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.

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 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.

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.

Microtubules associate with actin-containing filaments at discrete sites along the ventral surface of Allogromia reticulopods

1988 ◽  
Vol 89 (3) ◽  
pp. 297-307
Author(s):  
S.S. Bowser ◽  
J.L. Travis ◽  
C.L. Rieder
Keyword(s):  
Electron Microscopy ◽  
Cell Body ◽  
High Voltage ◽  
Ventral Surface ◽  
Filamentous Actin ◽  
High Voltage Electron Microscopy ◽  
Fluorescence Studies ◽  
Voltage Electron ◽  
Tubulin Antibodies ◽  
Double Label

We have investigated the distribution of actin and microtubules in pseudopodial networks (reticulopods) of the protozoan Allogromia sp., strain NF, in order to help elucidate the respective roles these components play in network organization and motility. Double-label fluorescence studies with tubulin antibodies and tetramethyl-rhodamine (TMR)-phalloidin reveal that microtubules and filamentous actin co-localize in regions where trunk pseudopods contact the substratum and splay to form the pseudopodial network; distal to these regions the network contains numerous microtubules but little or no F-actin. Similar results were obtained using various commercial actin antibodies. Correlative anti-actin immunofluorescence and high-voltage electron microscopy of serial 0.25 micron sections reveal that actin is contained within discrete electron-opaque, fan-shaped structures distributed along the cytoplasmic aspect of the ventral reticulopodial membrane. Electron microscopy of serial 100 nm sections from conventionally fixed specimens confirms that these actin-rich plaques are composed of a felt of roughly parallel, 5 nm diameter filaments. A subset of parallel and often bundled microtubules is enmeshed within, or contacts the periphery of, these filament plaques. Upon leaving a plaque, bundled microtubules frequently splay into smaller bundles. These observations are consistent with the hypothesis that interactions between microtubules and actin-containing microfilaments, particularly at substratum adhesion points, are involved in various aspects of reticulopodial motility, particularly network morphogenesis and cell body locomotion.

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.

High Voltage Electron Microscopy of Cultured Muscle Cells

1976 ◽  
Vol 34 ◽  
pp. 76-77
Author(s):  
Jerry W. Shay
Keyword(s):  
High Voltage ◽  
Three Dimensional ◽  
Muscle Differentiation ◽  
Biological Research ◽  
Muscular Tissue ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron

It is known from earlier observations that the in vitro rat L6 muscle cell line exhibits many of the features characteristic of in vivo muscle differentiation. The appearance of multinucleated cells, the development of highly organized contractile proteins and the phenomenon of muscle contraction are all easily recognizable features unique to muscular tissue. The L6 myoblasts fuse in a rather homogeneous and synchronous fashion to form myotubes and offer an excellent in vitro system to study the general mechanisms of muscle differentiation.The availability of the high voltage electron microscope (H.V.E.M.) in recent years for biological research has brought about a new interest in the three dimensional organization of components within whole cells, and the present study on L6 cells was undertaken, using the Jeol-1000 facility, at the University of Colorado in Boulder.

scholarly journals A functional in vitro model for studies of intracellular motility in digitonin-permeabilized erythrophores.

1982 ◽  
Vol 94 (3) ◽  
pp. 727-739 ◽  
Author(s):  
M E Stearns ◽  
R L Ochs
Keyword(s):  
Small Molecules ◽  
In Vitro Model ◽  
Inhibitory Effects ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Stabilizing Solution ◽  
Vitro Model ◽  
Cytoskeletal Elements ◽  
Intracellular Motility

Phase contrast cine results demonstrate that erythrophores maintain saltatory particle motion for hours after permeabilization with 0.001% digitonin in a cytoskeletal stabilizing solution at 23 degrees C. High voltage electron microscopy (HVEM) studies reveal that cytoskeletal elements are retained intact, except in immediate subplasmalemmal regions where the plasma membrane is punctured by digitonin. During digitonin treatments, cells are permeable to ions, small molecules, and antibodies. We find that motion is Ca2+ and ATP-sensitive, and optimal in PIPES buffer (pH 7.2 containing 1 mM Mg2+/ATP and EGTA-CA2+ (10(-7) M Ca2+) at 37 degrees C. Experiments testing the inhibitory effects of vanadate (0.4-10 microM), ouabain (100-600 microM), N-ethyl maleimide, and the cytochalasins B and D indicate that a dyneinlike ATPase may provide the motive force for driving saltatory pigment motion in erythropores.

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.

Toward a Better Understanding of 3-Dimensional Cell Structure Utilizing High Voltage Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 62-63
Author(s):  
Jerome J. Paulin
Keyword(s):  
Cell Body ◽  
High Voltage ◽  
Cell Structure ◽  
Stereo Pair ◽  
Anterior Portion ◽  
High Voltage Electron Microscopy ◽  
3 Dimensional ◽  
Voltage Electron ◽  
Longitudinal Orientation ◽  
Flagellar Axoneme

High voltage microscopy combined with stereoscopic photography of material embedded in epon-araldite and thick sectioned can be effectively utilized in studying complex organellar systems. The spatial distribution of the subpellicular and flagellar microtubules found in species of the Trypanosomatidae (Phylum Protozoa, class Zoomastigophorea) and their unique labyrinthine mitochondrial-kinetoplast complex lend themselves to this analysis.Figure 1 is a stereo-pair of a 0.5μM section (thicknesses are approximations based on interference colors) through the anterior portion of the cell body and flagellum of Trypanosoma equiperdum. Profiles of the subpellicular microtubules in both transverse and longitudinal orientation are observed. The dense plaques which form the attachment sites between the flagellum and cell body can be depicted. The arms on the A microtubules of the flagellar axoneme and a ring of dense bodies around the central pair of axonemal microtubules are resolvable in thick sections. Tilting of the specimen (± 5° from the horizontal axis) reveals the crystalline-like structure of the paraxial rod found in juxtaposition to the flagellar axoneme.

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