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Localized depolymerization of the major sperm protein cytoskeleton correlates with the forward movement of the cell body in the amoeboid movement of nematode sperm.

Italiano JE, Stewart M, Roberts TM - J. Cell Biol. (1999)

Bottom Line: At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued.The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer.These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida 32306, USA.

ABSTRACT
The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.

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Treatment of sperm with acetate buffer, pH 6.75, uncouples protrusion from retraction. The positions of the cell body and the leading edge, when the cell was perfused with acetate buffer (a), are outlined in white in a–d. During this sequence (elapsed time, 30 s) part of the leading edge protruded slowly (3 μm/min; b–d) while an adjacent portion, toward the top of the frame (c), retracted slightly. Thus, treatment with acetate buffer slowed cytoskeletal assembly dramatically. However, cytoskeletal disassembly was not inhibited and so the lamellipodium and the fiber complexes within shortened and the cell body continued to move forward at 15 μm/min. The black arrow indicates a kink in a fiber complex that moved rearward with respect to the leading edge during the sequence. This indicates that cytoskeletal treadmilling persists even when the rate of protrusion slowed. Note that the distance between the cell body and this kink decreases throughout the sequence, due to continued disassembly at the base of the lamellipodium. The white arrows indicate refringent spots that maintained their position in the cell body during retraction. This shows that the cell body moves forward without rolling. Interval between frames, 10 s. Bar, 10 μm.
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Figure 3: Treatment of sperm with acetate buffer, pH 6.75, uncouples protrusion from retraction. The positions of the cell body and the leading edge, when the cell was perfused with acetate buffer (a), are outlined in white in a–d. During this sequence (elapsed time, 30 s) part of the leading edge protruded slowly (3 μm/min; b–d) while an adjacent portion, toward the top of the frame (c), retracted slightly. Thus, treatment with acetate buffer slowed cytoskeletal assembly dramatically. However, cytoskeletal disassembly was not inhibited and so the lamellipodium and the fiber complexes within shortened and the cell body continued to move forward at 15 μm/min. The black arrow indicates a kink in a fiber complex that moved rearward with respect to the leading edge during the sequence. This indicates that cytoskeletal treadmilling persists even when the rate of protrusion slowed. Note that the distance between the cell body and this kink decreases throughout the sequence, due to continued disassembly at the base of the lamellipodium. The white arrows indicate refringent spots that maintained their position in the cell body during retraction. This shows that the cell body moves forward without rolling. Interval between frames, 10 s. Bar, 10 μm.

Mentions: To probe the mechanism of cell body retraction and its role in sperm locomotion, we used pH to uncouple the cytoskeletal assembly and disassembly that occur at opposite ends of the lamellipodium. The MSP cytoskeleton is sensitive to intracellular pH (Roberts and King 1991; King et al. 1994). We found that treating sperm with HKB buffer containing 20 mM sodium acetate (HKB-acetate) at different pH values caused cytoskeletal assembly and protrusion of the leading edge to either slow dramatically or stop entirely, while cytoskeletal disassembly and retraction of the cell body continued unaltered. The cell shown in Fig. 3, for example, was crawling at 15 μm/min until it was perfused with HKB-acetate, pH 6.75. This treatment caused protrusion of the leading edge to slow to <3 μm/min. However, retraction of the cell body continued at 15 μm/min for a further 30 s, then stopped. This continuing retraction of the cell body appeared to be correlated with localized disassembly of the fiber complexes near the cell body because the distance between the cell body–lamellipodium junction and distinctive morphological features of the fiber complexes decreased as the cell body moved forward. Moreover, the cell body appeared to move forward due to shortening of the lamellipodium rather than rolling forward over the rear of the lamellipodium. If the cell body was rolling, the organelles within it should also roll, but this was not observed. Instead, the organelles maintained their position in the cell body as it retracted (Fig. 3).


Localized depolymerization of the major sperm protein cytoskeleton correlates with the forward movement of the cell body in the amoeboid movement of nematode sperm.

Italiano JE, Stewart M, Roberts TM - J. Cell Biol. (1999)

Treatment of sperm with acetate buffer, pH 6.75, uncouples protrusion from retraction. The positions of the cell body and the leading edge, when the cell was perfused with acetate buffer (a), are outlined in white in a–d. During this sequence (elapsed time, 30 s) part of the leading edge protruded slowly (3 μm/min; b–d) while an adjacent portion, toward the top of the frame (c), retracted slightly. Thus, treatment with acetate buffer slowed cytoskeletal assembly dramatically. However, cytoskeletal disassembly was not inhibited and so the lamellipodium and the fiber complexes within shortened and the cell body continued to move forward at 15 μm/min. The black arrow indicates a kink in a fiber complex that moved rearward with respect to the leading edge during the sequence. This indicates that cytoskeletal treadmilling persists even when the rate of protrusion slowed. Note that the distance between the cell body and this kink decreases throughout the sequence, due to continued disassembly at the base of the lamellipodium. The white arrows indicate refringent spots that maintained their position in the cell body during retraction. This shows that the cell body moves forward without rolling. Interval between frames, 10 s. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Treatment of sperm with acetate buffer, pH 6.75, uncouples protrusion from retraction. The positions of the cell body and the leading edge, when the cell was perfused with acetate buffer (a), are outlined in white in a–d. During this sequence (elapsed time, 30 s) part of the leading edge protruded slowly (3 μm/min; b–d) while an adjacent portion, toward the top of the frame (c), retracted slightly. Thus, treatment with acetate buffer slowed cytoskeletal assembly dramatically. However, cytoskeletal disassembly was not inhibited and so the lamellipodium and the fiber complexes within shortened and the cell body continued to move forward at 15 μm/min. The black arrow indicates a kink in a fiber complex that moved rearward with respect to the leading edge during the sequence. This indicates that cytoskeletal treadmilling persists even when the rate of protrusion slowed. Note that the distance between the cell body and this kink decreases throughout the sequence, due to continued disassembly at the base of the lamellipodium. The white arrows indicate refringent spots that maintained their position in the cell body during retraction. This shows that the cell body moves forward without rolling. Interval between frames, 10 s. Bar, 10 μm.
Mentions: To probe the mechanism of cell body retraction and its role in sperm locomotion, we used pH to uncouple the cytoskeletal assembly and disassembly that occur at opposite ends of the lamellipodium. The MSP cytoskeleton is sensitive to intracellular pH (Roberts and King 1991; King et al. 1994). We found that treating sperm with HKB buffer containing 20 mM sodium acetate (HKB-acetate) at different pH values caused cytoskeletal assembly and protrusion of the leading edge to either slow dramatically or stop entirely, while cytoskeletal disassembly and retraction of the cell body continued unaltered. The cell shown in Fig. 3, for example, was crawling at 15 μm/min until it was perfused with HKB-acetate, pH 6.75. This treatment caused protrusion of the leading edge to slow to <3 μm/min. However, retraction of the cell body continued at 15 μm/min for a further 30 s, then stopped. This continuing retraction of the cell body appeared to be correlated with localized disassembly of the fiber complexes near the cell body because the distance between the cell body–lamellipodium junction and distinctive morphological features of the fiber complexes decreased as the cell body moved forward. Moreover, the cell body appeared to move forward due to shortening of the lamellipodium rather than rolling forward over the rear of the lamellipodium. If the cell body was rolling, the organelles within it should also roll, but this was not observed. Instead, the organelles maintained their position in the cell body as it retracted (Fig. 3).

Bottom Line: At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued.The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer.These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida 32306, USA.

ABSTRACT
The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.

Show MeSH
Related in: MedlinePlus