Tipo de investigación - Prácticas de laboratorio para la enseñanza aprendizaje de conceptos bás

3. METODOLOGÍA

3.1 Tipo de investigación

Hallmarks of BCR signaling are protein phosphorylation and an increase in cellular Ca2+

concentration upon BCR stimulation. BCR cross-‐linking in LCLs, generated from wt EBV infection, induced protein phosphorylation but not Ca2+_{-‐influx, whereas in}_Δ_LMP2A

LCLs, both processes were activated (Medele, 2010). Why LMP2A blocks Ca2+_{-‐influx but}

not protein phosphorylation is not known. Stable transfection of BJAB cells with a K1 expression plasmid decreased the surface IgM expression suggesting a downregulation of BCR signaling (Lee et al., 2000). Similarly, transient transfection of a K15 expression plasmid into BJAB cells and subsequent BCR cross-‐linking with an α-‐hIgM antibody

reduced the Ca2+_{-‐influx compared to vector transfected cells (Pietrek et al., 2010). Choi}

and colleagues transiently expressed a CD8-‐K15 chimera into BJAB cells, in which the cytoplasmic tail of K15 replaced the cytoplasmic tail of CD8. Cross-‐linking of the BCR with α-‐hIgM antibody slightly decreased tyrosine phosphorylation in Western blot

analysis compared to cells, which express CD8-‐Δ missing the K15 cytoplasmic tail (Choi

Results

Figure 5-23: The KSHV proteins K1 and K15 do not inhibit BCR signaling.

Cross-‐linking with an α-‐hIgG/M antibody can induce BCR signaling and phosphorylation of signaling

molecules, e.g. Syk and PLCγ2, or an influx of calcium into the cytoplasm. A) The increase of Syk and PLCγ2

phosphorylation after BCR stimulation was investigated in established LCLs, which were generated through infection of adenoid B cells with wt EBV, ΔLMP2A EBV, K1 EBV, or K15 EBV strains. An LCL clone,

which was generated from infection with wt EBV and unable to express a BCR (clone A16), was included as negative control. The BCR of the four LCLs was cross-‐linked for 10min with 25µg/ml α-‐hIgG/M

antibody or left untreated. Cells were fixed, permeabilized, and stained with phosphospecific α-‐pSyk-‐

Alexa647 or with α-‐pPLCγ2-‐Alexa647 antibodies. Histograms of flow cytometry analysis are shown.

Numbers next to the graphs display the mean fluorescent intensities for not cross-‐linked (blue) and cross-‐ linked (red) cells. Numbers below the graphs show the fold increases in mean fluorescent intensity of the pSyk-‐ and pPLCγ2-‐signals after BCR stimulation. BCR-‐_{wt EBV-‐infected LCLs (clone A16) did not show an}

increase in Syk and PLCγ2 phosphorylation after antibody addition, as expected. In LCLs generated from

wt (BCR+_),_Δ_{LMP2A EBV, K1 EBV, and K15 EBV strains, BCR cross-‐linking induced a significant increase in}

Syk and PLCγ2 phosphorylation indicating that neither LMP2A nor K1 and K15 inhibited phosphorylation

of BCR signaling molecules. B) The same LCLs were loaded with 3µM Indo-‐1 AM, and the ratio of Indo-‐1 in the violet vs. blue channel (y-‐axis) was measured over time (x-‐axis). After one minute of base line measurement the BCR was cross-‐linked with 25µg/ml α-‐hIgG/A/M F(ab)2-‐fragment. An influx of calcium

into the cytoplasm shifts the Indo-‐1 emission to the violet channel increasing the violet/blue ration. BCR-‐

wt EBV-‐infected LCLs (clone A16) did not show an increase in cytoplasmic calcium levels after antibody mediated BCR cross-‐linking, as expected. LCLs generated from wt but not ΔLMP2A EBV strain showed

Results

impaired calcium mobilization after BCR cross-‐link confining that LMP2A blocked BCR’s calcium signaling. In contrast, BCR cross-‐linking induced calcium influx in LCLs generated with K1 EBV and K15 EBV strains suggesting that K1 and K15 proteins did not block BCR-‐mediated calcium signaling.

I infected unsorted cells with wt EBV, ΔLMP2A EBV, K1 EBV, and K15 EBV and

generated LCLs, which expressed high levels of IgG (wt EBV infection) or IgM (ΔLMP2A

EBV, K1 EBV and K15 EBV). I investigated whether K1 and K15 can block BCR signaling by a change in phosphorylated Syk (pSyk) and phosphorylated PLCγ2 (pPLCγ2) levels

and an alteration in Ca2+_{-‐influx. A wt EBV LCL, unable to express a BCR due to crippled}

mutations in the Ig genes (referred as clone A16 in Mancao et al., 2005), was used as negative control for BCR cross-‐linking induced signaling. The BCR was either cross-‐ linked with an α-‐hIgG/M antibody for 10min at 37°C or left untreated. Cells were fixed,

permeabilized, stained with antibodies specific for pSyk or pPLCγ2, and analyzed by

flow cytometry. BCR cross-‐linking increased pSyk and pPLCγ2 levels in all tested cell

lines but the negative control cell line A16 indicating that LMP2A, K1, and K15 did not block BCR induced protein-‐phosphorylation.

The same LCLs were loaded with 3µM Indo-‐1 AM. Indo-‐1 is a fluorescent dye, which changes its emission wavelength from 475nm (blue) to 400nm (violet) upon calcium binding. An increase in cellular Ca2+_{can be measured by an increase in the violet/blue}

wavelength ratio. The baseline violet/blue ratio of Indo-‐1 loaded LCLs was measured for 1min. Subsequently, the BCR was cross-‐linked with an α-‐hIgG/A/M F(ab’)2 fragment and

the Ca2+_{-‐influx was measured by the change in the violet/blue ratio for 5min. The Ca}2+_-‐

influx was impaired in both wt EBV LCLs but not in ΔLMP2A, K1, and K15 EBV LCLs

indicating that LMP2A but not K1 or K15 blocked BCR induced calcium signaling.

Discussion

In document Prácticas de laboratorio para la enseñanza aprendizaje de conceptos básicos de la electrostática con estudiantes de grado once del Colegio Nuevo Reino De Granada (página 48-52)