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Cell Dynamics Group

Frequency and duration of intracellular signaling are associated with distinct biological responses. A major focus of the Cell Dynamics Group is to understand how dynamic control of Ca2+ and PI3K signaling determine negative B-cell selection. We are using optogenetic approaches to examine the mechanisms by which B cells can interpret differences in: (i) frequency of Ca2+ oscillations; and (ii) duration of PI3K activation.

(i) Regulation of Ca2+ oscillations as determinant of central tolerance and B-cell selection
In B cells, SYK is the most proximal tyrosine kinase that initiates BCR signaling (Beitz et al., 1999) to activate diverging downstream pathways towards Ca2+-NFAT signaling via BLNK (Baba et al., 2001), or Src kinase signaling via phosphorylation of CD19 and VAV1 (Okada et al., 2000). Instead of SYK, T cells express the highly homologous tyrosine kinase ZAP70, which initiates proximal TCR signaling in T cells. Our group has recently discovered that aberrant ZAP70 expression contributes to B-cell leukemogenesis by subverting SYK-dependent mechanisms of negative B-cell selection (Sadras et al., 2021).
As summarized in Figure 1, our BioID interactome and functional assays showed that ZAP70 competes with SYK and exerts a selective dominant-negative effect on SYK-dependent Ca2+-NFAT signaling, but not other substrates (e.g. CD19, VAV1). By occupying BLNK and preventing its phosphorylation, ZAP70 dramatically reduces Ca2+ release and Ca2+ oscillations. Fast oscillations in the sole presence of SYK (>15 mHz) are decoded by NFAT and trigger central tolerance and cell death (Peng et al., 2001, Barrington et al., 2006; Märklin et al., 2017). In contrast, slow Ca2+ oscillations in the presence of ZAP70 (<5 mHz) promote activation of NF-κB and B-cell survival (Dolmetsch et al., 1998).
To study the consequences of experimental frequency modulation of Ca2+ oscillations in B cells, we have engineered murine B cells and patient-derived B-ALL cells with a LOV2-based optogenetic tool termed light-operated Ca2+ channel (LOCa) (He et al., 2021) to control Ca2+ oscillations (Figure 1). While slow Ca2+ oscillations (0.5 mHz) did not impact viability, fast Ca2+ oscillations (50 mHz) induced cell death. To identify the changes in signal transduction underpinning central tolerance mechanisms in normal and malignant B cells, we will perform global and phospho-Y and S/T-proteomic analyses established in our laboratory.

(ii) Central tolerance mechanisms sense pathological B cells based on abnormal signaling patterns

Normal antigen encounter of B cells results in a short transient PI3K signal, which is distinct from persistent activation of PI3K signaling in the context of pathological BCR signaling (autoreactive BCR or transforming oncogene). We test the hypothesis that central tolerance mechanisms sense and eliminate (pre-) malignant B-cells based on protracted activation of the PI3K pathway.

In collaboration with Derek Toomre (Yale), we engineered human B-ALL and mantle cell lymphoma (MCL) cells with Opto-PI3K consisting of the light-responsive Arabidopsis thaliana transcription factor CIB1 and cryptochrome 2 (CRY2) fused to the inner SH2 (iSH2) region of the p85α regulatory subunit of PI3K (Idevall-Hagren et al., 2012). Exposure to blue light pulse induces a conformational change of CRY2 and its interaction with the membrane anchored N-terminus of CIB1 (CIBN). This interaction results in membrane-recruitment of the CRY2-iSH2 that binds the endogenous p110α catalytic subunit of PI3K, and eventually transient PI3K-activation (Figure 3).

While a single transient pulse of PI3K activity (to mimic normal antigen encounter) has minimal effects on cell size and did not impact viability, repetitive activation (to mimic pathological signaling) increased cell size and induced cell death. We will study biophysical aspects of PI3K signaling and cell mass accumulation in B-ALL, MCL and CLL cells (collaboration with Scott Manalis, MIT). Employing metabolomic and proteomic approaches established in our laboratory, we examine how dynamic control of PI3K signaling determine energy abundance, cell size and B-cell survival. To identify the changes in signal transduction underpinning central tolerance mechanisms in MCL cells, we will perform global and phospho-Y and S/T-proteomic analyses.