Megakaryocytes from guinea pig bone marrow were isolated and maintained in liquid culture and were treated with ADP, thrombin, arachidonic acid, or collagen . Megakaryocytes spread with an active ruffled membrane in response to ADP (1-100 tLM), thrombin (1 .0 U/ml), and arachidonic acid (50 tiM) but responded to collagen surfaces only if fibronectin was added to the cultures . Spreading could be blocked completely by dibutyryl cyclic AMP (dibutyryl CAMP) or isobutylmethylxanthine at 1 mM, as well as by cytochalasin D (2 ttg/ml), but not by colchicine up to 1 mg/ml .The distribution of contractile proteins was examined by immunofluorescence . In untreated, spherical cells, staining with antimyosin, antifilamin, anti-a-actinin, or with fluorescein-labeled subfragment 1 (FITC-S1) was diffuse and unpatterned. With antitubulin antibody, however, microtubules were seen in a dense array throughout the unspread cells. In actively ruffling spreading cells, myosin, filamin, and actin were visualized in the region of the ruffled membrane while a-actinin was seen most prominently in a band located proximal to the inner part of the ruffle . In fully spread cells, actin, myosin, filamin, and a-actinin were seen in filaments that filled the cytoplasm . Antimyosin and anti-a-actinin staining of the filaments was periodic witĥ "1 tAm center-to-center spacing. Actin, filamin, and a-actinin were also identified in punctate spots throughout the spread cytoplasm. Microtubules were absent from the ruffle but filled the cytoplasm of fully spread cells. Rings, 1 .5-2 .5 tim in diameter, were seen with antitubulin in 13% of the spread cells. Our results show that megakaryocytes respond to platelet agonists, but typically by spreading, rather than extending, filopodia. From the changes in localization of contractile proteins and from time-lapse cinematography, we propose a model for cell spreading.Megakaryocytes are bone marrow cells of mammalian species that, by fragmentation of their cytoplasm, give rise to new blood platelets . Platelet formation requires both external and internal morphological changes in the megakaryocytes . As the megakaryocytes mature, they extend processes into venous sinusoids in the bone marrow which may be released as platelets (2,19,31) . An internal structural change that must occur during platelet formation is the organization of microtubules into the characteristic circumferential coil seen in platelets (3,29) . The presence of microfilaments and microtubules in megakaryocytes has been established by electron microscope studies (2, 4, 9, 34), but an overall picture of the distribution of these structures throughout the cell is lacking . Since indirect immunofluorescence is most useful for this purpose, we used this method to study the arrangement of the contractile and struc-THE JOURNAL OF CELL BIOLOGY " VOLUME 92 FEBRUARY 1982 313-323 ©The Rockefeller University Press -0021-9525/82/02/0313/11 $1 .00 tural proteins of this interesting cell type . It had been reported earlier (11) that megakaryo...