Cytoskeletal Organization ofLimulusAmebocytes Pre- and Post-Activation: Comparative Aspects

Abstract

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.

New explanations for old observations: marginal band coiling during platelet activation

“…In support of this notion, highly similar microtubule coils have been described upon activation of fish thrombocytes, which are large, nucleated cells with a discoid shape in the resting state . Similarly, amoebocytes of the horseshoe crab, which are flat ellipsoid clotting cells in the hemolymph, have twisting MBs when activated . Another surprising example is nucleated erythrocytes of mollusks, which have an ellipsoid morphology and a MB formed by centrosomal organized microtubules.…”

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).…”

Transcriptomic Signature of Horseshoe Crab Carcinoscorpius rotundicauda Hemocytes’ Response to Lipopolysaccharides

“…Enrichment of other cytoskeleton-related GO terms (synapse, synapse part and structural molecule activity) was also reported after the infection of T. tridentatus with gram-negative bacteria [ 15 ]. The cytoskeleton function is implicated in various important processes of HSC protective responses, including phagocytosis and exocytosis of a wide array of defensive molecules upon interaction with LPS [ 13 , 24 ].…”