Shape transformation and cytoskeletal reorganization in activated non‐mammalian thrombocytes

Abstract

The nucleated thrombocytes of non-mammalian vertebrates are partially flattened, ovoid cells morphologically distinct from mammalian platelets, and the extent of their functional equivalence is unknown. To test whether they resemble platelets in having similar F-actin-based post-activation stages, rapid fixation/extraction/labeling methods were developed to reveal cytoskeletal organization in dogfish thrombocytes by confocal microscopy. Unactivated cells contained cortical F-actin plus denser F-actin co-localizing with outer marginal band (MB) microtubules. In the post-activation sequence, determined for the first time by continuous observation of individual thrombocytes following thrombin perfusion, cells rounded and blebbed, spread, and eventually flattened extensively. The MB twisted and then became disorganized, with microtubule bundles remaining centrally located and associated with nuclear clefts. In contrast, F-actin occupied blebs and outward-spreading cytoplasm, initially in spiky projections, then predominantly in stress fibers, and inhibitors of F-actin assembly or myosin ATPase blocked shape changes. Thus, the post-activation stages and cytoskeletal events observed in nucleated thrombocytes were found to parallel those of platelets.

Motor-driven marginal band coiling promotes cell shape change during platelet activation

“…Although it has long been recognized that the MB is essential to keep the dis coid shape of resting platelets (White and Rao, 1998), this is the first description of MB coiling inducing the rapid disc to sphere transition of activating platelets, first described in 1965 (Bull and Zucker, 1965). Interestingly, highly similar microtubule coils have been described in limulus amebocytes and nucleated throm bocytes of nonmammalian vertebrates, indicating that this process is evolutionarily conserved (Conrad et al, 2004;Lee et al, 2004).…”

Calcium oscillation and phosphatidylinositol 3-kinase positively regulate integrin αIIbβ3-mediated outside-in signaling

“…About 200 ll of cell suspension was added to the chamber and incubated for 15 min before microscopic observation. Confocal microscopy [39][40][41] and time-lapse recording [42][43][44][45][46] of living platelets were performed using a Leica TCS SP2 AOBS confocal microscope and a Nikon Diaphot 300 inverted microscope, respectively [5,47]. The processed images were recorded on an optical memory laser disk (Sony Model LVR-5000) and redigitized for data analysis and image processing prior to dyesublimation printer output (Kodak XLS 8600PS).…”

PI3-kinase is essential for ADP-stimulated integrin αIIbβ3-mediated platelet calcium oscillation, implications for P2Y receptor pathways in integrin αIIbβ3-initiated signaling cross-talks

“…About 200 ll of cell suspension was added to the chamber and incubated for 15 min before microscopic observation. Confocal microscope [4,26,27] and time-lapse recording [28][29][30][31][32] of living platelets was performed using a Leica TCS-SP2-AOBS confocal microscope and a Leica DM-IRE2 inverted microscope respectively [4,16].…”