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First published online 19 December 2006
doi: 10.1242/jcs.03328

Journal of Cell Science 120, 219-228 (2007)
Published by The Company of Biologists 2007
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Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration

Agnieszka Masztalerz1,*, Ingrid S. Zeelenberg1,*, Yvonne M. Wijnands1, Rosalie de Bruijn1, Angelika M. Drager2, Hans Janssen1 and Ed Roos1,{ddagger}

1 Division of Cell Biology, The Netherlands Cancer Institute, 121 Plesmanlaan, 1066CX Amsterdam, The Netherlands
2 Department of Hematology, VU University Medical Center, 1081HV Amsterdam, The Netherlands


Figure 1
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  		Fig. 1. Expression of synaptotagmins. (A) RT-PCR of synaptotagmins 1–11 on cDNA from mouse brain (B), TAM2D2 cells (T), or negative control (water added instead of cDNA; c). (B) RT-PCR of Syt3 on cDNA from murine T cells, TAM2D2 cells, mouse brain and negative control.

 

Figure 2
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  		Fig. 2. The SYT1 C2B domain inhibits migration and invasion by TAM2D2 cells. (A) FACS analysis of GFP in the C2B transfectants. Filled areas represent the fluorescence in the transfectants; open areas, the fluorescence in the untransfected control cells. C2B(h), cells sorted for high GFP; C2B(m), sorted for medium GFP levels. (B) Migration towards 100 ng/ml CXCL12. Results are given as the mean ± s.e.m. of the migration index, which is set to 1 for the control cells in each individual experiment. Shown are averages of 14 experiments. (C) Invasion into REF monolayers. Results are given as the mean ± s.e.m. of the invasion index, which is set to 1 for the control cells in each individual experiment. Shown are averages of eight experiments. In the absence of CXCL12, no migration of TAM2D2 cells was observed at all.

 

Figure 3
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  		Fig. 3. Synaptotagmin 3 downregulation by antisense transfection impairs migration. Western blot analysis of SYT3 protein levels in Syt3 transfectants (S, TAM2D2-Syt3), antisense Syt3 transfectants (AS, TAM2D2-anti-sense SYT3), and untransfected cells (T, TAM2D2). After SYT3 staining, blots were stripped and re-probed with actin antibody. Migration assay shows migration towards 100 ng/ml CXCL12, as in Fig. 2B. Data are averages of three experiments. Shown are results shortly after transfection (i.e. after sorting of GFP-high cells and growing of sufficient cells) and 1 month later, in two separate experiments (A and B). Experiments were done with separate sets of cells, transfected on two different occasions.

 

Figure 4
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  		Fig. 4. CXCL12 stimulation induces actin polymerization, associated with local CXCR4 accumulation, in TAM2D2 cells but not in C2B transfectants. Beads coated with fibronectin and CXCL12 (arrows) were allowed to adhere to cells on coverslips, fixed after 10 minutes and stained with (A) phalloidin (for actin) or (B) CXCR4 antibody. At least 30 cells per condition were analyzed using confocal microscopy. Beads coated with fibronectin but not CXCL12 were used as a negative control and did not have an effect on TAM2D2 cells (Fig. 4C). Experiments were repeated three times.

 

Figure 5
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  		Fig. 5. Adhesion of TAM2D2 cells and C2B transfectants to rat embryo fibroblasts (REF) monolayers under flow conditions in the absence or presence of the CXCR4-blocking peptide TC14012. In the latter case, TAM2D2 cells and REF monolayers were both pre-incubated with the peptide. Results are given as the mean ± s.e.m. of the adhesion index, which is set to 1 for the control cells in each individual experiment. Shown are averages of four experiments.

 

Figure 6
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  		Fig. 6. Synaptotagmin 3 localization in TAM2D2 cells. (A) TAM2D2-SYT3-GFP transfectants and (B) TAM2D2 cells were stained with SYT3 antibody and observed using confocal microscopy. Three cells are shown. (C) TAM2D2-SYT3-GFP transfectants were stained with GFP antibody bound to the gold particles and observed with the electron microscope. (Right panel) Magnified view of a multi-vesicular body. (D) Number of gold particles per µm2 in MVBs, cytosol, mitochondria, perimembrane area and Golgi apparatus. Images of 25 cells were examined.

 

Figure 7
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  		Fig. 7. CXCR4 levels, recycling and migration. (A) Western blot analysis of CXCR4 protein levels in TAM2D2 cells and C2B transfectants. After CXCR4 staining, blots were stripped and reprobed with actin antibody. (B), AMFR (autocrine motility factor receptor), IGF1R (insulin-like growth factor 1 receptor) and LFA-1 (aLb2 integrin) in TAM2D2 cells and C2B transfectants. (C) Cell surface levels of CXCR4 in TAM2D2 cells and C2B and C2B/CXCR4-GFP transfectants. (D) Migration towards 100 ng/ml CXCL12. Results are given as the mean ± s.e.m. of the migration index, which is set to 1 for the control cells in each individual experiment. Shown are averages of three experiments. (E) Recycling of CXCR4 in TAM2D2 cells and C2B transfectants. Cells were stained with CXCR4 antibody before or after 2 hours treatment with 100 ng/ml CXCL12; or after CXCL12 treatment and subsequent overnight recovery in medium without CXCL12, supplemented with cycloheximide. The experiment was repeated three times; representative results of one experiment are shown. (F) CXCR4 levels in TAM2D2 cells, assessed by western blotting at 2 and 4 hours in the presence of cycloheximide, after treatment with 100 ng/ml CXCL12, in both TAM2D2 cells and C2B transfectants. Staining with actin antibody was used as loading control.

 

Figure 8
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  		Fig. 8. TAM2D2 cells, and SYT3-GFP and C2B/SYT3-GFP transfectants were stained with antibodies to (A) EEA1 or both SYT3 and EEA1; (B) M6PR or both SYT3 and M6PR; (C) LAMP1 or both SYT3 and LAMP1, visualized with Cy5-labeled secondary antibodies; or (D) both CXCR4 and LAMP1 and Alexa Fluor 568- and Cy5-labeled secondary antibodies, respectively, and observed by confocal microscopy. (E) CXCR4-GFP and C2B/CXCR4-GFP transfectants were stained with GFP antibody and examined by electron microscopy.

 




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