Distribution of F-actin-vinculin or F-actin-myosin

For visualizing the distribution of vinculin or myosin with respect to F-actin, the platelets were fixed and double stained with specific antibodies and phalloidin at various time points of their spreading (5, 10, 15, 30, 60 and 120 minutes) and the qualitative distribution patterns of these proteins formed during platelet spreading is imaged. Examples of two different platelets showing the typical distribution patterns for that particular time point are shown in Figure 4.3 and Figure 4.4.

75 The F-actin is distributed in the filopodia and lamellipodia and later gets reorganized into the stress-fiber like structures (Cy5 channels in Figure 4.3 and Figure 4.4) in the same manner as described previously (see section 4.1.1).

In the initial stages of spreading (5-15 minutes in Figure 4.3), when the platelets have expanded via their lamellipodia, the focal adhesion protein vinculin is seen to be distributed evenly all over the cytoplasm although the staining signal is more intense at the plasma membrane and in the central region where the vinculin is arranged in a ring-like pattern (platelets 1 and 2 at 5 minutes and platelet 1 at 10 minutes in Figure 4.3). Interestingly, these vinculin ring-like patterns do not appear to coincide with the F-actin 'ring-like' structures that are described previously but rather seem to encircle the granulomere (section 4.1.1 and Figure 4.1 and Figure 4.2). A look at the BF images in the earlier spreading stages confirms this observation (platelet 1 at 10 minutes in Figure 4.5). As the platelets spread further (30 minutes and beyond), the vinculin gets distributed all over the cytoplasm but shows a predominantly higher signal at the tips of the stress fiber-like structure bundles that the platelets form (indicated by cyan arrows in Figure 4.3). At the later time points of spreading (60-120 minutes) the vinculin is associated with the circular F-actin stress fiber-like structures that the platelets form (platelet 2 at 60 minutes, platelets 1 and 2 at 120 minutes in Figure 4.3).

Over the course of the platelet spreading, the myosin distribution is comparable with that of the F-actin, except for its absence in the cortex (5-120 minutes in Figure 4.4). As the platelets spread and form filopodia and lamellipodia, the F-actin and myosin are co-distributed in these structures. Strikingly, myosin highly coincides with the F-actin 'ring-like' structures seen in the first 15 minutes of spreading (indicated by yellow arrows in platelets at 5, 10 and 15 minutes in Figure 4.4). A closer inspection of the corresponding BF images shows that these F-actin-myosin 'ring-like' structures are associated with the platelet granulomeres (platelet 1 at 10 minutes in Figure 4.5). As the platelets spread further (30-120 minutes) and reorganize into bundles of stress fiber-like structures, the myosin distribution too coincides with these bundles. During this later stage of spreading, when the granulomeres have flattened out and are no longer visible, the F-actin-myosin 'ring-like' structures too are not visible (platelet 1 at 30 minutes in Figure 4.5).

76 Figure 4.3: Qualitative distribution of F-actin-vinculin post-fixation

Epifluorescence images of platelets fixed at different time points of their spreading (5-120 minutes) and stained for their actin (Cy5) and vinculin (FITC). During the course of platelet spreading, the F-actin is seen in the filopodia, lamellipodia, cortex, and ring-like structures and gradually forms the stress-fiber like structures, whereas the vinculin is distributed all over the cytoplasm but shows a predominant distribution in the center in a ring-like pattern in the first 5-15 minutes and is later predominant at the tips of the bundles of F-actin stress fiber-like structures (indicated by cyan arrows and the white areas in the overlayed fluorescence images at time points 30, 60 and 120 minutes). All images were taken by Tim Dullweber during his Bachelor thesis.

77 Figure 4.4: Qualitative distribution of F-actin-myosin post-fixation

Epifluorescence images of platelets fixed at different time points of their spreading (5-120 minutes) and stained for their actin (Cy5) and myosin (FITC). During the course of platelet spreading, the F-actin is seen in the filopodia, lamellipodia, cortex, and ring-like structures and gradually forms the stress-fiber like structures. The myosin too is associated in all of these structures with the F-actin, except in the cortex. Myosin predominantly coincides with the F-actin ring like structures seen in the first 5-15 minutes (indicated by yellow arrows) and later coincides with the bundles of F-actin stress fiber-like structures that develop at the later time points (white areas in the overlayed fluorescence images at time points 30, 60 and 120 minutes). All images were taken by Tim Dullweber during his Bachelor thesis.

78 Figure 4.5: Distribution of vinculin and myosin at platelet granulomeres

Distribution of vinculin (left panel) and myosin (right panel) in the platelet granulomere zone are shown with respect to F-actin during the early (top panel, 10 minutes) and later (bottom panel, 30 minutes) time points of platelet spreading. A) In the early time point, vinculin seems to encircle the granulomere (indicated by yellow arrow) and is not associated with the F-actin ring-like structure formed by the platelets and shows no defined association at the later time point of spreading when the granulomere has flattened. B) In contrast, myosin highly coincides with the F-actin ring-like structure (indicated by yellow arrow and white area in overlayed fluorescence image) in the early time point of spreading. However, there is no sign of the F-actin-myosin ring-like structure at the later time point of spreading when the granulomere has flattened.

In general, during platelet spreading on glass, the sites of focal adhesion (vinculin) are distributed all over the platelets with prominent distribution at their centers and at the tips of their F-actin bundles, whereas the sites of force generation (myosin) overlap with the sites of F-actin distribution, the only exception being the cortical regions.