The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.
Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.
The major sperm protein (MSP) motility system in nematode sperm is best known for propelling the movement of mature sperm, where it has taken over the role usually played by actin in amoeboid cell motility. However, MSP filaments also drive the extension of filopodia, transient organelles composed of a core bundle of MSP filaments, that form in the late in sperm development but are not found on crawling cells. We have reconstituted filopodial extension in vitro whereby thin bundles of MSP filaments, each enveloped by a membrane sheath at their growing end, elongated at rates up to 17 microm/min. These bundles often exceeded 500 microm in length but were comprised of filaments only 1 microm long. The reconstituted filopodia assembled in the same cell-free sperm extracts that produced MSP fibers, robust meshworks of filaments that exhibit the same organization and dynamics as the lamellipodial filament system that propels sperm movement. The filopodia and fibers that assembled in vitro both had a membranous structure at their growing end, shared four MSP accessory proteins, and responded identically to agents that alter MSP-based motility by modulating protein phosphorylation. However, filopodia grew three- to four-fold faster than fibers. The reconstitution of filopodial extension shows that, like the actin cytoskeleton, MSP filaments can adopt two architectures, bundles and meshworks, each capable of pushing against membranes to generate protrusion. The reconstitution of both forms of motility in the same in vitro system provides a promising avenue for understanding how the forces for membrane protrusion are produced.
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