Abstract. We have examined the cortex of Caenorhabditis elegans eggs during pseudocleavage (PC), a period of the first cell cycle which is important for the generation of asymmetry at first cleavage (Strome, S. 1989. Int. Rev. Cytol. 114: 81-123). We have found that directed, actin dependent, cytoplasmic, and cortical flow occurs during this period coincident with a rearrangement of the cortical actin cytoskeleton (Strome, S. 1986 . J. Cell Biol. 103: 2241-2252. The flow velocity (4-7 #m/min) is similar to previously determined particle movements driven by cortical actin flows in motile cells. We show that directed flows occur in one of the daughters of the first division that itself divides asymmetrically, but not in its sister that divides symmetrically. The cortical and cytoplasmic events of PC can be mimicked in other cells during cytokinesis by displacing the mitotic apparatus with the microtubule polymerization inhibitor nocodazole. In all cases, the polarity of the resulting cortical and cytoplasmic flows correlates with the position of the attenuated mitotic spindle formed. These cortical flows are also accompanied by a change in the distribution of the cortical actin network. The polarity of this redistribution is similarly correlated with the location of the attenuated spindle. These observations suggest a mechanism for generating polarized flows of cytoplasmic and cortical material during embryonic cleavages. We present a model for the events of PC and suggest how the poles of the mitotic spindle mediate the formation of the contractile ring during cytokinesis in C. elegans. T rIE phenomenon of cortical flow has been described in many systems including the lamellipodia of motile cells (Abercrombie et al., 1970; Heath, 1983; Fisher et al., 1988) , the growth cones of neurons (Forscher and Smith, 1990) , during amoeboid locomotion (Grebecki, 1984) , and during cytokinesis (Dan, 1954) . In these cases, material adjacent to the plasma membrane flows in a continual and directed manner away from one region to a distant region. For example, in the case of motile cells, patches of aggregated membrane proteins (Taylor et al., 1971; Holifield and Jacobson, 1991) , or particles (Abercrombie et al., 1970; Fisher et al., 1988) applied to the dorsal surface of the lamellipodia, flow away from the leading edge of the cell. There is now significant evidence, derived from a number of observations, that there is continual movement of the underlying cortical actin network (Heath, 1983; Wang, 1985; Forscher and Smith, 1988) in motile cells. Membrane proteins may be associated with such moving cortical networks and so may be swept along in the same direction (Holifield et al., 1990) , and they may in turn carry along extracellular particles. Alternative hypotheses predict that the cortical flows are driven by flows of membrane lipid (Bretscher, 1976 (Bretscher, , 1984 . AIPlease address all correspondence to S. N. Hird, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, England. though it has been suggested that this type of flow is not solely responsible for cortical flow during capping (Sheetz et al., 1989; Lee et al., 1990; Kucik et al., 1990) , it is still possible that it may contribute to cortical movements. There is a period in the first cell cycle of the Caenorhabditis elegans embryo during which a number of cytoskeletal and cytoplasmic rearrangements occur which bear some similarities to the cortical flows seen in other organisms. During the interval known as pseudocleavage (PC) ~ the anterior cortex of the fertilized egg undergoes a series of contractions; cytoplasm flows towards the posterior end of the egg and a transient cleavage furrow (the pseudocleavage furrow) forms and then regresses (Nigon, 1960) . It is believed that at least some of these events are crucial for the correct partitioning of developmental determinants in the egg Strome, 1988, 1990; Strome, 1989; Kirby et al., 1990) . In particular, the cytoplasmic flow could carry determinants to the posterior end of the egg in a nonspecific way where they could then be anchored before the first determinative cleavage. Germ-line specific granules (P granules), may be localized to the posterior periphery of the egg in this way (Strome and Wood, 1982) . There is a striking reorganization of the cortical actin network concomitant with the events of PC (Strome, 1986) . Initially, actin fibers and foci are uniformly distributed throughout the egg cortex, but the foci become progressively concentrated in the anterior cortex. There is also immunocytological evidence that cortical myosin is redistributed similarly at this time (Strome, S., personal communication). It has therefore been suggested that during PC, cortical actin and, possibly, myosin form a contractile network that becomes localized to the anterior cortex displacing cytoplasm (and determinants) posteriorly (Strome, 1989) . We are interested in understanding how this fundamental reorganization of the egg cortex is brought about, as it may reveal how the polarity of an asymmetric cleavage is determined. In this paper we show that there is a directed flow of cortical material during PC in C. elegans by using a time lapse microscopy system that records multiple focal levels. We show that there is a similar cortical flow before cleavage in another blastomere which partitions cytoplasmic components before division but no directed flow in its sister which subsequently divides symmetrically. Furthermore, the cytoplasmic and cortical flows of PC, the formation of the transient furrow and the accumulation of cortical actin can be mimicked in other blastomeres during cytokinesis by treatment with the microtubule polymerization inhibitor nocodazole. This drug reduces the size of the mitotic spindle and often causes it to lie close to the cell cortex. In all cases, the orientation of the flows and the site of the accumulation of cortical actin are correlated with the location of the mitotic spindle. This suggests a general mechanism for generating directed flows of cytoplasm. Materials and Methods Wild type (N2) Caenorhabditis elegans strain Bristol was cultured on agar plates at 20°C with Escherichia coli strain OP50 as a food source (Brenner, 1974) . Embryos were obtained from young gravid adults by dissection into egg salts (118 mM NaC1, 40 mM KCI, 3.4 mM MgCI2, 3.4 mM CaC12, 5 mM Hepes, pH 7.4; Edgar and McGhee, 1986) . Early embryos were identified using a dissecting microscope, and then grouped together using the tip of a scalpel blade. They were moved for observation using a drawn out capillary tube. Embryos which had completed the second meiotic division before dissection survived this procedure; those isolated before the completion of meiosis usually arrested. Embryos were deposited onto a glass coverslip coated with a solution of 0.1% polylysine (Sigma Chem. Co., St. Louis, MO). The coverslip was then inverted over a depression slide containing egg salts. The embryos were observed using a Zeiss Axioplan microscope equipped with Nomarskl differential interference optics and a Hamamatsu C2400 video camera with analogue black-level subtraction. A time-lapse microscopy system was used to record events taking place on the surface and in the cytoplasm simultaneously (see below). A system was developed that allows a user to make time-lapse recordings of multiple focal planes, allowing three-dimensional events to be followed in time. The system uses a Sony LVR-LVS/6000 optical disk system controlled via a serial link from a lap-top computer. The computer is also used to control a motorized focus drive unit (as used in a BioRad MRC 500/600 confocal microscope) by means of a second serial port. The software system allows the user to specify the number of focal planes per scan, the separation between planes, the time interval between focus scans, the total number of focus scans and a file name for referencing the data on playback. All the parameters of a recording run are stored on a dedicated digital area on the video disk, and a directory may be obtained of all the video "files ~ existing on the currently mounted disk cartridge. When recording, a single video frame is captured and recorded at each focal level in a scan; scans are repeated at the specified intervals. On playback a user can replay the recording forwards and backwards in time at a specified focal level or go up and down through the focal levels at a given point in time. In this way it is possible to track objects in three dimensions over the period in which the recording was made. The programs were written by Mike Thomson of the Laboratory of Molecular Biology Electronics Workshop. As early pronuclear stage embryos are permeable to nocodazole, treatment of these embryos with this drug was accomplished by using the appropriate concentration of nocodazole (from a 5 mg/ml in DMSO stock solution) in egg salts. Older embryos were treated by laser permeabilizing the chitinous eggshell in the presence of egg salts containing nocodazole. Embryos were also permeabilized to cytochalasin D (from a 1 mg/ml in DMSO stock solution) using the laser. Laser microbeam permeabilization was performed as described previously . Granules were traced directly from the video monitor onto acetate sheets at suitable time intervals. Partially synchronized young adults were washed from NGM plates into M9 buffer (Brenner, 1974) . The worms were treated with 3% hypochlorite, 0.8 M NaOH in M9 which causes them to break open and release their embryos. The embryos were collected by centrifugation in a benchtop centrifuge (1,500 rpm, 30 s). The embryos were washed twice in M9 with centrifugation, and then resuspended in 10/~g/ml nocodazole in egg salts. The resuspended embryos were then permeabilized by being forced with moderate pressure through a 30 g needle to remove the eggshells. The permeabilized embryos were incubated for ,o5-10 min at room temperature in the nocodazole solution. Microfilament fixation and staining was performed as described in Hill and Strome (1988) . To fix, an equal volume of freshly prepared 3.0% paraformaldehyde, 0.2% glutaraldehye in egg salts was added and mixed quickly. Only permeabilized embryos were fixed by this procedure; such embryos would have therefore been permeable to the nocodazole. Fixation was allowed to proceed for 3 h at 16"C with shaking. After fixation, the embryos were collected by centrifugation, resuspended in a solution of 0.5 % Triton X-100 in egg salts, and extracted for 1 h at room temperature. The embryos were collected by centrifugation and resuspended in 6.6 nM Bodipy-phallacidin (Molecular Probes Inc., Eugene, OR) in PBS containing 0.25% Triton X-100. They were then stained overnight at 16°C with agitation. The stained embryos were collected by centrifugation, resuspended in a small volume of PBS containing 1 ~g/ml DAPI to stain the nuclei, and placed on a polylysine-coated slide. A coverslip with two greased edges was laid over the embryos, and PBS was wicked underneath to wash. Finally, the embryos were mounted in DABCO/glycerol and examined using a BioRad MRC-600 laser-scanning confocal microscope.
For example, in the very early stages of pronuclear migration in the C. elegans embryo, the female pronucleus moves from the anterior side toward the male pronucleus, presumably because of streaming flows along the cell periphery REF produced by motions of the actin-myosin cortex.
Cortical and cytoplasmic flow polarity in early embryonic cells of Caenorhabditis elegans
The Journal of Cell Biology
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