participate in regulating the phase separation assembly of specific adaptor proteins or signaling complexes? For example, under growth factor stimulation, could PLCγ1 also promote the phase separation of its corresponding adaptor proteins, thereby amplifying pro-proliferative signals? This provides a new perspective for understanding the common organizational principles of multiple receptor signal transductions. At the same time, it suggests that drug design targeting PLCγ1's protein-protein interaction interfaces (such as its SH2 domains) rather than its catalytic pocket may become a potential new strategy for regulating specific signaling pathways.

6. Which manufacturers provide recombinant rabbit monoclonal antibodies against PLCγ1 (Tyr783)?

Hangzhou Start Biotech Co., Ltd. has independently developed the "Phospho-PLCγ1 (Tyr783) Recombinant Rabbit Monoclonal Antibody" (product name: Phospho-PLCγ1 (Tyr783) Recombinant Rabbit mAb (S-1184-12). This is a highly specific, sensitive, and stable tool for detecting the activity of key signaling enzymes. The product was developed using recombinant rabbit monoclonal antibody technology and has been rigorously validated across multiple platforms, including Western Blot (WB) and immunofluorescence (IF). It holds critical application value in growth factor signaling, immune receptor activation, calcium mobilization research, and other fields.

Professional technical support: We provide detailed product technical information, including examples of phosphorylation time courses under different growth factor stimulations, methods for co-using with total PLCγ1 antibodies to assess activation ratios, and professional technical consultation, fully assisting customers in achieving precise and reliable discoveries in the fields of cell signal transduction and immunology research.

Hangzhou Start Biotech Co., Ltd. is committed to providing high-quality, high-value biological reagents and solutions to global innovative pharmaceutical companies and research institutions. For more information about the "Phospho-PLCγ1 (Tyr783) Recombinant Rabbit Monoclonal Antibody" or to request sample testing, please contact us.

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