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[
International C. elegans Meeting,
2001]
The kinetochore is a complex and dynamic macromolecular structure that attaches and moves chromosomes within the spindle during mitotic and meiotic chromosome segregation. A partial list of kinetochore proteins now exists and attempts are being made to identify their functions. To define additional kinetochore proteins, we turned to C. elegans because the holocentric structure of its mitotic chromosomes is well-suited to identification and localization of these molecules. To characterize kinetochore structure at a macromolecular level of resolution, we are using electron microscopy to investigate chromosomes of wild-type worms and worms depleted of kinetochore proteins. An EM procedure optimized for the preservation of structure revealed that in wild-type spindles, the kinetochore ultrastructure is similar to that seen on mammalian chromosomes, i.e., a plaque represented by a ribosome exclusion zone and an underlying fibrous mat on the poleward face of the chromatin. Microtubules originating from the spindle poles insert orthogonally into this plaque. We have shown that HIM-10 is an evolutionarily conserved component of the kinetochore required for attachment of C. elegans holocentric chromosomes to mitotic spindles. HIM-10 is a coiled-coil protein related to the Nuf2 kinetochore proteins in yeast and humans, and is more poleward facing on mitotic chromosomes than the C. elegans centromere protein HCP-3. Depletion of HIM-10 disrupts kinetochore ultrastucture, causes a failure of bipolar spindle attachment, and results in embryonic lethality associated with chromosome nondisjunction or loss in mitosis. Electron microscopy of HIM-10-depleted spindles reveals that the plaque structure seen in wild-type chromosomes is greatly diminished or absent. This correlates exactly with the loss of HIM-10 immunofluorescence seen in the light microscope, and indicates that HIM-10 is necessary for the proper assembly and function of the kinetochore. Meiotic chromosomes viewed by EM have a similar kinetochore structure to mitotic chromosomes. This meiotic structure, together with the localization and function of HIM-10 in meiosis, provides the first demonstration that meiotic chromosomes of a holocentric organism possess kinetochores that share molecular, morphological and functional features with those of mitotic chromosomes. Conservation of kinetochore proteins in C. elegans extends to KCM-1, a kinesin-like protein that couples ATP hydrolysis with depolymerization of microtubule plus-ends. KCM-1 localizes to mitotic and meiotic chromosomes in a pattern related to that of HIM-10. Reduction of KCM-1 causes a failure of polar body extrusion, floppy spindles in mitosis and embryonic lethality. The conservation in kinetochore structure and proteins makes the extended kinetochores characteristic of holocentric chromosomes in C. elegans a guide to the structure, molecular architecture, and function of conventional kinetochores in organisms such as humans.
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[
Neuronal Development, Synaptic Function, and Behavior Meeting,
2006]
Mechanotransduction underlies an array of physiological processes, including hearing, pain, and touch. In C. elegans, mechanical stimulation of the body wall opens MEC-4 DEG/ENaC transduction channels in touch receptor neurons (TRN). Touch sensation also requires components of a characteristic 15-protofilament (pf) microtubule bundle and extracellular matrix (ECM). Genetic interactions between the genes encoding the MEC-4 channel, 15-pf microtubules, and ECM led to the hypothesis that MEC-4 channel gating occurs in a tethered mode. We tested this hypothesis by using post-embedding immunoelectron microscopy (IEM) to establish the subcellular distribution of the MEC-4 channel relative to the 15-pf microtubules and the ECM.
First, we developed antibodies against the MEC-4 channel subunits, MEC-2 and MEC-4. At the light level, these antibodies label the TRNs of adult animals with a punctate pattern similar in organization and spacing to previously published findings1. The MEC-2 and MEC-4 antibodies fail to label TRNs in their respective null mutants, confirming the specificity of both antibodies. Second, we used these antibodies to label TRNs in 50 nm serial sections. Consistent with published genetic, molecular, and functional observations, post-embedding IEM reveals that the MEC-2 and MEC-4 antibodies label the 15-pf microtubules and plasma membrane of TRNs. Microtubule-associated MEC-2 and MEC-4 label is observed on 15-pf microtubules located near the center and the periphery of the microtubule bundle. Label near the periphery is >50 nm from the plasma membrane. These observations suggest that some MEC-2 and MEC-4 puncta observed at the light level represent channels in transit. Membrane-associated MEC-2 and MEC-4 label is distributed around the circumference of the TRN. All membrane-associated label is >100nm from the nearest intracellular contact point between the membrane and the 15-pf microtubule ends. Only a fraction of the membrane-associated label is in register with hemidesmosome-like structures. Thus, MEC-4 channels are not exclusively aligned with these structures. These findings indicate that MEC-4 channels are not consistently associated with either 15-pf microtubule termination points or hemidesmosome-like structures. We speculate, therefore, that MEC-4 channels can be opened independently of intracellular and extracellular tethers.
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[
East Coast Worm Meeting,
2000]
Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.
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[
Midwest Worm Meeting,
2000]
Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.
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[
International C. elegans Meeting,
2001]
We continue to test alternate methods for preparing worms for transmission electron microscopy. We will describe new protocols, and will demonstrate what makes them better [or different] in comparison to previous methods (Hall, 1995). We still like simple immersion fixation and chopping open the animals by knife blade, and have made minor changes in the starting solutions to get optimum results. For early larval stages, which have never fixed well by immersion, and which are too little to chop open easily, we have adapted a new microwave protocol which gives very good results on intact worms. The resulting fixation looks equivalent to our immersion preparations of adults. Microwave fixation is proving very useful in the analysis of arrested animals from RNAi preparations, and should be excellent for looking at late embryos or dauers. Fast freezing methods offer a quite different approach, and the quality of tissue preservation can be superb. Both metal mirror freezing and high pressure freezing can produce excellent results, and they are achieving wider use over the past few years (Mohler et al., 1998; Rappleye et al., 1999). The inherent contrast after freeze substitution is often much greater, in part because the primary fixation contains only osmium, or a combination of osmium and aldehyde together. These methods allow much more rapid fixation. We can capture more "life-like" views of biological events in action, particularly for events such as vesicle fusions at the plasma membrane. Delicate cytoskeletal elements such as microtubules are also well preserved. We continue to try new combinations of fixatives and solvents to improve the appearance of nerve processes and synapses by fast freezing. Kent McDonald has been very helpful in suggesting improvements to these protocols. Laserhole fixations of embryos are technically rather difficult to accomplish, but can facilitate the passage of fixatives and embedding resins through the eggshell. We are continuing to use the protocol worked out by Carolyn Norris. See our website for details.
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[
International C. elegans Meeting,
2001]
C. elegans is an exciting model system for studying the molecular mechanisms of cell division. It is well suited for microscopic analysis, because each hermaphrodite contains a 'row' of early embryos of various stages. We apply a method which allows us to analyze high pressure frozen, plastic embedded embryos in whole mounted worms by transmission electron microscopy. We have started to cut serial sections through entire C. elegans embryos to reconstruct the early C. elegans mitotic spindle . Using three-dimensional reconstruction we have started to model the spatial organization of microtubules, chromatin, centrosomes and the nuclear envelope. We are analyzing embryos which contain about 20-30 cells, because these embryos are much smaller compared to one-cell stage embryos. We have also started to look at the structure of the C. elegans kinetochore. In embryos we found a ribosome-free zone which extends along the length of the chromosome. We will present preliminary three-dimensional models.
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[
East Coast Worm Meeting,
2004]
Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. C. elegans sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using major sperm protein (MSP) as a signaling molecule. MSP promotes meiotic maturation and activates MAP kinase in oocytes in part by binding the VAB-1 Eph receptor protein-tyrosine kinase. The discovery of MSP's signaling role raised the question of how sperm release MSP to signal oocytes and sheath cells at a distance. MSP is a cytoskeletal protein that nematode sperm utilize for motility and it does not possess a hydrophobic leader sequence. In addition, C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. Thus, the release of MSP may depend on a novel mechanism. Using specific antibodies, we detect MSP as far as 90 microns outside of spermatids and spermatozoa in vivo, consistent with its function as an extracellular signal. Labeling with vital dyes and sperm specific antibodies rules out sperm lysis as a potential mechanism. Wide-field and confocal microscopy shows extracellular MSP to be punctate with large (<0.5 microns) MSP-staining puncta near spermatids or spermatozoa. Confocal microscopy shows that MSP localizes to apparent membrane blebs at the surface of the sperm cell body, suggesting that the free puncta might originate from the membrane blebs. In the spermatheca, extracellular MSP appears to be more finely punctate suggesting that the large puncta may lose their integrity in this region to form a diffusible signal. Neither a pseudopod nor motility is required for MSP release because MSP puncta are located near wild-type or
spe-8 mutant spermatids. However, MSP puncta produced by spermatids appears to provide a longer acting more local signal. Using high-pressure freezing and freeze substitution to prepare samples for transmission electron microscopy, we have identified unusual free 150-300 nm double-layered vesicles located near spermatozoa in extracellular spaces of the spermatheca and uterus. We are currently testing if MSP is localized within these structures using immuno-EM. Since, MSP release appears not to occur from spermatids within or dissected from males, we reasoned a cue from the hermaphrodite must initiate release. To test this hypothesis we devised an in vitro release assay. Spermatids treated in vitro with a female extract rapidly (20s to 2 min) form MSP-containing blebs at their cell surface and MSP appears to be lost from the cell upon further incubation. This activity is distinct from activation during spermiogenesis during which the sperm forms a pseudopod. Scanning electron microscopy also shows a difference in the cell morphology of spermatids treated with the extract. Activity from this extract is abundant, soluble, heat stable, yet abolished by boiling. MSP puncta are found within the uterus of mated germline deficient
glp-4(
bn2 ts ) animals, suggesting the proposed cue may originate from the soma. Based on these results we purpose a model in which spermatids and spermatozoa receive a signal from the hermaphrodite soma and shed vesicles containing MSP in response to trigger meiotic maturation. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical and cell biological tests of this model are underway.
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[
Mid-west Worm Meeting,
2004]
Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. C. elegans sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using major sperm protein (MSP) as a signaling molecule. MSP promotes meiotic maturation and activates MAP kinase in oocytes in part by binding the VAB-1 Eph receptor protein-tyrosine kinase. The discovery of MSP's signaling role raised the question of how sperm release MSP to signal oocytes and sheath cells at a distance. MSP is a cytoskeletal protein that nematode sperm utilize for motility and it does not possess a hydrophobic leader sequence. In addition, C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. Thus, the release of MSP may depend on a novel mechanism. Using specific antibodies, we detect MSP as far as 90 microns outside of spermatids and spermatozoa in vivo, consistent with its function as an extracellular signal. Labeling with vital dyes and sperm specific antibodies rules out sperm lysis as a potential mechanism. Wide-field and confocal microscopy shows extracellular MSP to be punctate with large (<0.5 microns) MSP-staining puncta near spermatids or spermatozoa. Confocal microscopy shows that MSP localizes to apparent membrane blebs at the surface of the sperm cell body, suggesting that the free puncta might originate from the membrane blebs. In the spermatheca, extracellular MSP appears to be more finely punctate suggesting that the large puncta may lose their integrity in this region to form a diffusible signal. Neither a pseudopod nor motility is required for MSP release because MSP puncta are located near wild-type or
spe-8 mutant spermatids. However, MSP puncta produced by spermatids appears to provide a longer acting more local signal. Using high-pressure freezing and freeze substitution to prepare samples for transmission electron microscopy, we have identified unusual free 150-300 nm double-layered vesicles located near spermatozoa in extracellular spaces of the spermatheca and uterus. We are currently testing if MSP is localized within these structures using immuno-EM. Since, MSP release appears not to occur from spermatids within or dissected from males, we reasoned a cue from the hermaphrodite must initiate release. To test this hypothesis we devised an in vitro release assay. Spermatids treated in vitro with a female extract rapidly (20s to 2 min) form MSP-containing blebs at their cell surface and MSP appears to be lost from the cell upon further incubation. This activity is distinct from activation during spermiogenesis during which the sperm forms a pseudopod. Scanning electron microscopy also shows a difference in the cell morphology of spermatids treated with the extract. Activity from this extract is abundant, soluble, heat stable, yet abolished by boiling. MSP puncta are found within the uterus of mated germline deficient
glp-4(
bn2 ts ) animals, suggesting the proposed cue may originate from the soma. Based on these results we purpose a model in which spermatids and spermatozoa receive a signal from the hermaphrodite soma and shed vesicles containing MSP in response to trigger meiotic maturation. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical and cell biological tests of this model are underway.
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[
West Coast Worm Meeting,
2000]
Several recent reports (see below) have demonstrated that C. elegans tissues can be very well preserved for electron microscopy by high pressure freezing (HPF) followed by freeze substitution, perhaps substantially better than by standard chemical immersion fixation. HPF shows the potential to capture a more "life-like" view of the worm's ultrastructure. We have been testing both HPF and a related technique, rapid freezing on a metal mirror (MMF) followed by freeze substitution. Both methods obtain similar high quality fixation, although there are some freezing artifacts using the metal mirror device that are eliminated in HPF. For MMF, live animals are concentrated on a small piece of filter paper and plunged against a metal mirror at liquid nitrogen temperature. While freezing damage often occurs about 5-15 microns into the worms, some animals are very well frozen throughout. The frozen samples are held at low temperature and freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. For HPF, we have tried two methods to concentrate live animals into small metal planchette, either holding the animals within fine strands of dialysis tubing (C. Lavin, pers. comm.), or mixing them into a slurry of yeast paste to form a space-filling solid support (McDonald, 1999). Examination of fast-frozen specimens by TEM reveals excellent views of membrane events and organelles. For instance, we see many omega figures on coelomocytes which are indicative of active endocytosis, events which are not commonly captured by chemical fixation. Synaptic active zones and vesicles are well preserved, as are their relationships to microtubules. A network of microtubules can also been seen extending to the periphery of hypodermis. Basal laminae look strikingly different, much looser and more mesh-like when compared to chemical fixation. Sample images are shown on our website [www.aecom.yu.edu/wormem/new.html]. These two preparation methods, HPF and MMF, also hold great promise for high resolution immuno-EM. By reducing the osmium content and adding a dilute aldheyde fixation to the freeze substitution medium, we can obtain better resolution than is currently possible by our microwave technique. We have successfully localized epitopes in thin sections from HPF samples. MMF equipment is available here at Einstein campus. We are conducting HPF trials with the help of Stan Erlandson and Ya Chen at the University of Minnesota. As our skills improve, we will be happy to offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/caoting.html Martin Muller's website at www.em.bio.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Human Press) 1999. In the U.S., there are HPF machines open to the outside users in Madison, Berkeley, Minneapolis and Albany.
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[
International Worm Meeting,
2005]
Intercellular signaling facilitates multiple steps of the fertilization process. In many species, sperm prepare the oocyte for fertilization by providing signals for meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, nuclear envelope breakdown, and meiotic spindle assembly. C. elegans sperm signal oocyte meiotic maturation using the major sperm protein (MSP) as a hormone. Interestingly, the MSP also functions as the central cytoskeletal protein required for the amoeboid motility of nematode sperm. The discovery of MSPs signaling role raised the question of how sperm export MSP to signal oocytes at a distance. MSP lacks a hydrophobic leader sequence and C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. We used correlative light and electron microscopy to analyze the mechanism of MSP release from sperm. We will present evidence showing that sperm bud novel MSP vesicles to signal distant oocytes. These 150-300 nm MSP vesicles contain both an inner and an outer membrane, with MSP sandwiched in between. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly, suggesting that MSP may generate the protrusive force for its own vesicular export. MSP vesicles are labile structures that generate long-range MSP gradients for signaling at oocyte and sheath cell surfaces. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Spermatozoa generate a long-range, short-acting signal, whereas spermatids generate a long-acting signal. EM results suggest that differential vesicle stability affects the physical and temporal range of signaling.