Dissection of the<i>Ascaris</i>Sperm Motility Machinery Identifies Key Proteins Involved in Major Sperm Protein-based Amoeboid Locomotion

Buttery, Shawnna M.; Ekman, Gail C.; Seavy, Margaret; Stewart, Murray; Roberts, Thomas M.

doi:10.1091/mbc.e03-04-0246

Cited by 48 publications

(54 citation statements)

References 24 publications

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“…Whereas MSP is concentrated at these budding sites, the MSD proteins are absent. In vitro studies of MSP-based motility in Ascaris identified MFD1, the ortholog of the MSD proteins, as having an activity that decreases the rate of MSP fiber growth (Buttery et al, 2003). Thus, the absence of the MSD proteins at the vesicle-budding sites may favor MSP filament assembly and membrane protrusion.…”

Section: Discussionmentioning

confidence: 99%

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C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

et al. 2005

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The major sperm protein (MSP) is the central cytoskeletal element required for actin-independent motility of nematode spermatozoa. MSP has a dual role in Caenorhabditis elegans reproduction, functioning as a hormone for both oocyte meiotic maturation and ovarian muscle contraction. The identification of the signaling function of MSP raised the question, how do spermatozoa, which are devoid of ribosomes, ER and Golgi, release a cytoplasmic protein lacking a signal sequence? Here, we provide evidence that MSP export occurs by the budding of novel vesicles that have both inner and outer membranes with MSP sandwiched in between. MSP vesicles are apparently labile structures that generate long-range MSP gradients for signaling at the oocyte cell surface. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly. We propose that MSP generates the protrusive force for its own vesicular export.

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Section: Discussionmentioning

confidence: 99%

“…The Ascaris ortholog, MFP1, is a component of the MSP cytoskeleton that decreases the rate of MSP fiber assembly in vitro (Buttery et al, 2003). We examined the localization of MSP and MSD in mated females by confocal microscopy and generated 3D-image reconstructions of the data.…”

Section: Specificity Of Msp Release and Apparent Budding From Spermatmentioning

confidence: 99%

C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

et al. 2005

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“…Previous analyses of fibers assembled in vitro have identified five accessory proteins in the MSP motility system [Buttery et al, 2003;LeClaire et al, Fig. 4.…”

Section: Msp Motility Proteins In Reconstituted Filopodiamentioning

confidence: 99%

“…Primary antibodies were added at 5 lg/ml in KPM containing 1% BSA. We tested mouse monoclonal antibodies 6F9 and 3A10 directed against MSP fiber proteins (MFP) 1 and 2, respectively [Buttery et al, 2003], rabbit polyclonal antisera directed against p34 kinase (our unpublished observations) and MFP3 (our unpublished data). Monoclonal anti-phosphotyrosine antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA).…”

Section: Immunofluorescence Labeling and Confocal Microscopymentioning

confidence: 99%

“…In each case, the distribution of the protein in reconstituted filopodia was similar to that observed in fibers. For example, MFP2 is a family of three closely related polypeptides that is located in the filament meshwork of fibers and required for fiber assembly [Buttery et al, 2003]. Anti-MFP2 antibody labeling of filopodia produced a punctuate distribution of fluorescence along the filamentous shaft (Fig.…”

Section: Msp Motility Proteins In Reconstituted Filopodiamentioning

confidence: 99%

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

Liu

Mackey

et al. 2006

Cell Motil. Cytoskeleton

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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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Vesicle Transport Assay

Wöllert

Langford

2015

Encyclopedia of Life Sciences

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Eukaryotic cells contain an extensive cytoplasmic network of actin filaments and microtubules that function as tracks for vesicle transport. The ability to visualise vesicle movement along both sets of filaments advanced rapidly when in vitro vesicle transport (motility) assays were developed. These assays utilise super‐resolution, contrast‐enhanced DIC microscopy to detect structures below the resolution of the light microscope including vesicles as small as 100 nm and microtubules 25 nm in diameter. Images are captured in real time and stored digitally for subsequent analysis. The standard motion analysis parameters include vesicle type, velocity, distances and path length. In vitro motility assays enabled the identification of important factors that are essential for the regulation of intracellular transport including motor proteins and their membrane receptors, the formation of hetero‐motor complexes and transported cargoes. Key Concepts The cytoskeleton contains actin filaments and microtubules, both of which serve as tracks for vesicle transport. Vesicle transport is the directed movement of membrane vesicles on filaments of the cytoskeleton. Microtubules function as intracellular tracks for transport of vesicles by two classes of motor proteins, kinesins and dyneins. Actin filaments serve as tracks for vesicle transport by myosin motors. Motor proteins are mechanochemical nanomachines that use the energy of ATP for muscle contraction, cell motility, cell division and transport of different cargoes along the cytoskeleton. The superfamily of myosin motor proteins found in eukaryotic cells is known to contain over 20 different classes and of these, 12 classes are found in vertebrates, including humans. The kinesin superfamily is subdivided into 15 classes and the dynein family is grouped into axonemal and cytoplasmic dyneins. Motor protein defects are associated with diseases including Griscelli syndrome, Usher syndrome, myopathies, deafness, tumour progression and metastasis. Differential interference microscopy, also known as Nomarski microscopy, uses polarised light to enhance gradients in the optical path length and phase shifts in unstained, transparent biological specimens. Cell‐free extracts are important tools for cell biologists and have a variety of applications including cell cycle studies, intracellular transport mechanisms, signal transduction events and maintenance of cell architecture.

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

Cited by 48 publications

References 24 publications

C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes

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

Vesicle Transport Assay

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