Reconstitution in vitro of MSP‐based filopodium extension in nematode sperm

Abstract

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.

Structure, development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida)

“…Rhabditid sperm generally displays a conserved pattern during spermatogenesis known as the "rhabditid" pattern (Slos et al, 2015;Yushin et al, 2016;Yushin & Malakhov, 2014 A congruence test between specific traits shows a remarkable and strongly supported, correlated evolution between the presence of filopodia and the absence of MO (BF > 6). Both MOs and filopodia play a role in MSP assembly: filopodia appear to represent an intermediate stage in sperm activation, and agents that interfere with fibre growth likewise affect filopodia assembly (Miao, Yi, Mackey, & Roberts, 2007;Singaravelu & Singson, 2011); MOs meanwhile play an essential role in regulating MSP cytoskeleton assembly (Zhao et al, 2012). However, despite their similarities in function, a functional explanation for this correlated evolution remains elusive.…”

New Insights into the Mechanism of Fertilization in Nematodes

“…Similarly, spermatids from C. briggsae and C. remanei can also be activated by pronase treatment in vitro (Geldziler et al, 2006). Treatment of Ascaris spermatids with vas deference extract causes the extension of spikes or filopodia within 30 seconds (Miao et al, 2007). The spikes or filopodia appears to represent intermediate stage of the sperm activation.…”

“…Like C. elegans , extension of filopodia is immediately succeeded by the formation of pseudopod in Ascaris as well. Miao et al (2007) reconstituted filopodial growth in vitro and analyzed their dynamics and ultra structure. Electron microscopy of in vitro reconstituted filopodia revealed that the MSP fibers are bundled together to form a filament and the tip of the filaments are covered with membrane envelope.…”

“…Filopodia and MSP fibers share many common features. Several proteins found in MSP fibers, such as MFP1, MFP2, MFP3 (See sperm motility) are also found in filopodia; agents that interfere with fiber growth likewise affects filopodia assembly (Miao et al, 2007). …”

The physiological acquisition of amoeboid motility in nematode sperm: Is the tail the only thing the sperm lost?

“…Curiously, MPAK shares homology with the Casein kinase 1 SPE‐6 from C. elegans (Yi et al, 2007). MPAK, MFP2, and a tyrosine‐phosphorylated protein (putatively MPOP) are also involved in the extension of spikes or blebs during sperm activation and in the in vitro reconstitution of filopodia in A. suum (Rodriguez et al, 2005; Miao et al, 2007).…”