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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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Cytoskeletal organization at proplatelet bends. (a) Representative micrograph showing the cytoskeleton of a proplatelet shaft. Parallel bundles of microtubules line the tube and prospective branch points are defined by outpouchings of 10-nm filaments. (b) Micrograph showing a platelet-sized swelling along the length of a proplatelet that has spread on the surface. This spread region contains a dense meshwork of F-actin, with ends that are collapsed and bound to the sides of the microtubule bundles. Microtubule bundles are bent and appear to have separated. A meshwork of spread actin filaments is observed extending from the platelet-sized segment. (c) Micrograph showing a pronounced bend in a proplatelet. The elbow of the bend contains aggregates of filamentous material, which contact the substratum in the elbow region. Bar, 1 μm.
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Figure 8: Cytoskeletal organization at proplatelet bends. (a) Representative micrograph showing the cytoskeleton of a proplatelet shaft. Parallel bundles of microtubules line the tube and prospective branch points are defined by outpouchings of 10-nm filaments. (b) Micrograph showing a platelet-sized swelling along the length of a proplatelet that has spread on the surface. This spread region contains a dense meshwork of F-actin, with ends that are collapsed and bound to the sides of the microtubule bundles. Microtubule bundles are bent and appear to have separated. A meshwork of spread actin filaments is observed extending from the platelet-sized segment. (c) Micrograph showing a pronounced bend in a proplatelet. The elbow of the bend contains aggregates of filamentous material, which contact the substratum in the elbow region. Bar, 1 μm.

Mentions: The separation of proplatelet morphogenesis into stages characterized by specific alterations of the microtubule cytoskeleton and the recognition of distinct dynamic features provide approaches to investigation of the underlying molecular mechanisms. Megakaryocytes cultured in the presence of cytochalasin B, an inhibitor of actin assembly, retain the capacity to extend long, slender proplatelet-like projections (Fig. 7 a) but show specific abnormal features. The same results were observed with cytochalasin D. The cell body fails to spread, proplatelets extend from multiple points around the cell margin instead of the typical single erosion site, branching is completely inhibited, and although proplatelet processes retain bulbous ends, they harbor many fewer intermediate swellings. Thus, actin assembly is not required for megakaryocytes to extend proplatelets but is essential for proplatelet branching. Interestingly, one process associated with proplatelet bending is the attachment of a small region to the substratum over a period of 5–10 min and robust ruffling activity of this portion (data not shown), movements classically thought to be mediated by actin. In the electron microscope, the microtubule bundles composing the shaft routinely reveal small filamentous outpouchings (Fig. 8 a). At sites of bending, meshworks of actin filaments extrude processes that connect the microtubule bundles much like tendons attaching muscle to bone (Fig. 8 b). At more pronounced bends, the apparent sites of proplatelet branching, filamentous aggregates form a cusp between the microtubule bundles (Fig. 8 c). Considered together, these observations demonstrate that actin filaments are enriched at the sites of proplatelet bifuraction and probably required to execute this process.


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)

Cytoskeletal organization at proplatelet bends. (a) Representative micrograph showing the cytoskeleton of a proplatelet shaft. Parallel bundles of microtubules line the tube and prospective branch points are defined by outpouchings of 10-nm filaments. (b) Micrograph showing a platelet-sized swelling along the length of a proplatelet that has spread on the surface. This spread region contains a dense meshwork of F-actin, with ends that are collapsed and bound to the sides of the microtubule bundles. Microtubule bundles are bent and appear to have separated. A meshwork of spread actin filaments is observed extending from the platelet-sized segment. (c) Micrograph showing a pronounced bend in a proplatelet. The elbow of the bend contains aggregates of filamentous material, which contact the substratum in the elbow region. Bar, 1 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2168104&req=5

Figure 8: Cytoskeletal organization at proplatelet bends. (a) Representative micrograph showing the cytoskeleton of a proplatelet shaft. Parallel bundles of microtubules line the tube and prospective branch points are defined by outpouchings of 10-nm filaments. (b) Micrograph showing a platelet-sized swelling along the length of a proplatelet that has spread on the surface. This spread region contains a dense meshwork of F-actin, with ends that are collapsed and bound to the sides of the microtubule bundles. Microtubule bundles are bent and appear to have separated. A meshwork of spread actin filaments is observed extending from the platelet-sized segment. (c) Micrograph showing a pronounced bend in a proplatelet. The elbow of the bend contains aggregates of filamentous material, which contact the substratum in the elbow region. Bar, 1 μm.
Mentions: The separation of proplatelet morphogenesis into stages characterized by specific alterations of the microtubule cytoskeleton and the recognition of distinct dynamic features provide approaches to investigation of the underlying molecular mechanisms. Megakaryocytes cultured in the presence of cytochalasin B, an inhibitor of actin assembly, retain the capacity to extend long, slender proplatelet-like projections (Fig. 7 a) but show specific abnormal features. The same results were observed with cytochalasin D. The cell body fails to spread, proplatelets extend from multiple points around the cell margin instead of the typical single erosion site, branching is completely inhibited, and although proplatelet processes retain bulbous ends, they harbor many fewer intermediate swellings. Thus, actin assembly is not required for megakaryocytes to extend proplatelets but is essential for proplatelet branching. Interestingly, one process associated with proplatelet bending is the attachment of a small region to the substratum over a period of 5–10 min and robust ruffling activity of this portion (data not shown), movements classically thought to be mediated by actin. In the electron microscope, the microtubule bundles composing the shaft routinely reveal small filamentous outpouchings (Fig. 8 a). At sites of bending, meshworks of actin filaments extrude processes that connect the microtubule bundles much like tendons attaching muscle to bone (Fig. 8 b). At more pronounced bends, the apparent sites of proplatelet branching, filamentous aggregates form a cusp between the microtubule bundles (Fig. 8 c). Considered together, these observations demonstrate that actin filaments are enriched at the sites of proplatelet bifuraction and probably required to execute this process.

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