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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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Structure of the megakaryocyte cytoskeleton before and during early proplatelet formation. (a and b) Structure of a megakaryocyte cytoskeleton lacking extensions. (a) Electron micrograph showing the structure of the cortical cytoskeleton in a megakarytocyte. A meshwork of actin filaments and microtubules is present in the cell cortex. Individual microtubules radiate outward from the cell center. (b) Electron micrograph showing the structure of the megakaryocyte cytoskeleton near its nuclear mass (N). Microtubules gather into arrays in the cell center. (right inset) Morphology of megakaryocyte in the light microscope photographed with phase contrast optics. (left inset) Confocal photograph of an early stage megakaryocyte stained with antitubulin antibody. Arrays of microtubules originate near the cell center and radiate out into the cell cortex. (c) Electron micrograph of a more mature megakaryocyte cytoskeleton before pseudopodia formation. Microtubules become densely packed into cortical bundles situated parallel to and just beneath the plasma membrane. (d) Organization of microtubules within pseudopodia. These structures contain dense rims composed of large bundles of microtubules. (Inset) Antitubulin immunofluorescent staining reveals the concentration of microtubules at the margins of the pseudopodia. Bars, 1 μm.
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Figure 4: Structure of the megakaryocyte cytoskeleton before and during early proplatelet formation. (a and b) Structure of a megakaryocyte cytoskeleton lacking extensions. (a) Electron micrograph showing the structure of the cortical cytoskeleton in a megakarytocyte. A meshwork of actin filaments and microtubules is present in the cell cortex. Individual microtubules radiate outward from the cell center. (b) Electron micrograph showing the structure of the megakaryocyte cytoskeleton near its nuclear mass (N). Microtubules gather into arrays in the cell center. (right inset) Morphology of megakaryocyte in the light microscope photographed with phase contrast optics. (left inset) Confocal photograph of an early stage megakaryocyte stained with antitubulin antibody. Arrays of microtubules originate near the cell center and radiate out into the cell cortex. (c) Electron micrograph of a more mature megakaryocyte cytoskeleton before pseudopodia formation. Microtubules become densely packed into cortical bundles situated parallel to and just beneath the plasma membrane. (d) Organization of microtubules within pseudopodia. These structures contain dense rims composed of large bundles of microtubules. (Inset) Antitubulin immunofluorescent staining reveals the concentration of microtubules at the margins of the pseudopodia. Bars, 1 μm.

Mentions: Microtubule assembly has been previously implicated in platelet formation (Handagama et al. 1987; Radley and Hatshorn 1987; Tablin et al. 1990), and our studies confirm its essential role in the progression of initial pseudopodia into proplatelets. The microtubule disrupting agent nocodazole (1–10 μM) completely inhibits formation of all megakaryocyte projections (Tablin et al. 1990). When nocodazole is added to megakaryocytes after they have formed proplatelet processes, particle transport is completely blocked (data not shown), indicating the requirement for a microtubule-based motile apparatus in this process. To understand the structural basis of the transitions between the megakaryocyte cell body, pseudopodia, proplatelet processes, and nascent platelets, we examined the microtubule cytoskeletons of proplatelet-producing megakaryocytes. Early in the maturation process, before spreading and erosion of the cell body begin, the megakaryocyte cytoplasm is replete with long individual arrays of microtubules that cluster around the nucleus and radiate toward the cell margins (Fig. 4, a and b). As large and blunt pseudopodia are formed in the area of erosion, the microtubules consolidate into cortical bundles situated just beneath the membrane surface of the protrusion (Fig. 4 c). As the pseudopodia extend and become thinner, they display a prominent band of microtubules along their edges; in some cases, these bundles are seen to curl or spiral inside the pseudopodia (Fig. 4 d). The next recognizable step is the conversion of the blunt pseudopodia, via elongation, into cytoplasmic extensions that continue to harbor thick bundles of microtubules (Fig. 5 a). These processes always have bulbous ends, and electron microscopy reveals a microtubule bundle that loops just beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure (Fig. 5b and Fig. c).


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)

Structure of the megakaryocyte cytoskeleton before and during early proplatelet formation. (a and b) Structure of a megakaryocyte cytoskeleton lacking extensions. (a) Electron micrograph showing the structure of the cortical cytoskeleton in a megakarytocyte. A meshwork of actin filaments and microtubules is present in the cell cortex. Individual microtubules radiate outward from the cell center. (b) Electron micrograph showing the structure of the megakaryocyte cytoskeleton near its nuclear mass (N). Microtubules gather into arrays in the cell center. (right inset) Morphology of megakaryocyte in the light microscope photographed with phase contrast optics. (left inset) Confocal photograph of an early stage megakaryocyte stained with antitubulin antibody. Arrays of microtubules originate near the cell center and radiate out into the cell cortex. (c) Electron micrograph of a more mature megakaryocyte cytoskeleton before pseudopodia formation. Microtubules become densely packed into cortical bundles situated parallel to and just beneath the plasma membrane. (d) Organization of microtubules within pseudopodia. These structures contain dense rims composed of large bundles of microtubules. (Inset) Antitubulin immunofluorescent staining reveals the concentration of microtubules at the margins of the pseudopodia. Bars, 1 μm.
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Related In: Results  -  Collection

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Figure 4: Structure of the megakaryocyte cytoskeleton before and during early proplatelet formation. (a and b) Structure of a megakaryocyte cytoskeleton lacking extensions. (a) Electron micrograph showing the structure of the cortical cytoskeleton in a megakarytocyte. A meshwork of actin filaments and microtubules is present in the cell cortex. Individual microtubules radiate outward from the cell center. (b) Electron micrograph showing the structure of the megakaryocyte cytoskeleton near its nuclear mass (N). Microtubules gather into arrays in the cell center. (right inset) Morphology of megakaryocyte in the light microscope photographed with phase contrast optics. (left inset) Confocal photograph of an early stage megakaryocyte stained with antitubulin antibody. Arrays of microtubules originate near the cell center and radiate out into the cell cortex. (c) Electron micrograph of a more mature megakaryocyte cytoskeleton before pseudopodia formation. Microtubules become densely packed into cortical bundles situated parallel to and just beneath the plasma membrane. (d) Organization of microtubules within pseudopodia. These structures contain dense rims composed of large bundles of microtubules. (Inset) Antitubulin immunofluorescent staining reveals the concentration of microtubules at the margins of the pseudopodia. Bars, 1 μm.
Mentions: Microtubule assembly has been previously implicated in platelet formation (Handagama et al. 1987; Radley and Hatshorn 1987; Tablin et al. 1990), and our studies confirm its essential role in the progression of initial pseudopodia into proplatelets. The microtubule disrupting agent nocodazole (1–10 μM) completely inhibits formation of all megakaryocyte projections (Tablin et al. 1990). When nocodazole is added to megakaryocytes after they have formed proplatelet processes, particle transport is completely blocked (data not shown), indicating the requirement for a microtubule-based motile apparatus in this process. To understand the structural basis of the transitions between the megakaryocyte cell body, pseudopodia, proplatelet processes, and nascent platelets, we examined the microtubule cytoskeletons of proplatelet-producing megakaryocytes. Early in the maturation process, before spreading and erosion of the cell body begin, the megakaryocyte cytoplasm is replete with long individual arrays of microtubules that cluster around the nucleus and radiate toward the cell margins (Fig. 4, a and b). As large and blunt pseudopodia are formed in the area of erosion, the microtubules consolidate into cortical bundles situated just beneath the membrane surface of the protrusion (Fig. 4 c). As the pseudopodia extend and become thinner, they display a prominent band of microtubules along their edges; in some cases, these bundles are seen to curl or spiral inside the pseudopodia (Fig. 4 d). The next recognizable step is the conversion of the blunt pseudopodia, via elongation, into cytoplasmic extensions that continue to harbor thick bundles of microtubules (Fig. 5 a). These processes always have bulbous ends, and electron microscopy reveals a microtubule bundle that loops just beneath the plasma membrane and reenters the shaft to form a teardrop-shaped structure (Fig. 5b and Fig. c).

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