A 48 kDa integral membrane phosphoprotein orchestrates the cytoskeletal dynamics that generate amoeboid cell motility in Ascaris sperm

Abstract

Protrusion of the lamellipod in the crawling sperm of Ascaris is tightly coupled to the localized vectorial assembly and bundling of the major sperm protein cytoskeleton. In cell-free extracts of sperm, vesicles derived from the leading edge membrane reconstitute protrusion by directing the assembly of columnar meshworks of major sperm protein filaments that push the vesicle forward as they elongate. Treatment with proteases or a tyrosine phosphatase abolished vesicle activity, suggesting the involvement of a membrane phosphoprotein. Fractionation of vesicle proteins by sequential detergent lysis, size exclusion chromatography and immunoprecipitation with antiphosphotyrosine antibody identified a 48 kDa integral membrane phosphoprotein as the only sperm membrane component required to nucleate major sperm protein polymerization under physiological conditions. Immunolabeling assays showed that this protein is distributed uniformly in the sperm plasma membrane, but that its active phosphorylated form is located only at sites of major sperm protein polymerization at the leading edge. Because this protein specifies sites of cytoskeletal assembly, we have named it major sperm protein polymerization organizing protein (MPOP). The phosphorylation of MPOP is pH sensitive and appears to require a soluble tyrosine kinase. Comparison of the activity of MPOP to that of analogous membrane proteins in actin-based systems emphasizes the importance of precise transmission of information from the membrane to the cytoskeleton in amoeboid cell motility.

Cytosolic Ca2+ as a multifunctional modulator is required for spermiogenesis inAscaris suum

“…sperm are terminally differentiated cells and are quiescent transcriptionally and translationally, their maturation is highly dependent on post-translational modifi cations to the existing protein components. Protein phosphorylation and dephosphorylation of MSP cytoskeletal accessory proteins are necessary for modulating the assembly and disassembly of the MSP cytoskeleton at the leading and rear edges of the pseudopod, respectively (LeClaire et al, 2003;Miao et al, 2003;Yi et al, 2007;Yi et al, 2009). Phosphorylation sites in the MSP have also been identifi ed in C. elegans (Fraire-Zamora et al, 2011).…”

The geometry and motion of nematode sperm cells

“…The pH gradient also plays a role in the spatial separation of protrusion and contraction by acting as an on/off switch for the critical proteins involved in these processes. A tyrosine kinase phosphorylates MPOP (the membrane protein involved in protrusion) at the leading edge where the pH is higher, thereby activating the protein and enabling protrusion to occur [5]. Though not actively triggering cytoskeletal disassembly and retraction, the pH gradient may play an indirect role by activating a phosphatase that would in turn activate the protein involved in depolymerization, YOP [7].…”

Dissection of theAscarisSperm Motility Machinery Identifies Key Proteins Involved in Major Sperm Protein-based Amoeboid Locomotion

“…Ascaris were dissected and sperm activated as described (Sepsenwol et al, 1989). S100 was prepared by centrifugation of sperm extracts obtained by freeze-thaw lysis as described by LeClaire et al (2003).…”

“…The cell-free extract of Ascaris sperm (S100) that produces fibers upon addition of ATP can be separated into cytosolic and vesicle fractions by centrifugation; components from both fractions are required to assemble fibers (Italiano et al, 1996;LeClaire et al, 2003). To identify soluble proteins involved in MSP assembly dynamics, we fractionated cytosol by using a range of biochemical techniques and identified active fractions by their ability to generate fiber assembly when combined with vesicles, MSP, and ATP.…”

“…Previous work showed that in vitro reconstitution of motility involves polymerization of MSP at the vesicle surface (Italiano et al, 1996;Roberts et al, 1998) that is orchestrated by a 48-kDa membrane phosphoprotein (LeClaire et al, 2003). However, cytosolic proteins determine the rate of cytoskeletal assembly (Italiano et al, 1996), and information on these components is lacking.…”