Limits...
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.

Show MeSH

Related in: MedlinePlus

Inhibition of polymerization and depolymerization with PAO completely stops motility. a and b, A crawling sperm before and 15 s after treatment with 30 μM PAO. After 3 min in PAO (c), there was no movement of the cell or treadmilling of its cytoskeleton. For example, the arrows in b and c indicate a bend in a fiber complex that did not change position while the cell was incubated in PAO. However, within 15 s after washing the cell with HKB containing 5 mM dimercaptopropanol (a PAO antagonist), locomotion and cytoskeletal treadmilling resumed (d). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169480&req=5

Figure 6: Inhibition of polymerization and depolymerization with PAO completely stops motility. a and b, A crawling sperm before and 15 s after treatment with 30 μM PAO. After 3 min in PAO (c), there was no movement of the cell or treadmilling of its cytoskeleton. For example, the arrows in b and c indicate a bend in a fiber complex that did not change position while the cell was incubated in PAO. However, within 15 s after washing the cell with HKB containing 5 mM dimercaptopropanol (a PAO antagonist), locomotion and cytoskeletal treadmilling resumed (d). Bar, 10 μm.

Mentions: Manipulation of intracellular pH allowed us to uncouple cytoskeletal assembly from disassembly and thereby examine the separate roles of these processes in locomotion. We sought to determine if other factors are involved in locomotion by identifying a method for keeping the cytoskeleton intact, but blocking both polymerization and depolymerization. Previously, we had shown that antiphosphotyrosine antibodies stained the leading edge of the pseudopod preferentially (Italiano et al. 1996) and so we treated sperm with 30 μM phenylarsine oxide (PAO), a protein tyrosine phosphatase inhibitor. In PAO-treated cells, the fiber complexes remained clearly visible, but we were unable to detect either cytoskeletal flow or locomotion (Fig. 6). However, the effect of the drug was completely reversible; cytoskeletal treadmilling and locomotion resumed within 10 s after washing the cells with a PAO antagonist, dimercaptopropanol, at 5 mM in HKB buffer. Thus, without the forces associated with cytoskeletal polymerization and depolymerization, sperm exhibit no detectable motility.


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)

Inhibition of polymerization and depolymerization with PAO completely stops motility. a and b, A crawling sperm before and 15 s after treatment with 30 μM PAO. After 3 min in PAO (c), there was no movement of the cell or treadmilling of its cytoskeleton. For example, the arrows in b and c indicate a bend in a fiber complex that did not change position while the cell was incubated in PAO. However, within 15 s after washing the cell with HKB containing 5 mM dimercaptopropanol (a PAO antagonist), locomotion and cytoskeletal treadmilling resumed (d). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169480&req=5

Figure 6: Inhibition of polymerization and depolymerization with PAO completely stops motility. a and b, A crawling sperm before and 15 s after treatment with 30 μM PAO. After 3 min in PAO (c), there was no movement of the cell or treadmilling of its cytoskeleton. For example, the arrows in b and c indicate a bend in a fiber complex that did not change position while the cell was incubated in PAO. However, within 15 s after washing the cell with HKB containing 5 mM dimercaptopropanol (a PAO antagonist), locomotion and cytoskeletal treadmilling resumed (d). Bar, 10 μm.
Mentions: Manipulation of intracellular pH allowed us to uncouple cytoskeletal assembly from disassembly and thereby examine the separate roles of these processes in locomotion. We sought to determine if other factors are involved in locomotion by identifying a method for keeping the cytoskeleton intact, but blocking both polymerization and depolymerization. Previously, we had shown that antiphosphotyrosine antibodies stained the leading edge of the pseudopod preferentially (Italiano et al. 1996) and so we treated sperm with 30 μM phenylarsine oxide (PAO), a protein tyrosine phosphatase inhibitor. In PAO-treated cells, the fiber complexes remained clearly visible, but we were unable to detect either cytoskeletal flow or locomotion (Fig. 6). However, the effect of the drug was completely reversible; cytoskeletal treadmilling and locomotion resumed within 10 s after washing the cells with a PAO antagonist, dimercaptopropanol, at 5 mM in HKB buffer. Thus, without the forces associated with cytoskeletal polymerization and depolymerization, sperm exhibit no detectable motility.

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