Studying Organelle Dynamics in B Cells During Immune Synapse Formation

In this review, we will discuss both imaging and biochemical techniques used to study organelle positioning and composition, as well as cytoskeleton rearrangements observed during the formation of an immunological synapse. Initial stages of active remodeling allow B cells to increase their cell surface and maximize the quantity of antigen BCR complexes gathered at the synapse. On the other hand, local recruitment of lysosomes, together with the centrosome at the synaptic membrane, can be coupled to lysosome secretion, which is known to facilitate the extraction of immobilized antigens.

Uptaken antigen is further processed within endolysosome compartments into peptides, which are loaded onto MHC class II molecules for presentation to T helper cells. Therefore, studying organelle dynamics associated to the immune synapse is crucial to understand how B cells are fully activated. Immunofluorescence of B cells activated with immobilized antigens allows us to follow different cell components and how they can be recruited to the immune synapse.

B cells can be activated through immobilized antigens at a bead surface or a coverslip surface. Antigen-coated beads are prepared by incubating specific ligands with amino-beads previously treated with glutaraldehyde to activate amino groups. Resuspend activated beads in 100 microliters of PBS and vortex.

Meanwhile, prepare the antigen solution by adding 100 microgram per mL of BCR ligand in 150 microliters of PBS. Next, add 50 microliters of activated beads to the antigen solution and incubate overnight at four degrees Celsius. Remember, also use an unrelated ligand as negative control.

After incubation, wash beads with PBS. Aspirate the supernatant and replace with PBS. Repeat three times.

Next, block the three amino groups by resuspended beads in 500 microliters of a solution of BSA. Finally, resuspend beads in 70 microliters of PBS. To calculate the final concentration, you can use a hemocytometer to count the beads.

For B cell activation, use a 50 mL tube and resuspend cells to a concentration of 1.5 million per mL in CLICK supplemented with 5%fetal bovine serum. Next, add 150, 000 B cells, corresponding to a 100 microliters of the cell bead mixture to a poly-L-lysine-coated coverslip ad incubate at 37 degrees for the different time points. A second approach to activate B cells is seeding them onto antigen-coated coverslips.

For this purpose, coat slides with anti-BCR and B220 with improves the cell adhesion. Prepare the antigen solution by adding anti-BRC and B220 in PBS to a final concentration of 10 micrograms per mL and 0.5 micrograms per mL, respectively. Next, set the coverslip over a lid of 24 well plate covered with parafilm film and add 40 microliters of antigen solution.

Seal the plate and then incubate at 4 degrees overnight. To start the activation in coverslip, first wash them with PBS and let them dry for a few minutes. Next, add 150, 000 B cells and incubate at 37 degrees for the different time points.

In both cases, when activating either with beads or antigen-coated slides, we recommend starting with the longest time point and then proceeding to the shorter ones. Once you have mounted the dry coverslip onto microscope slide, you can use an epifluorescence or a confocal microscope to view your samples, depending on the resolution requirements. Focus the sample using the transmitted light.

You should search for fields where cells and beads are interacting at one and one ratio. For B cells activated on antigen-covered coverslips, use the 100x objective for better resolution. For parameters, consider taking a z-stack that covers the entire cell.

You can label actin to delimit the lower and upper boundaries of the cell. For B cells activated with antigen-coated beads and label it for organelles confined to one point, first delimit two areas:the cell area and the bead area. Next, identify the position or the organelle by a point and obtain its localisation coordinates.

Draw two vectors:one between the cell center and the organelle, and other between the cell center and the bead center. Measure the length of both and measure the angle between the two vectors, now referred as alpha. Make a projection with the vector between the centrosome and the cell center using the Guassian of alpha and then calculate the polarity index of the organelle by dividing the projected vector, a, by b.

Thus a polarity index between zero and one indicates a polarized phenotype. Values between zero and minus one indicate a non-polarized phenotype. In order to determine the polarity index for more disperse organelles, such as lysosomes, you can use the same algorithm mentioned before, changing the organelle localisation coordinates for the mass center's coordinates of the labels, in this case, lysosomes.

Similarly to the analysis described before, a polarity index between zero and one indicates a polarized phenotype, whereas values between zero and minus one indicates a non-polarized phenotype. To quantify the amount of each organelle closely recruited to the new synapse, you can calculate the the percentage of the organelle fluorescence at the bead. For this, you must delimit the bead and cell area, measure the respective fluorescence intensity and then divide the bead fluorescence by the sum of the bead and the cell fluorescence.

You will obtain the amount of each organelle that is in contact with the new synapse. Alternatively, draw a rectangle opposite to the bead. Use a weight corresponding to a quarter of the cell length, then measure the fluorescence intensity within the rectangle and divide it by the total fluorescence.

This algorithm will always to obtain the percentage of the organelle that is closely to the new synapse, but not necessarily in direct contact with the interface. To quantify the centrosome-associated components in resting and activated B cells. Briefly, we determine three parameters.

The cell and bead area, and the centrosomal localisation coordinates. Next, we define a circle surrounding the centrosome and run a radial profile analysis from the center of the centrosome, previously defined. Once you have the plot, determine the radius at which 70%of the maximum fluorescence is maintained.

Use this radius to draw a circle surrounding the centrosome. Finally, obtain fluorescence density of the component of interest at the centrosome area and the cell area, and divide both values to obtain the fluorescent density ratio. To measure the distribution of organelles at the immune synapse interface, first identify the area of the B cells in contact with the antigen-coated coverslip.

You can label actin to help you define this area. Next, measure the area in contact with the slide, the height and the width of the cell. The area facing the antigen-coated cover slip is a measurement of B cell spreading upon B cell activation.

Use the height and width values to delimit a central area in the cell, which is separated from the boundaries by a quarter of the height and the width values. Usually these are correspond to approximately a third of the total cell area. Finally, calculate the organelle recruitment at the center of the new synapse, dividing the fluorescence density at the central area by the fluorescence density of the whole cell.

Positive values of this index indicate an organelle enrichment at the center of the new synapse. On the contrary, negative values indicate a peripheral distribution. To measure the distribution of organelles across the z-planes of B cells activated on antigen-coated coverslips, first delimit the synaptic interface and the upper limit of the cell.

Next, draw a line across the cell center and reslice the image to obtain an XZ image. Measure the head of the cell and divide the image into 10 fractions of the same area, from the immune synapse to the upper limit of the cell. Measure the fluorescence in each z-fraction and calculate the percentage of fluorescence by dividing the fluorescence at each z-fraction by the total fluorescence of the cell.

Finally, plot the percentage of fluorescence intensity per z-fraction. Fraction one and two represent the synaptic interface. Here is the workflow or the centrosome isolation procedure.

Use 20 million B cells per condition. Pre-treat cells with Cytochalasin D and Nocodazole according to the required protocol to facilitate centrosome detachment. Next, watch cells by resuspending them in 5 mL of TBS buffer.

Repeat by using 1 mL of 0.1x TBS buffer supplemented with 8%sucrose. Resuspend the cell pellet with 150 microliters of lysis buffer. Centrifuge at 10, 000 g for 10 minutes at 4 degrees Celsius to separate the cytosol and organelles from the nucleus.

Carefully recover the cell supernatant and place on top of a 1.5 mL tube. Fill it with 300 microlitres of gradient buffer supplemented with 60%sucrose. Centrifuge samples at 10, 000 g for 30 minutes at 4 degrees Celsius.

This centrifugation step is important to concentrate the centrosomes. After centrifugation, carefully remove the supernatant until you reach the interface. Vortex the sample remaining in the tube.

Prepare discontinuous sucrose gradient in an ultracentrifuge tube by overlaying 0.45 mL of 70%sucrose, with 0.27 mL of 50%sucrose, and then 0.27 mL of 40%sucrose in gradient buffer. Place the centrosome-enriched samples on the discontinuous gradient and calibrate the weight of each sample in the respective adapters. Centrifuge the tubes at 40, 000 g for 1 hour at 4 degree Celsius, with minimal acceleration and without break to avoid perturbing the gradient.

After centrifugation, carefully collect 12 fractions with equal volume. Resuspend the sample with loading buffer and run an SDS-PAGE. To study organelle distribution in B cells during the formation of an immune synapse, we used 2A1.6 B cells activated with antigen-coated beads for different time points.

As a control, B cells were activated with beads containing an unrelated BCR-ligand. We then, using immunofluorescence staining, do detect different organelles, which change their localisation upon activation with immobilized antigens. We show here the centrosome, labeled with alpha-tubulin and golgi apparatus labeled with Rab6a.

In the graph, we show the polarity index for centrosome and golgi apparatus polarity verus each time point of activation, in which each dot represents one cell. During the activation, we can observe that the polarity indexes for these organelles reach values closer to 1, suggesting an increase in their recruitment to the IS.Organelles which display a more dispersed distribution, such as lysosomes, can also be quantified using a polarity index. We can observe that the polarity index of the lyososome will reach more positive values upon activation, which results from the recruitment to the IS.We show a complementary approach to quantify the recruitment of a lysosome toward the immune synapse.

Using the same protocol of activation with antigen-coated beads, lysosome recruitment to the IS was calculated by quantifying the fluorescence label of lyosomes recruited to the area defined around the bead or within the synaptic area. We can observe that open activation. There is an increase of lysosomes coupled to the site of synapse.

Here we present the quantification of actin at the centrosome in B cells activated with specific and non-specific antigen-coated beads. The pool of actin is indicated with an arrow. Quantification of centrosome-associated components, such as actin in this case, can be represented by dot plot, in which each dot represents one cell.

After the activation, the B cell decrease in the amount of actin around the centrosome. This step is important to detach the centrosome from the perinuclear region to allow its polarization to the new synapse. For B cell activated on antigen-coated surface, you can study actin remodeling as well as the recruitment of organelles to the synaptic membrane.

Here we show the analysis of organelles, such as the golgi apparatus and endoplasmic reticulum, which display a central and peripheral distribution, respectively. Additionally, we can calculate the spreading area of B cells after different time points of activation. For this purpose, we label actin to be able to define the cell limit in the XY plane.

In the graph, you can observe the increase in the cell area after 30 and 60 minutes of activation. The distribution of an organelle can also be calculated in the XZ plane in respective to the synaptic interface. The graph represents the distribution of actin in z-plane, where the first two planes correspond to the synaptic interface.

We can observe that actin becomes enriched in this area upon activation. In addition to imaging analysis, biochemical approach can also be used to study change in centrosome composition upon B cell activation. We show here a representative Western blot of centrosome-containing fraction highlighted by the beige rectangle.

Centrosome fractions were isolated on a discontinuous sucrose gradient and detected by gamma-tubulin leveling. The Western blot below shows actin and Arp2 in centrosome fractions from resting B cells, which become depleted upon activation. B lymphocytes undergo dynamic changes at the membrane interface in order to form a functional immune synapse.

This process can be studied by imaging and biochemical techniques described here in this video. We believe that this analysis can provide value information on the molecular mechanisms involved on how B cells can become efficiently activated.