scholarly journals Localized Depolymerization of the Major Sperm Protein Cytoskeleton Correlates with the Forward Movement of the Cell Body in the Amoeboid Movement of Nematode Sperm

1999 ◽  
Vol 146 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Joseph E. Italiano ◽  
Murray Stewart ◽  
Thomas M. Roberts
Keyword(s):  
Cell Body ◽  
Body Movement ◽  
Leading Edge ◽  
Amoeboid Movement ◽  
Major Sperm Protein ◽  
Membrane Protrusion ◽  
Forward Movement ◽  
Sperm Protein ◽  
Amoeboid Motility ◽  
Tightly Coupled

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.

scholarly journals Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm

2009 ◽  
Vol 20 (14) ◽  
pp. 3200-3208 ◽  
Author(s):  
Kexi Yi ◽  
Xu Wang ◽  
Mark R. Emmett ◽  
Alan G. Marshall ◽  
Murray Stewart ◽  
...  
Keyword(s):  
Cell Body ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Leading Edge ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
In Vitro Motility ◽  
Fiber Protein ◽  
Phosphatase 2A

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.

Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum

1992 ◽  
Vol 101 (4) ◽  
pp. 847-857 ◽  
Author(s):  
K.L. King ◽  
M. Stewart ◽  
T.M. Roberts ◽  
M. Seavy
Keyword(s):  
Polyethylene Glycol ◽  
Biochemical Analysis ◽  
Critical Concentration ◽  
Cytoskeletal Proteins ◽  
Amoeboid Movement ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Macromolecular Assembly ◽  
Amoeboid Motility ◽  
Simple Alternative

Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.

Supramolecular assemblies of the Ascaris suum major sperm protein (MSP) associated with amoeboid cell motility

1994 ◽  
Vol 107 (10) ◽  
pp. 2941-2949
Author(s):  
K.L. King ◽  
M. Stewart ◽  
T.M. Roberts
Keyword(s):  
Ascaris Suum ◽  
Leading Edge ◽  
Intrinsic Property ◽  
Basic Unit ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Amoeboid Cell ◽  
Left Handed ◽  
Self Association

Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of locomotion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.

scholarly journals A Ser/Thr Kinase Required for Membrane-associated Assembly of the Major Sperm Protein Motility Apparatus in the Amoeboid Sperm of Ascaris

2007 ◽  
Vol 18 (5) ◽  
pp. 1816-1825 ◽  
Author(s):  
Kexi Yi ◽  
Shawnna M. Buttery ◽  
Murray Stewart ◽  
Thomas M. Roberts
Keyword(s):  
Membrane Surface ◽  
Leading Edge ◽  
Accessory Proteins ◽  
Major Sperm Protein ◽  
Actin Assembly ◽  
Casein Kinase 1 ◽  
Sperm Protein ◽  
Fiber Protein ◽  
Sperm Extract

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.

How nematode sperm crawl

2002 ◽  
Vol 115 (2) ◽  
pp. 367-384 ◽  
Author(s):  
Dean Bottino ◽  
Alexander Mogilner ◽  
Tom Roberts ◽  
Murray Stewart ◽  
George Oster
Keyword(s):  
Cell Motility ◽  
Molecular Motors ◽  
Leading Edge ◽  
Element Model ◽  
Basic Mechanism ◽  
Actin Binding ◽  
Major Sperm Protein ◽  
Cellular Functions ◽  
Amoeboid Motility ◽  
Filament Bundles

Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple ‘stripped down’ version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.Movies available on-line.

Amoeboid Motility Without Actin: Insights into the Molecular Mechanism of Locomotion Using the Major Sperm Protein (MSP) of Nematodes

1998 ◽  
Vol 194 (3) ◽  
pp. 342-344 ◽  
Author(s):  
M. Stewart ◽  
T. M. Roberts ◽  
J. E. Italiano ◽  
K. L. King ◽  
R. Hammel ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Amoeboid Motility

scholarly journals Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

1998 ◽  
Vol 140 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Thomas M. Roberts ◽  
E.D. Salmon ◽  
Murray Stewart
Keyword(s):  
Plasma Membrane ◽  
Hydrostatic Pressure ◽  
Leading Edge ◽  
Major Sperm Protein ◽  
Sperm Protein ◽  
Fiber Assembly ◽  
Fiber Growth ◽  
Filament Assembly

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.

scholarly journals A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum.

1989 ◽  
Vol 108 (1) ◽  
pp. 55-66 ◽  
Author(s):  
S Sepsenwol ◽  
H Ris ◽  
T M Roberts
Keyword(s):  
Cell Body ◽  
Ascaris Suum ◽  
Leading Edge ◽  
Major Sperm Protein ◽  
Differential Interference Contrast Microscopy ◽  
Membrane Turnover ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Male Gametes

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.

scholarly journals The Role of Myosin II Motor Activity in Distributing Myosin Asymmetrically and Coupling Protrusive Activity to Cell Translocation

2006 ◽  
Vol 17 (10) ◽  
pp. 4435-4445 ◽  
Author(s):  
John Kolega
Keyword(s):  
Motor Activity ◽  
Cell Body ◽  
Body Movement ◽  
Myosin Ii ◽  
Time Lapse ◽  
The Body ◽  
Myosin Filament ◽  
Forward Movement ◽  
Myosin Iia ◽  
And Function

Nonmuscle myosin IIA and IIB distribute preferentially toward opposite ends of migrating endothelial cells. To understand the mechanism and function of this behavior, myosin II was examined in cells treated with the motor inhibitor, blebbistatin. Blebbistatin at ≥30 μM inhibited anterior redistribution of myosin IIA, with 100 μM blebbistatin causing posterior accumulation. Posterior accumulation of myosin IIB was unaffected. Time-lapse cinemicrography showed myosin IIA entering lamellipodia shortly after their formation, but failing to move into lamellipodia in blebbistatin. Thus, myosin II requires motor activity to move forward onto F-actin in protrusions. However, this movement is inhibited by myosin filament assembly, because whole myosin was delayed relative to a tailless fragment. Inhibiting myosin's forward movement reduced coupling between protrusive activity and translocation of the cell body: In untreated cells, body movement followed advancing lamellipodia, whereas blebbistatin-treated cells extended protrusions without displacement of the body or with a longer delay before movement. Anterior cytoplasm of blebbistatin-treated cells contained disorganized bundles of parallel microfilaments, but anterior F-actin bundles in untreated cells were mostly oriented perpendicular to movement. Myosin II may ordinarily move anteriorly on actin filaments and pull crossed filaments into antiparallel bundles, with the resulting realignment pulling the cell body forward.

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