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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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Effect of cytoskeletal disrupting agents on proplatelet growth. (a) Gallery of phase-contrast micrographs of megakaryocytes treated with 2 μM cytochalasin B. Extensions from the megakaryocyte surface show reduced beading and absence of branching. (b) Images showing a time series at a higher magnification of processes extending from the megakaryocyte surface in the presence of 2 μM cytochalasin B. The white arrowheads indicate the tip of one extension, which remains almost stationary while the diameter of the loop formed by the extension increases with time. (c) Effect of taxol on proplatelet formation. Phase-contrast images showing a time series of a tubelike extension growing from a taxol-treated megakaryocyte. In the presence of 10 μM taxol, the megakaryocyte forms a single pseudopodia. As this tube elongates, its distal tip bends and curves back on itself (0–1 min) until its tip contacts its shaft and appears to fuse (2 min). This forms a teardrop-shaped structure on the end of the projection. (d) Antitubulin immunofluorescence staining of a megakaryocyte cultured in the presence of 10 μM taxol. Taxol treatment reduces the number of extensions made by cells. Microtubule staining concentrates at the cell edge and in the proplatelet-like extensions grown from the cell surface. The microtubule bundles in the shafts are thickened compared with those extended by untreated cells, and are observed to curl within the bulbous ends. Bars: (b) 10 μm; (c) 25 μm.
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Figure 7: Effect of cytoskeletal disrupting agents on proplatelet growth. (a) Gallery of phase-contrast micrographs of megakaryocytes treated with 2 μM cytochalasin B. Extensions from the megakaryocyte surface show reduced beading and absence of branching. (b) Images showing a time series at a higher magnification of processes extending from the megakaryocyte surface in the presence of 2 μM cytochalasin B. The white arrowheads indicate the tip of one extension, which remains almost stationary while the diameter of the loop formed by the extension increases with time. (c) Effect of taxol on proplatelet formation. Phase-contrast images showing a time series of a tubelike extension growing from a taxol-treated megakaryocyte. In the presence of 10 μM taxol, the megakaryocyte forms a single pseudopodia. As this tube elongates, its distal tip bends and curves back on itself (0–1 min) until its tip contacts its shaft and appears to fuse (2 min). This forms a teardrop-shaped structure on the end of the projection. (d) Antitubulin immunofluorescence staining of a megakaryocyte cultured in the presence of 10 μM taxol. Taxol treatment reduces the number of extensions made by cells. Microtubule staining concentrates at the cell edge and in the proplatelet-like extensions grown from the cell surface. The microtubule bundles in the shafts are thickened compared with those extended by untreated cells, and are observed to curl within the bulbous ends. Bars: (b) 10 μm; (c) 25 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)

Effect of cytoskeletal disrupting agents on proplatelet growth. (a) Gallery of phase-contrast micrographs of megakaryocytes treated with 2 μM cytochalasin B. Extensions from the megakaryocyte surface show reduced beading and absence of branching. (b) Images showing a time series at a higher magnification of processes extending from the megakaryocyte surface in the presence of 2 μM cytochalasin B. The white arrowheads indicate the tip of one extension, which remains almost stationary while the diameter of the loop formed by the extension increases with time. (c) Effect of taxol on proplatelet formation. Phase-contrast images showing a time series of a tubelike extension growing from a taxol-treated megakaryocyte. In the presence of 10 μM taxol, the megakaryocyte forms a single pseudopodia. As this tube elongates, its distal tip bends and curves back on itself (0–1 min) until its tip contacts its shaft and appears to fuse (2 min). This forms a teardrop-shaped structure on the end of the projection. (d) Antitubulin immunofluorescence staining of a megakaryocyte cultured in the presence of 10 μM taxol. Taxol treatment reduces the number of extensions made by cells. Microtubule staining concentrates at the cell edge and in the proplatelet-like extensions grown from the cell surface. The microtubule bundles in the shafts are thickened compared with those extended by untreated cells, and are observed to curl within the bulbous ends. Bars: (b) 10 μm; (c) 25 μm.
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Related In: Results  -  Collection

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Figure 7: Effect of cytoskeletal disrupting agents on proplatelet growth. (a) Gallery of phase-contrast micrographs of megakaryocytes treated with 2 μM cytochalasin B. Extensions from the megakaryocyte surface show reduced beading and absence of branching. (b) Images showing a time series at a higher magnification of processes extending from the megakaryocyte surface in the presence of 2 μM cytochalasin B. The white arrowheads indicate the tip of one extension, which remains almost stationary while the diameter of the loop formed by the extension increases with time. (c) Effect of taxol on proplatelet formation. Phase-contrast images showing a time series of a tubelike extension growing from a taxol-treated megakaryocyte. In the presence of 10 μM taxol, the megakaryocyte forms a single pseudopodia. As this tube elongates, its distal tip bends and curves back on itself (0–1 min) until its tip contacts its shaft and appears to fuse (2 min). This forms a teardrop-shaped structure on the end of the projection. (d) Antitubulin immunofluorescence staining of a megakaryocyte cultured in the presence of 10 μM taxol. Taxol treatment reduces the number of extensions made by cells. Microtubule staining concentrates at the cell edge and in the proplatelet-like extensions grown from the cell surface. The microtubule bundles in the shafts are thickened compared with those extended by untreated cells, and are observed to curl within the bulbous ends. Bars: (b) 10 μm; (c) 25 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