3.3.1 Generation of a TALEN-mediated CIN85/SLP65 double-deficient DG75 sub-line
As the SLP65 M23 variant does not only circumvent binding to CIN85 but also to its homolog CD2AP, the exclusive impact of CIN85 on SLP65 cannot be determined. In addition, it has to be noted that there might be further interactors of both proline-arginine motifs that influence SLP65 function in the here performed experimental systems.
Figure 3.8: Generation of CIN85/SLP65 double-deficient DG75 cells. A) To test the activity of the TALEN constructs, the targeted exon 6 region of TALEN-treated CIN85-deficient DG75 cells and DG75 control cells was amplified by PCR and a BmrI restriction was performed. The BmrI binding site overlaps with the TALEN cutting site and hence allows for activity test of the TALEN constructs. B) Cleared cellular lysates of DG75 parental (par.), SLP65-deficient (SLP65-/-) and DKO cell clones #1, #2, #3, #4, #5, #6, #7 and #8 were subjected to SDS-PAGE and the immunoblot was probed with anti-SLP65 and anti-Actin antibodies. Cell clone
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#6 was used in the following experiments (marked in red). The respective molecular weight of marker proteins (in kDa) is indicated on the left.
To investigate the relevance of the CIN85-SLP65 interaction for SLP65 function, a DG75 sub-line deficient for CIN85 and SLP65 was generated. Therefore, I applied the TALEN method to target SLP65 in the CIN85-deficient DG75 cell clone #34. The TALEN constructs targeting exon 6 of the human SLP65 gene were kindly provided by Christoffer Hitzing. Transient transfection of TALEN constructs, cell sorting and subsequent activity test (figure 3.8.A) were performed as described in chapter 3.2.1. The cells were consecutively subcloned and cleared cellular lysates were prepared to test for loss of SLP65 expression. As shown in figure 3.8.B, efficiency of the TALEN constructs was very high as almost every tested cell clone lacked SLP65 expression. The CIN85/SLP65 double-deficient DG75 cell clone #6 was used in all following experiments and is hereafter referred to as DKO.
To investigate whether the CIN85 enhancing function in Ca2+ signaling depends exclusively on the support of SLP65, I compared DKO DG75 cells with SLP65-deficient cells reconstituted with either SLP65wt or EGFP in Ca2+ mobilization experiments (figure 3.9.A). The DKO cells had a reduced Ca2+ signaling that matched the phenotype of the sole SLP65-deficient cells expressing EGFP. This indicates that SLP65 deficiency already mediates a severe phenotype in terms of Ca2+ mobilization, which is not further impaired by an additional loss of CIN85.
Next, reconstitution experiments were performed to prove that the observed effects for the DKO (figure 3.9.A) are based on the absence of CIN85 and SLP65 and are no unwanted site effects. Ca2+ mobilization experiments with these cells revealed that SLP65wt expressing DKO cells were capable of mediating more Ca2+ influx compared to DKO cells, but it was less compared to SLP65-deficient cells expressing SLP65wt (figure 3.9.B, C). In contrast, expression of CIN85wt in the DKO DG75 cells did not restore the diminished Ca2+ flux, confirming the supporting function of CIN85 for its constitutive binding partner SLP65 in this context.
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Figure 3.9: CIN85 and SLP65 act in concert to enhance BCR-induced Ca2+ mobilization. A) The DKO DG75 cell clone #6 and SLP65-deficient DG75 cells expressing CitSLP65wt or EGFP were subjected to Ca2+
flux measurements upon BCR stimulation with 10 µg/ml (upper panel) and 1 µg/ml (lower panel) as before. B) The DKO cells were reconstituted with citrine-tagged wild-type SLP65 (CIN85-/- SLP65-/- CitSLP65wt) or wild-type CIN85 (CIN85-/- SLP65-/- CitCIN85wt). Expression of analyzed proteins was tested by flow cytometry. C) Ca2+ mobilization measurement of DKO cells reconstituted with either CitSLP65wt or CitCIN85wt, and SLP65 -/-CitSLP65wt cells after BCR crosslinking with 1 µg/ml of α-IgM was done as before.
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3.3.2 Plasma membrane recruitment of SLP65 is supported by CIN85
I could show that CIN85 supports SLP65 function in the context of BCR-induced Ca2+
signaling in the human DG75 B cell line. Hence, I tested whether it also influences SLP65 membrane recruitment irrespective of CD2AP and other possible binding partners of both described proline-rich motifs (SLP65M23) in human DG75 cells. Therefore, reconstituted DKO cells with either SLP65wt or CIN85wt were used, which have the advantage that endogenous SLP65 and CIN85 proteins cannot interfere with the plasma membrane recruitment of ectopic expressed variants. At first, recruitment of citrine-tagged wild-type SLP65 expressed in either SLP65-deficient or DKO cells was compared. Citrine-tagged SLP65wt was rapidly (~2 min) translocated to the plasma membrane in SLP65-deficient DG75 cells like already shown in figure 3.7.C. In contrast, in DKO cells citrine-tagged SLP65 was recruited to the plasma membrane delayed in time (~5 min) (figure 3.10.B).
Hence, CIN85 had an accelerating function in terms of SLP65 membrane recruitment.
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Figure 3.10: CIN85 enhances SLP65 plasma membrane recruitment. A) DG75 DKO and SLP65-deficient DG75 cells were retrovirally transduced with constructs encoding citrine-tagged wild-type SLP65 and the expression was tested by flow cytometry. B) The analyzed cells in A) were subjected to confocal laser scanning microscopy and images were taken before as well as 2 min (2´) and 5 min (5´) after stimulation with 5 µg/ml anti-IgM F(ab´)2 fragments. C) CIN85-deficient DG75 and DG75 DKO cells expressing CitCIN85wt were analyzed as described in B) with the difference that stimulation was induced with 2 µg/ml anti-IgM F(ab´)2 fragments.
Vice versa, the impact of SLP65 on CIN85 plasma membrane recruitment was analyzed in DG75 DKO cells and compared to CIN85-deficient DG75 cells in figure 3.9.C. BCR stimulation induced rapid CIN85 plasma membrane recruitment with high amounts of antibody (10 µg/ml) in CIN85-deficient DG75 cells (compare figure 3.5.B). When stimulation was induced with reduced amounts of antibody (2 µg/ml), CIN85 was still recruited to the plasma membrane but delayed in time (figure 3.10.C). This decelerating effect was equalized when SLP65 was missing as in DKO cells expressing CIN85wt.
Here, CIN85 recruitment was visible shortly after BCR stimulation.
Taken together, CIN85 is a positive regulator ofSLP65 membrane recruitment whereas the presence of SLP65 seems to slow CIN85 recruitment down.