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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 along the shaft of proplatelets. Swellings along the proplatelet (a) contain detergent-insoluble membrane and vesicles that are similar to those found in released platelets (b) in these samples. (c) Low magnification electron micrograph showing the microtubules bundles lining the shaft of a typical proplatelet. Although proplatelet ends (arrowhead) have distinct microtubule coils, microtubules observed in swellings along the shaft (arrow) are not coiled. (d and g) Proplatelets stained for tubulin by immunofluorescence and photographed on a confocal microscope. Proplatelets show periodic segments connected by thin cytoplasmic bridges, but only the ends (arrowheads) have microtubule bundles arranged into teardrop-shaped loops. (e and f) Higher magnification electron micrograph of a swelling along a proplatelet shaft showing that these are points where the microtubule bundle separates for a short distance (arrows) but does not form a loop. Bars: (b and f) 0.5 μm; (c and e) 1 μm.
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Figure 6: Organization of microtubules along the shaft of proplatelets. Swellings along the proplatelet (a) contain detergent-insoluble membrane and vesicles that are similar to those found in released platelets (b) in these samples. (c) Low magnification electron micrograph showing the microtubules bundles lining the shaft of a typical proplatelet. Although proplatelet ends (arrowhead) have distinct microtubule coils, microtubules observed in swellings along the shaft (arrow) are not coiled. (d and g) Proplatelets stained for tubulin by immunofluorescence and photographed on a confocal microscope. Proplatelets show periodic segments connected by thin cytoplasmic bridges, but only the ends (arrowheads) have microtubule bundles arranged into teardrop-shaped loops. (e and f) Higher magnification electron micrograph of a swelling along a proplatelet shaft showing that these are points where the microtubule bundle separates for a short distance (arrows) but does not form a loop. Bars: (b and f) 0.5 μm; (c and e) 1 μm.

Mentions: Most released platelet-sized particles remain connected by cytoplasmic bridges, the most abundant being barbell forms composed of two platelet-like particles joined by one cytoplasmic strand. To ask whether the bulbous ends and thickenings along the shaft of proplatelets show mature cytoskeletal features, we examined representative released particles by antitubulin immunofluorescence confocal microscopy (Fig. 5 d) and electron microscopy (Fig. 5e and Fig. f). A microtubule bundle forms the core of the proplatelet shaft, and the ends have microtubule bundles forming teardrop-shaped loops that are similar in both size (1–3 μm) and appearance to those described in mature blood platelets (White 1968). Distorted microtubule bundles are also visible in platelet-sized swellings along the shaft of the proplatelet; however, in contrast to the terminal rings, those observed along the proplatelet shafts are uniformly less distinct (Fig. 6). Whereas these platelet-sized nodules superficially appear to include microtubule rings, close inspection reveals that they are actually points where microtubule bundles simply diverge for a short distance but fail to form the mature teardrop shape (Fig. 6, c–g). These nodules consistently reveal membrane and other detergent-insoluble materials, presumably destined for final platelet assembly elsewhere (compare Fig. 6, a and b). These results are entirely consistent with the dynamic activity that is observed at the sites of the platelet-sized nodules, including branching (Fig. 2 b), interconversion between condensed and lamellipodial forms (Fig. 1 and Fig. 2 a), and rapid translocation of particles (Fig. 2 c), and indicate that they are structurally unstable. More importantly, they establish that the mature cytoskeletal feature of a microtubule coil is only detected at the ends of proplatelets (Fig. 5) and not along their lengths.


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 along the shaft of proplatelets. Swellings along the proplatelet (a) contain detergent-insoluble membrane and vesicles that are similar to those found in released platelets (b) in these samples. (c) Low magnification electron micrograph showing the microtubules bundles lining the shaft of a typical proplatelet. Although proplatelet ends (arrowhead) have distinct microtubule coils, microtubules observed in swellings along the shaft (arrow) are not coiled. (d and g) Proplatelets stained for tubulin by immunofluorescence and photographed on a confocal microscope. Proplatelets show periodic segments connected by thin cytoplasmic bridges, but only the ends (arrowheads) have microtubule bundles arranged into teardrop-shaped loops. (e and f) Higher magnification electron micrograph of a swelling along a proplatelet shaft showing that these are points where the microtubule bundle separates for a short distance (arrows) but does not form a loop. Bars: (b and f) 0.5 μm; (c and e) 1 μm.
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Figure 6: Organization of microtubules along the shaft of proplatelets. Swellings along the proplatelet (a) contain detergent-insoluble membrane and vesicles that are similar to those found in released platelets (b) in these samples. (c) Low magnification electron micrograph showing the microtubules bundles lining the shaft of a typical proplatelet. Although proplatelet ends (arrowhead) have distinct microtubule coils, microtubules observed in swellings along the shaft (arrow) are not coiled. (d and g) Proplatelets stained for tubulin by immunofluorescence and photographed on a confocal microscope. Proplatelets show periodic segments connected by thin cytoplasmic bridges, but only the ends (arrowheads) have microtubule bundles arranged into teardrop-shaped loops. (e and f) Higher magnification electron micrograph of a swelling along a proplatelet shaft showing that these are points where the microtubule bundle separates for a short distance (arrows) but does not form a loop. Bars: (b and f) 0.5 μm; (c and e) 1 μm.
Mentions: Most released platelet-sized particles remain connected by cytoplasmic bridges, the most abundant being barbell forms composed of two platelet-like particles joined by one cytoplasmic strand. To ask whether the bulbous ends and thickenings along the shaft of proplatelets show mature cytoskeletal features, we examined representative released particles by antitubulin immunofluorescence confocal microscopy (Fig. 5 d) and electron microscopy (Fig. 5e and Fig. f). A microtubule bundle forms the core of the proplatelet shaft, and the ends have microtubule bundles forming teardrop-shaped loops that are similar in both size (1–3 μm) and appearance to those described in mature blood platelets (White 1968). Distorted microtubule bundles are also visible in platelet-sized swellings along the shaft of the proplatelet; however, in contrast to the terminal rings, those observed along the proplatelet shafts are uniformly less distinct (Fig. 6). Whereas these platelet-sized nodules superficially appear to include microtubule rings, close inspection reveals that they are actually points where microtubule bundles simply diverge for a short distance but fail to form the mature teardrop shape (Fig. 6, c–g). These nodules consistently reveal membrane and other detergent-insoluble materials, presumably destined for final platelet assembly elsewhere (compare Fig. 6, a and b). These results are entirely consistent with the dynamic activity that is observed at the sites of the platelet-sized nodules, including branching (Fig. 2 b), interconversion between condensed and lamellipodial forms (Fig. 1 and Fig. 2 a), and rapid translocation of particles (Fig. 2 c), and indicate that they are structurally unstable. More importantly, they establish that the mature cytoskeletal feature of a microtubule coil is only detected at the ends of proplatelets (Fig. 5) and not along their lengths.

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