Klein RD et al. (1989) Worm Breeder's Gazette "notes on galvanotaxis."

Kim SK, Klein RD, Meyer BJ

[Worm Breeder's Gazette, 1989]

As a joke, Stuart and I decided to put worms in an electric field. We did this by placing worms on an agarose mini-gel (0.3% in 1X M9 salts) with the running buffer just touching the sides of the gel. Interestingly, when we turned the power on, the worms rapidly moved toward the cathode. As a result of these observations, we have been following up this behavior in order to determine if it can tell us anything about worm biology. Galvanotaxis shows two distinct phases. The first occurs at relatively low voltage (~0.07 V/mm in M9 salts) and consists of animals moving somewhat haphazardly toward the cathode in a manner similar to that seen in chemotaxis assays. As the voltage increases, the response gets stronger until a new type of behavior is seen. At approximately 0.4 V/mm (in M9 salts) the worms move toward the cathode in a much more directed manner. They very quickly decide on the direction in which they are going and, once decided, rarely stop or change directions. Although the worms move in a straight line at the higher voltage, they do not move directly toward the cathode. Rather, worms initially placed in a single spot will move out of that spot in two streams, each of which makes an approximately 45 angle with a line between the initial spot and the cathode. An animal traveling along one of these angles will generally (but not always) have its ventral side closer to the cathode. Occasionally a worm will flip onto its other side causing it to change its angle of movement such that it now moves at an angle that is flipped relative to its starting angle (i.e. it makes a V-shaped pattern). The exact angle that a group of worms adopts is somewhat variable and appears to depend on the agarose concentration. Adult and L4 hermaphrodites as well as males show the strongest response. However, male worms tend to get distracted and make slower progress toward the cathode. L3 and L2 hermaphrodites show weaker responses in that younger animals spend a greater proportion of their time moving in a direction other than toward the cathode. L1 animals show little if any response to an electric field. Finally, dauer larvae tend to galvanotax fairly strongly over short periods of time. However, they often can be seen going in a direction other than toward the cathode. In order to determine if galvanotaxis might be a tractable genetic behavior, we have checked (and are continuing to check) existing mutations for the ability to galvanotax. These include a large number of unc, daf, che, lin, mec, and osm mutations. To date only three mutations appear to be defective (to varying extents) in galvanotaxis; lin-32(u282), 33), and n1754, a mutation isolated by L. Bloom as defective in thermal avoidance and also found to be chemotaxis deficient. All three of these mutations are quite pleiotropic in their defects and clearly are not specific for galvanotaxis. One possible explanation for this behavior could be that the worms are responding to a heat or salt gradient set up over time by the electric field. We do not believe this to be true for two reasons: (1) the response starts as soon as the power is turned on and stops immediately after the power is turned off, and (2) two mutations, n1937 and n1938, isolated by C. Bargmann as being completely chemotaxis deficient, are wild type for galvanotaxis. Therefore, it is likely that at least some components of galvanotaxis are distinct from chemotaxis. In order to find mutations that are specific for galvanotaxis, we have begun a mutant screen for animals that do not respond to an electric field. To date we have screened approximately 3000 haploid genomes without finding any galvanotaxis mutations. In order to try to increase the number of worms we can screen as well as make the assay easier to set up, we have adapted the assay to 9 cm tissue culture plates.

Charnay Y et al. (1980) Worm Breeder's Gazette "characterization of two sub populations in C elegans, Bergerac by bacteria in the digestive tractus."

Charnay Y, Brun J

[Worm Breeder's Gazette, 1980]

In our laboratory C. elegans, Bergerac strain,has been routinely cultivated in xenic conditions on nutrient agar since several years. Different populations were maintained separately. The intestinal tractus of the worm was studied cytologically by the 'frottis' technique followed by Feulgen reaction. We have distinguished two populations categories. Results are homogeneous in each of them what ever the larval and adult stages considered and are similar after 100 generations. - First category (majority of populations) - The intestine nuclei alone are Feulgen positive, intestine lumen is clear. - Second category - Feulgen positive material is found throughout the intestine lumen with the same intensity from the very anterior region to the rectum. This Feulgen positive material when observed under Nomarski differential interference contrast optics in the light microscope appears to be constituted by many thousand particles. Their size is just larger than resolution power allowed by microscope. These Feulgen positive particles are neither revealed in Nematodes cultivated in axenic conditions, nor when the hydrolysis is omitted during Feulgen reaction. All these results allowed us to consider that the positive Feulgen material originates in the staining of bacterial chromosomes. So, one or more bacteria strains would be able to colonize the intestine lumen of this Bergerac sub population and to be tolerated by the worm even in large amounts. This result is interesting in the perspective of better knowledge of Nematode-bacteria relations. Which and how much bacteria species are implicated in the nutritional requirements? Are these bacteria symbiot or not?

Politz SM (1984) Worm Breeder's Gazette "stage-specific surface antigens and antibodies."

Politz SM

[Worm Breeder's Gazette, 1984]

I am developing stage-specific cuticle antibodies to help identify genes controlling the stage transitions in C. elegans. I want to use these antibodies to select heterochronic mutants expressing stage- specific cuticle antigens at alternative times in development compared to wild-type. Rabbit anti-cuticle antibodies elicited by immunization with whole cuticles or solubilized cuticle collagens bind to the surface of live worms, making it fluoresce uniformly when fluorescein conjugated (FITC) second antibody is applied. Surface immunofluorescence was at first visualized in the compound microscope. Recently, I have developed an arrangement for viewing immunofluorescing worms on agarose plates under a low power dissecting microscope adapted for illumination by an FITC excitation source. This allows me to pick individual live fluorescing worms by the normal method. The basic strategy for using stage-specific antibodies to select heterochronic mutants is to bind antibody to worm populations under conditions of synchrony or genetic background where only mutants expressing stage-specific antigens heterochronically will fluoresce, enabling them to be recognized amongst thousands of nonfluorescent wild-type worms. Approaches involving adult-specific antibodies are in progress; characterization of adult-specific antibodies and surface antigens is described below. For antibody characterization, I devised a quantitative radioimmunoassay to measure surface binding of antibodies. Aliquots containing about 500 worms are incubated with antiserum, washed, incubated with [125I]-labeled Staph. aureus protein A, washed, and counted for total bound radioactivity. Binding is saturable by worms, antiserum, or protein A, indicating stoichiometric reaction. Pre-immune serum gives minimal binding. This assay has been used to study stage-specificity of antibodies and antigens. Rabbit anti-adult cuticle serum cross-reacts with the surface of live L4's. I have made the serum adult-specific by adsorption; after adsorption with saturating numbers of live L4's, serum no longer binds detectably to L4's, but binding to adults is reduced by only 50%. As expected, this adult-specific serum does not bind to adults of strain CB0912(lin-4), a retarded cell-lineage mutant that never develops adult cuticle. After a serum made to L4 cuticles was adsorbed with saturating numbers of adults, no binding to either L4's or adults was observed. These results indicate the existence of at least two distinct classes of surface antigens; i.e., those common to both L4 and adult cuticles, and those restricted to the adult cuticle (adult-specific surface antigens).

Schriefer LA et al. (1990) Worm Breeder's Gazette "PCR and the muscle tropomyosin gene in C."

Schriefer LA, Waterston RH

[Worm Breeder's Gazette, 1990]

As part of our ongoing effort to identify genes for known muscle proteins, we have used an approach that starts with the purified protein and works backwards to the gene. To test this approach and gain experience with it we selected tropomyosin (Tm) a relatively abundant, easily purified, actin associated protein. A candidate Tm clone had previously been identified by Hiro Kagawa, but it was not known whether this gene produced the major muscle form or another form of Tm. Tropomyosin from total worm homogenate was brought to near 100% purity by salt extractions, ethanol and low pH precipitations, and FPLC column chromatography. The N-terminal was blocked so this material was treated with CNBr, and the fragments fractionated on a Laemmli acrylamide gel. The peptides were then transferred to an Immobilon-P membrane and Coomassie stained. Cleavage was partial with about 10 bands present ranging in molecular weight from 39kd to 15kd. 5 major bands were excised and given to the Washington U. Protein Chemistry Lab. to sequence. These yielded protein sequence data on two separate regions of Tm. Both regions had high homology to rabbit alpha skeletal muscle Tm and are presented below: [See Figure 1] Two degenerate oligonucleotides were made to the arrowed regions in the upper fragment. These oligos were used to amplify the predicted 80 bp PCR band from a cDNA library and genomic DNA. This PCR fragment was gel purified and then cloned into M13 and sequenced. This sequence yielded the expected protein sequence and is presented below: [See Figure 1] The 80 bp PCR fragment was hexamer labelled and used to probe the YAC library. The positive clones Y38D6 and Y68B7 map to the left of unc-54. This position and amino acid sequence agreed with the results independently obtained by Hiro Kagawa (this issue and personal communication) and indicates that Hiro has cloned and sequenced the major muscle tropomyosin gene. Deficiencies that map to the left of unc-54 were obtained from the CGC stock center and these were used as a substrate for PCR amplification. The arrested homozygous deficiency animals were digested with proteinase K and amplified using the oligos to Tm. Our results indicate eDf10, elete Tm. Tentative data indicates that eDf15 does not delete Tm while eDf16 does. The genes lev-11 and let-205 are known to be in this region. This project has shown the power of micro-sequencing of immobilized proteins, PCR amplification, the C. elegans YAC library, and the C. elegans genetic map with its deficiencies. These tools can lead the C. elegans researcher from their favorite protein to localize its gene.

Byerly L et al. (1978) Worm Breeder's Gazette "voltage-clamp studies of the electrical currents of the Ascaris pharyngeal muscle."

Masuda M, Byerly L

[Worm Breeder's Gazette, 1978]

It has proven difficult to study the currents that generate the electrical activity in the somatic muscle cells of Ascaris due to the inability to control the voltage across the excitable membrane. Therefore, we have directed our attention to the pharyngeal muscle, where it is possible to directly measure the voltage and pass large currents across the excitable membrane. We have developed a system which allows us to do current-clamp and voltage-clamp experiments on an isolated segment of the pharyngeal membrane. We find that this membrane has no pacemaker activity. In the absence of nervous input the membrane potential is flat at a level near -40 mV. Two types of spontaneous postsynaptic potentials are frequently seen; one type has a reversal potential near -40 mV and the other has a reversal potential near -10 mV. When the membrane is at the resting level, this second type PSP triggers a positive-going action potential, which reaches a level between +30 and +50 mV. The membrane potential then falls to a plateau near O mV, where it remains until a negative-going PSP triggers a negative-going action potential that reaches about -50 mV (the potassium reversal potential). The membrane potential remains at the plateau level for periods ranging from 100 msec to several minutes. The positive-going action potential is produced by an inward current that appears to be carried by both Na+ and Ca++. This current is prolonged, showing little inactivation by 200 msec after a positive voltage step. Clamping the membrane to positive potentials elicits essentially no delayed-rectification K current, the current that normally repolarizes active membranes. However, stepping the membrane potential back to the resting level after a large positive pulse elicits a strong, transient outward K+ current; this is the current that produces the negative-going action potential. We have done a detailed analysis of this current and have shown that it is a voltage- inverted analogue of the Hodgkin-Huxley Na+ current. It is activated by negative steps in potential. It shows inactivation, being completely inactivated at the resting potential. Conditioning pulses to levels more positive than -10 mV are necessary to remove the inactivation from the channel. This is a new K+ conductance that has not been found in any other animal. This demonstration of unique mechanisms in nematode physiology should serve as a caution in trying to interpret the function of the nematode nervous system. The pharyngeal nervous system must not only generate the signal that triggers the pharynx to contract (open the lumen), but also the signal to relax (close the lumen). Since the relaxation of the pharynx is the 'power stroke' of this muscle, it is not surprising that the membrane has developed a special K+ current to produce a fast, separately-triggerable repolarization.

Waterston RH (1986) Worm Breeder's Gazette "reversed field gel electrophoresis of C elegans DNA."

Waterston RH

[Worm Breeder's Gazette, 1986]

Schwartz and Cantor (Cell 37, 67-75 (1984) and Carle and Olson (NAR 12, 5647-5664, 1984) have devised an orthogonal field agarose gel system that allows the separation of large DNA molecules (>2000 kb). A recent modification of the method by Carle et al. (Science 232, 65- 68, 1986) allows these separations to be obtained using a conventional gel box by periodically reversing the field. Control of the switch times through a microcomputer is convenient and allows the switch time to be varied at will during the run. This is important as the switch time controls the range of sizes that can be separated in a given run. Chris Bond of the MRC electronics laboratory designed and built an extremely convenient switch box employing Mosfett power bridges, which have an excellent response time and a long life time. Details of the set up I'm using are available to anyone interested (see also Carle and Olson's Science paper). To make high molecular weight (chromosomal?) DNA from C. elegans, I am currently using pure populations of L1's as starting material. These are embedded in 0.5% agarose in 0.125 M EDTA 0.125 M Tris pH9 and then lysed by overlayering with 1% sarcosyl, 1 mg/ml proteinase K and 7% -ME as described by Carle and Olson (1985) for yeast. Without restriction of the lysed worms very little ethidium stained material enters the gel under a variety of conditions with the exception of some low molecular weight material, assumed to be degraded RNA, and 2 or 3 ethidium stained bands with a mobility corresponding to about 100 kb but of unknown origin. After digestion with any of a series of restriction enzymes large amounts of DNA enter the gel. Two enzymes, NotI and SfiI which both have 8 bp recognition sequences composed entirely of G:C pairs yield a smear extending from about 100 kb to more than 1000 kb. A few distinct bands are visible against the background smear but it is impossible to directly estimate the number of fragments present. From parallel experiments using digests of random cosmid clones, NotI sites are estimated to occur every 600-700 kb on average, predicting be less than 150 bands. SfiI sites are slightly more frequent. After BglI digest, a 6 bp recognition site enzyme that does not cut within the ribosomal repeat sequence (Ellis et al. NAR 14, 2345-2364, 1986), most DNA appears to be about 50-150 kb although bands are visible at 300 and 350-400 kb and a single distinct band is present with a mobility between that of yeast chromosomes XI (~700 kb) and X. This band hybridizes strongly and specifically with an rDNA probe provided by R. Fishpool, and places an upper limit of 100 rDNA of 7.2 kb repeats. Current efforts are being directed toward using this methodology to characterize genome rearrangements in the sma-1 region of LGV. I am also trying to identify the free duplication fragments mnDp30 and eDp6 in unrestricted DNA preps. The methodology may provide a level of resolution intermediate between that of cytogenetics and the cosmid mapping.

Kennedy BP et al. (1987) Worm Breeder's Gazette "fairly large scale oocyte preparation."

McGhee JD, Ito K, Kennedy BP

[Worm Breeder's Gazette, 1987]

1. Preparation of fer-1 dauer larvae A temperature sensitive sperm defective mutant, fer-1 (hc-1) is grown at 20 C on 150mm NGM plates with 10 ml of whole egg homogenate ( 50 ml H20/egg) dried on top. Leave the plates for 10-14 days (or even longer - but the number of dauer begin to decrease) by which time most worms on plate are dauers. Wash worms from plate with M9 or H2O and preclean either by multiple washing or by filtration through a 44 micron Nytex screen. The worms are further cleaned by flotation in 35% w/v sucrose or if only dauers are required by 1st treating with 1% SDS for 30 minutes and then sucrose flotation. 2. Adult fer-1For each 150 mm NGM plate, add 10 ml whole egg homogenate plus 1-3 g of E. coli and dry onto plate in sterile hood. Once the plate is dry or semi-dry, add 0.7 ml of packed fer-1 dauers and incubate at 25.5 C for about 2 days. This is the non-permissive temperature and necessary for a synchronous shift out of dauer hood. After 2 days, worms are ready to harvest; by this time greater than 95% should be young adults full of oocytes. Worms are cleaned by 35% sucrose flotation. The yield from 10-12 150mm plates is about 60-80 ml packed worms. 3. Preparation of oocytes The clean worms are given a final wash in egg salts buffer (esb=110 mM NaCl, 4 mM KCl, 5 mM CaCl2, 5 mM MgCl2 and 5 mM Hepes pH 7.2). Resuspend each 10 ml of packed worms in a 50 ml tube with 40 ml of esb and sonicate on ice. We have a Braun Sonic 2000 with micro and medium probes. If micro probe is used, the settings are 60-80 watts for 30- 40 sec. and if the medium probe is used, the settings are 40 watts for 30-40 sec. You can test your probe by sonicating worms at various power settings and analyzing the sonicate - too high destroys oocytes - too low just tickles worms. After sonication, the worms are allowed to settle and the supernatant removed and stored on ice. 10ml of esb are added to the worms and mixed, the worms are allowed to settle again and the supernatant saved. The settled worms are again resuspended in esb to 40ml and the sonication and settling procedure repeated 4-6 times. The supernatants from all the sonications are centrifuged at 500 xg for 5 minutes. The pellet is taken up in 10 ml of cold esb and transferred to a clear 15ml polystyrene tube; the supernatant is discarded. By this time, the oocytes are at sufficient concentration that they aggregate and settle faster than the contaminating worms. Let the suspension settle and remove the supernatant. Repeat this procedure at least 20 times (nobody said this was elegant) or until no more worms can be seen. You may have to break up the oocyte aggregate a few times to get rid of all the worms. The yield from about 60ml packed adult fer-1 is about 1ml oocytes. 4. Purity check of oocytes Presence of worms or developing embryos can be detected under polarized light for their gut granules. Usually less than a few percent of the preparation shows gut granules.

Weyler W (1992) Worm Breeder's Gazette "Evidence for Monoamine Oxidase in Caenorhabditis elegans"

Weyler W

[Worm Breeder's Gazette, 1992]

Monoamine oxidase A and B are mitochondrial outer membrane flavoproteins best characterized in higher vertebrates, and their association with the nervous system has led to the belief that their most important function is a role in neurotransmitter amine metabolism. In spite of extensive efforts to understand the role of these enzymes in the nervous system, little progress has been made. This is in part due to the inherent complexity of the systems studied. Because the nervous system of Caenorhabditis elegans is relatively simple and well defined, I decided to investigate if MAO is present in C elegans. The biogenic amines octopamine, serotonin, and dopamine, presumptive neurotransmitters in the worm, are all substrates of MAO. It is therefore reasonable to believe that MAO is present in C. elegans. Although first experiments to spectrophotometrically detect MAO activity failed, immunological studies of whole worm extracts indicated its presence. Homogenates were examined by SDS-PAGE and Western blot analysis with sheep anti-human MAO A, rabbit anti-human MAO A and sheep anti-bovine MAO B antibodies. A strong and specific alkaline phosphatase reaction was obtained with both anti-human MAO A antibodies. No trace of a signal was detected with anti-bovine MAO B antibody or preimmune sera. The positive band migrated more slowly than the human control suggesting that the putative nematode MAO is about 5 to 7kDa larger than the human enzyme. Evidence for the presence of MAO in whole animals was found by culturing the nematode on agar plates seeded with E. coli OP50 and containing the specific MAO A suicide inhibitor clorgyline or the structurally similar specific MAO B inhibitor deprenyl. For assays, starved Bristol N2 hermaphrodites from a plate were transferred to give 5 to 6 individuals per test plate. Plates were sealed with paraffin film and incubated at 20 C and monitored for two weeks with a microscope at 100 and 200x power. Clorgyline inhibited growth of the worm at 2.5x10 -5Mwhile deprenyl showed inhibition at >1x10 but <1x10 -3M.The effect with clorgyline expressed at 25 M is a concentration 2000 times lower than was required to observe behavioral effects in similar incubation studies with exogenously added neurotransmitters (Horvitz et al., '82, Science 216,1014). The high concentration of amines required in these experiments was attributed to impermeability of the worm's cuticle. This suggests that inhibition of growth results from an even lower concentration of clorgyline entering the animal. In vitro, MAO A is rapidly inhibited by 1x10 -7Mclorgyline or 1x10 -5Mdeprenyl. In addition to the observation that worms stopped growing in the presence of clorgyline and bacteria, cessation of pharyngeal pumping was evident. It is possible that inactivation of MAO increases the concentration of octopamine in a neuron that controls inhibition of pharyngeal pumping (Horvitz et al., '82, ibid.). Growth may have ceased due to starvation. Treatment of octopamine-defective and other mutants with clorgyline and other drugs should shed light on this proposal. These preliminary experiments strongly suggest the C. elegans contains MAO, possibly of the A type, and that the enzyme plays an essential role in either the development or the normal functioning of this simple organism. We now plan to firmly establish the presence of MAO and its type and determine the mechanism of growth arrest as this most likely underlies the role of MAO and its mode of action in C. elegans.

Williams CJ et al. (1997) Worm Breeder's Gazette "Antisense knockouts of genes involved in RNA processing."

Xu L, Blumenthal TE, Huang T, Williams CJ

[Worm Breeder's Gazette, 1997]

By searching Genbank, we have identified C. elegans cDNAs encoding proteins known in mammals to be needed for RNA processing: 3' end formation and splicing. Full length antisense RNAs were generated by in vitro transcription from plasmids (kindly supplied by Y. Kohara) and injected into the gonads of adult worms in order to mimic the knockout phenotype of these genes. Injection of antisense RNA to the subunits of the 3' end formation protein, CstF, which binds RNA downstream of the cleavage site and is thought to be involved in cleavage site selection, resulted in an embryonic lethal phenotype. This is perhaps not surprising as null alleles of the Drosophila homolog of the 77K subunit of CstF are also embryonic lethal (1,2). To investigate the phenotypes of knockouts of genes involved in splicing, antisense RNAs were injected into smg-2 animals to prevent degradation of any resulting incompletely processed RNAs. Injection of antisense RNA to the integral U1-associated protein, U1C, resulted in a developmental delay phenotype. In contrast, injection of RNA antisense to another integral U1 protein, U170K, caused embryonic lethality. The difference in phenotype between knockouts of these two genes may simply reflect injection of different concentrations of antisense RNA or different maternal protein contributions. We have used RT-PCR to look at processing of the myo-3 gene in these delayed or dead embryos. The embryos from antisense-injected mothers accumulated large amounts of incompletely cis-spliced and unprocessed pre-mRNAs suggesting that the phenotype is indeed due to perturbation of splicing. Interestingly, only the mRNAs from which both introns had been removed were found to be trans-spliced, suggesting that either cis-splicing must occur in order for trans-splicing to occur in vivo or trans-splicing is even more sensitive to lack of U1 snRNP than is cis-splicing. The latter explanation would be quite surprising, since trans-splicing in Ascaris has been shown to be U1 snRNP-independent in vitro (3) (although the possibility of these proteins serving as components of the SL snRNP has not been investigated). We have recently identified two U1A protein homologs arranged in an operon. The two encoded proteins appear to be quite different from one another, particularly at key residues within the second RNA binding domain. Nevertheless, the antisense injection experiments indicate they are functionally redundant: neither antisense RNA on its own gave a detectable phenotype, but when both were injected together approximately half the embryos died and the remainder exhibited delayed development, hatching 48 hours after laying instead of the usual 12 hours. We have not yet tested for cis- or trans-splicing defects in these embryos. Overall, our results demonstrate that genes involved in mRNA processing are vital to embryonic development and also confirm the power of antisense technology in predicting the knockout phenotypes of genes. (1)Mitchelson, A., M. Simonelig, C. Williams and K. O'Hare (1993) Homology with Saccharomyces cerevisiae RNA14 suggests that phenotypic suppression in Drosophila melanogaster by suppressor of forked occurs at the level of RNA stability. Genes Dev 7: 241-9 (2)Takagaki, Y. and J. L. Manley (1994) A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein. Nature 372: 471-474 (3) Hannon, G. J., P. A. Maroney and T. W. Nilsen (1991) U small nuclear ribonucleoprotein requirements for nematode cis- and trans-splicing in vitro. J Biol Chem 266: 22792-5

Hird SN et al. (1992) Worm Breeder's Gazette "Cortical and Cytoplasmic Flow in Early C. elegans Embryos"

Hird SN, White JG

[Worm Breeder's Gazette, 1992]

The actin rich cortex of many animal cells forms a network subjacent to the plasma membrane that is capable of large scale movements in this plane. For example, in migrating fibroblasts and in nerve growth cones, the actin network continually flows away from the leading edge in a concerted manner at approximately 6um/min (1). It appears that monomeric actin is polymerised at the leading edge into filaments which join the network and then move away from this region. The filaments disassemble into monomers after moving away. A cytoplasmic flow in the opposite direction to the cortical flow (i.e towards the leading edge) could carry these actin monomers back to the leading edge to complete the cycle. The driving force for the movement of the cortical network could be gradients in myosin generated tension (2). If the mechanical tension of the network at the leading edge is lower than that elsewhere, then the network would tend to collapse back on itself towards the regions of higher tension. These gradients of tension could also power the compensatory cytoplasmic flow. We have been looking at pseudocleavage in C. elegans as a possible and novel example of this cortical flow phenomenon. During pseudocleavage (PC), the anterior of the egg cortex undergoes a series of contractions whilst the posterior cortex remains relatively quiescent. Cytoplasmic material ( and possibly cytoplasmic determinants) flow from the anterior to the posterior, P granules become localized in the posterior (3), the pronuclei move towards each other (4) and a PC furrow forms and regresses. In addition, foci of actin become concentrated in the anterior of the embryo (5). Using the 4D time lapse system, we have analyzed the behavior of granules associated with the cortex during PC. In other systems, the movement of such granules parallels that of the underlying actin cytoskeleton (1). Pseudocleavage begins with waves of contraction moving from the posterior end to the anterior end at the same time as the anterior cortex begins to contract. These waves cease, and cortical granules stream from posterior to the site of the forming PC furrow. At the same time, cytoplasmic material flows in the opposite direction from anterior to posterior. The cortical flow occurs at a rate of between 3-7um/min which is consistent with previously determined rates for cortical flow. It is restricted to the granules in the posterior of the embryo: those in the anterior exhibit random movement. Thus, PC seems to involve a directed and continual movement of cortical material away from the posterior pole and a cytoplasmic flow in the opposite direction. If a pronuclear stage embryo is treated with the microtubule polymerization inhibitor nocodazole, then no pronuclear migration occurs and a small spindle is formed around the centrosomes associated with the sperm pronucleus. This attenuated mitotic apparatus lies adjacent to the cortex at a variable position in the posterior part of the embryo. When such a drug treated embryo attempts to cleave, cortical material flows away from the region of the cortex next to the spindle at between 3-7um/min. At the same time, cytoplasm flows towards this region of the cortex and the anterior cortex contracts vigorously and repeatedly. A regressive cleavage furrow is formed between the zones of anterior contraction and posterior cortical flow just as in PC. These cortical and cytoplasmic flow patterns can also be seen in nocodazole treated AB and P1 blastomeres, again with the directions of the flows determined by the position of the attenuated spindle. In each case the flows and the position of the transient cleavage furrow mimic those seen during PC. This suggests that during PC some component of the sperm asters is inducing cortical material to move away form the asters, possibly by locally relaxing the mechanical tension of the actin network in the posterior part of the embryo. This may also occur during cytokinesis, with the asters of the mitotic spindle relaxing the cortical tension at the poles of the cell and hence inducing the network to flow into the equatorial region to form a cleavage furrow. A model for pseudocleavage is shown in Figure 1.