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Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.

Italiano JE, Lecine P, Shivdasani RA, Hartwig JH - J. Cell Biol. (1999)

Bottom Line: We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy.Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends.These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

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Organization of microtubules at the ends of proplatelet extensions (a–c) and in released proplatelet forms (d–g). (a) Antitubulin staining of a megakaryocyte and its early proplatelet extension. Microtubules concentrate along the edges of the megakaryocyte and enter into the proplatelet extensions. Bundles enter the projections from both sides and separate periodically within the extensions. The distal end of each proplatelet has a teardrop-shaped enlargement that contains a microtubule loop (arrowheads). (b and c) Representative electron micrographs showing the organization of microtubules within the tips of proplatelet termini. The end of each proplatelet contains a microtubule bundle that loops beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure. (d) Gallery of released platelet forms stained for tubulin by immunofluorescence confocal microscopy. Released platelet-sized particles are connected by thin cytoplasmic bridges, the most abundant being barbell shapes composed of two platelet-like particles connected by a single cytoplasmic strand. Microtubules line the shaft and rim the bulbous ends. (e) Low magnification electron micrograph showing the microtubule-based cytoskeleton of a representative released proplatelet form. A microtubule bundle lines the shaft of the proplatelet. Bar, 2 μm. (f) Higher magnification electron micrograph of one end of the proplatelet shown in e. Ends have microtubule bundles arranged into teardrop-shaped loops, similar to those at the ends of proplatelets extending from megakaryocytes. Within these loops are microtubule coils structurally similar to those observed in mature platelets isolated from the blood. Bar, 0.5 μm.
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Figure 5: Organization of microtubules at the ends of proplatelet extensions (a–c) and in released proplatelet forms (d–g). (a) Antitubulin staining of a megakaryocyte and its early proplatelet extension. Microtubules concentrate along the edges of the megakaryocyte and enter into the proplatelet extensions. Bundles enter the projections from both sides and separate periodically within the extensions. The distal end of each proplatelet has a teardrop-shaped enlargement that contains a microtubule loop (arrowheads). (b and c) Representative electron micrographs showing the organization of microtubules within the tips of proplatelet termini. The end of each proplatelet contains a microtubule bundle that loops beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure. (d) Gallery of released platelet forms stained for tubulin by immunofluorescence confocal microscopy. Released platelet-sized particles are connected by thin cytoplasmic bridges, the most abundant being barbell shapes composed of two platelet-like particles connected by a single cytoplasmic strand. Microtubules line the shaft and rim the bulbous ends. (e) Low magnification electron micrograph showing the microtubule-based cytoskeleton of a representative released proplatelet form. A microtubule bundle lines the shaft of the proplatelet. Bar, 2 μm. (f) Higher magnification electron micrograph of one end of the proplatelet shown in e. Ends have microtubule bundles arranged into teardrop-shaped loops, similar to those at the ends of proplatelets extending from megakaryocytes. Within these loops are microtubule coils structurally similar to those observed in mature platelets isolated from the blood. Bar, 0.5 μm.

Mentions: Platelet-sized particles released by cultured mouse megakaryocytes exhibit each of the hallmark features of blood-derived platelets. They are disc-shaped, 2–3 μm in diameter, lack discernible membrane topology or protrusions (Fig. 3 a), and show periodic surface invaginations (Zucker-Franklin 1970) that demarcate entrances into the open canalicular system (Fig. 3 a, arrowheads). Removal of the plasma membrane exposes an elaborate membrane skeleton composed of 3–5-nm strands identical in appearance to the spectrin-based network of human platelets (Fig. 3 b), and a prominent marginal band (Haydon and Taylor 1965; White and Krivit 1967; Kenney and Linck 1985) derived from the apparent coiling of a single microtubule (Fig. 3 c). A complex three-dimensional network of actin filaments, similar to that observed in mature blood platelets (Fox et al. 1988; Nachmias and Yoshida 1988; Hartwig and DeSisto 1991; Fox et al. 1996), is also found beneath the membrane skeleton (Fig. 3 d). 200–225 platelet-sized microtubule coils may be seen extending from typical proplatelet-producing cells (see Fig. 5g and Fig. h), a value that is in good agreement with the proposed theoretical range of platelets produced per megakaryocyte (Kaufman et al. 1965; Harker and Finch 1969; Trowbridge et al. 1984; Stenberg and Levin 1989) and with observations in cultured human cells (Choi et al. 1995). Furthermore, the released particles respond to stimulation with thrombin by extending lamellipodia and filopodia (Fig. 3 e), forming microaggregates (Fig. 3 f), and exhibiting a twofold increase in their proportion of filamentous actin (data not shown), which is similar to observations in activated blood platelets (Carlsson et al. 1979; Fox and Phillips 1981; Fox et al. 1984; Karlsson et al. 1984). These particles showed increased expression of the activation-dependent antigens P-selectin and the functional fibrinogen receptor (data not shown), as previously reported for human proplatelets (Choi et al. 1995). Hence, platelets released in cell culture display all the morphological and functional criteria that distinguish blood platelets.


Blood platelets are assembled principally at the ends of proplatelet processes produced by differentiated megakaryocytes.

Italiano JE, Lecine P, Shivdasani RA, Hartwig JH - J. Cell Biol. (1999)

Organization of microtubules at the ends of proplatelet extensions (a–c) and in released proplatelet forms (d–g). (a) Antitubulin staining of a megakaryocyte and its early proplatelet extension. Microtubules concentrate along the edges of the megakaryocyte and enter into the proplatelet extensions. Bundles enter the projections from both sides and separate periodically within the extensions. The distal end of each proplatelet has a teardrop-shaped enlargement that contains a microtubule loop (arrowheads). (b and c) Representative electron micrographs showing the organization of microtubules within the tips of proplatelet termini. The end of each proplatelet contains a microtubule bundle that loops beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure. (d) Gallery of released platelet forms stained for tubulin by immunofluorescence confocal microscopy. Released platelet-sized particles are connected by thin cytoplasmic bridges, the most abundant being barbell shapes composed of two platelet-like particles connected by a single cytoplasmic strand. Microtubules line the shaft and rim the bulbous ends. (e) Low magnification electron micrograph showing the microtubule-based cytoskeleton of a representative released proplatelet form. A microtubule bundle lines the shaft of the proplatelet. Bar, 2 μm. (f) Higher magnification electron micrograph of one end of the proplatelet shown in e. Ends have microtubule bundles arranged into teardrop-shaped loops, similar to those at the ends of proplatelets extending from megakaryocytes. Within these loops are microtubule coils structurally similar to those observed in mature platelets isolated from the blood. Bar, 0.5 μm.
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Related In: Results  -  Collection

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Figure 5: Organization of microtubules at the ends of proplatelet extensions (a–c) and in released proplatelet forms (d–g). (a) Antitubulin staining of a megakaryocyte and its early proplatelet extension. Microtubules concentrate along the edges of the megakaryocyte and enter into the proplatelet extensions. Bundles enter the projections from both sides and separate periodically within the extensions. The distal end of each proplatelet has a teardrop-shaped enlargement that contains a microtubule loop (arrowheads). (b and c) Representative electron micrographs showing the organization of microtubules within the tips of proplatelet termini. The end of each proplatelet contains a microtubule bundle that loops beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure. (d) Gallery of released platelet forms stained for tubulin by immunofluorescence confocal microscopy. Released platelet-sized particles are connected by thin cytoplasmic bridges, the most abundant being barbell shapes composed of two platelet-like particles connected by a single cytoplasmic strand. Microtubules line the shaft and rim the bulbous ends. (e) Low magnification electron micrograph showing the microtubule-based cytoskeleton of a representative released proplatelet form. A microtubule bundle lines the shaft of the proplatelet. Bar, 2 μm. (f) Higher magnification electron micrograph of one end of the proplatelet shown in e. Ends have microtubule bundles arranged into teardrop-shaped loops, similar to those at the ends of proplatelets extending from megakaryocytes. Within these loops are microtubule coils structurally similar to those observed in mature platelets isolated from the blood. Bar, 0.5 μm.
Mentions: Platelet-sized particles released by cultured mouse megakaryocytes exhibit each of the hallmark features of blood-derived platelets. They are disc-shaped, 2–3 μm in diameter, lack discernible membrane topology or protrusions (Fig. 3 a), and show periodic surface invaginations (Zucker-Franklin 1970) that demarcate entrances into the open canalicular system (Fig. 3 a, arrowheads). Removal of the plasma membrane exposes an elaborate membrane skeleton composed of 3–5-nm strands identical in appearance to the spectrin-based network of human platelets (Fig. 3 b), and a prominent marginal band (Haydon and Taylor 1965; White and Krivit 1967; Kenney and Linck 1985) derived from the apparent coiling of a single microtubule (Fig. 3 c). A complex three-dimensional network of actin filaments, similar to that observed in mature blood platelets (Fox et al. 1988; Nachmias and Yoshida 1988; Hartwig and DeSisto 1991; Fox et al. 1996), is also found beneath the membrane skeleton (Fig. 3 d). 200–225 platelet-sized microtubule coils may be seen extending from typical proplatelet-producing cells (see Fig. 5g and Fig. h), a value that is in good agreement with the proposed theoretical range of platelets produced per megakaryocyte (Kaufman et al. 1965; Harker and Finch 1969; Trowbridge et al. 1984; Stenberg and Levin 1989) and with observations in cultured human cells (Choi et al. 1995). Furthermore, the released particles respond to stimulation with thrombin by extending lamellipodia and filopodia (Fig. 3 e), forming microaggregates (Fig. 3 f), and exhibiting a twofold increase in their proportion of filamentous actin (data not shown), which is similar to observations in activated blood platelets (Carlsson et al. 1979; Fox and Phillips 1981; Fox et al. 1984; Karlsson et al. 1984). These particles showed increased expression of the activation-dependent antigens P-selectin and the functional fibrinogen receptor (data not shown), as previously reported for human proplatelets (Choi et al. 1995). Hence, platelets released in cell culture display all the morphological and functional criteria that distinguish blood platelets.

Bottom Line: We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy.Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends.These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.

Show MeSH
Related in: MedlinePlus