The dependence of the growth rate and meat content of young boars on semen parameters and conception rate.

Boars have a decisive impact on the progress in pig production, however, there is no recent information about the optimal growth parameters during the rearing period for modern breed later used in artificial insemination (AI) stations. Therefore, the objective of the research was to conduct semen parameter and conception rate analyses on the basis of growth rate and meat content assessments made during the rearing of AI boars of different genotypes. The study was carried out between 2010 and 2014 and included 184 boars in five breed combinations: 46 Polish Large White, 50 Polish Landrace, 27 Pietrain, 36 Duroc×Pietrain and 25 Hampshire×Pietrain. Boars were qualified by daily gains and meat content assessment (between 170 and 210 days of life). A total number of 38 272 ejaculates were examined (semen volume (ml), spermatozoa concentration (×106 ml-1), total number of spermatozoa (×109) and number of insemination doses from one ejaculate (n)). The fertility was determined by the conception rate (%). Semen volume, spermatozoa concentration and conception rate (P<0.01), followed by the total number of spermatozoa and insemination doses (P<0.05) were characterized by the highest variability in relation to breed of boars. The effect of daily gains was reported for spermatozoa concentration, number of insemination doses, conception rate (all P<0.01) and total number of spermatozoa (P<0.05). The peak of growth for spermatozoa concentration, total number of spermatozoa, insemination doses and conception rate was achieved for 800 to 850 g gains. Meat content affected semen volume, number of insemination doses and conception rate (P<0.05). Rearing boars while maintaining daily gains at the 800 to 850 g level and 62.5% to 65% meat content helps AI stations to increase the efficiency and economic profitability, and the number of insemination doses to increase by up to 300 doses/boar within a year. The analyses of growth parameters may help increase the efficiency and economic viability of AI stations.

Factors affecting the tissues composition of pork belly.

Bellies derived from the commercial population of pig carcasses are diverse in terms of tissue composition. Knowledge of the factors influencing it and the expected results, permits quick and easy evaluation of raw material. The study was designed to determine the factors affecting the tissues composition of pork bellies and to estimate their lean meat content. The research population (n=140 pig carcasses) was divided into groups according to sex (gilts, barrows), half-carcass mass (<40, 40 to 43.9, 44 to 46.9, ⩾47 kg) and lean meat content class: S (⩾60%), E (55% to 60%), U (50% to 55%), R (<50%). Bellies were subjected to a detailed dissection. Half-carcass mass affected the levels of all the analysed parameters. The only exception was the mass of the fat with the skin in the 40 to 43.9 kg group, for which the value did not differ statistically between the two groups <40 and 44 to 46.9 kg. Decrease in lean meat content affected the growth of the fat and skin mass in a linear way. No differences were observed between class S and E in terms of belly muscle mass. A 0.37% higher share of belly in the half-carcass was found for barrows (P<0.001), although bellies issued from barrows were characterized by a higher proportion of fat with skin compared with gilts (P=0.02). Interactions were observed between sex and half-carcass mass, so the sex of heavy half-carcasses becomes an important determinant for conditioning the muscle content. Equations were calculated and allow a fast and highly accurate determination of the lean meat content in bellies, suggesting they may be used directly in the production line.

The influence of endoparasites on selected production parameters in pigs in various housing systems.

The aim of the study was to determine the level of lean meat content and daily gains of 400 fatteners infected by endoparasites and kept in two systems (shallow and deep litter). Slaughter evaluation of the pigs was conducted according to the EUROP carcass classification. In order to evaluate the average daily gains (g) during finishing period, body weights were investigated twice: at the beginning and at the end of the finishing period. The housing system affected the presence of Ascaris suum and Oesophagostomum spp. Infestation was found to be higher on shallow than on deep litter, and it significantly affected selected fattening and slaughter parameters of the fatteners. Infected animals were characterized by gains approximately 60 g lower than those of uninfected ones, while meatiness was higher in fatteners which were not infected at the end of the fattening period compared to animals with parasites (55.2% vs. 52.0%).

Does a boar's season of birth determine semen parameters and reproductive performance?

This article studies the effect of a boar's birth season and breed on semen parameters and its further reproductive performance. Research material consisted of 72 boars from three breeds (24 Polish Large White PLW, 24 Polish Landrace PL, 24 Duroc × Pietrain D × P). During the whole period of the study, selected semen parameters were analysed: semen volume, spermatozoa concentration, total number of spermatozoa, total number of motile spermatozoa, number of insemination doses and also reproductive indicators: farrowing rate, total born litter size, total number of piglets born live and still, and average piglet weight. Boars born in the winter and summer months demonstrated the highest spermatozoa concentrations (383.25 and 392.37 × 10(6)/ml), total number of spermatozoa (91.75 and 93.21 × 10(9)), total number of motile spermatozoa (76.10 and 77.99 × 10(9)) and number of insemination doses (24.53 and 24.89; p ≤ 0.01). Statistically lower values for these parameters were observed for boars born in the spring and especially in autumn (p ≤ 0.01). The significant impact of birth season on farrowing rate (p ≤ 0.05) and average piglet weight (p ≤ 0.05) was confirmed for PLW boars. For the PL breed, only the total number of piglets born live was proven to be significantly affected (p ≤ 0.05). No impact of birth season was shown on semen quality or reproductive performance for D × P boars. In our study, we showed that the birth season of a boar had a more impact on the level of semen parameters, and less on the reproductive performance indicators. The results indicated that both the quality of semen and reproductive performance varied in terms of the study factors, as well as between individual breeds of boars involved in the experiment.

ReAsH: another viable option for in vivo protein labelling in Dictyostelium.

Biarsenical-tetracysteine fluorescent protein tagging has been effectively used in a variety of cell types. It has the advantage of requiring a much smaller peptide alteration to existing proteins than fusion to green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP). However, there are no reports of the tetracysteine tagging system being used in Dictyostelium. In order to establish this tagging system in Dictyostelium, the filamin gene (FLN) was modified to express a C-terminal tetracysteine sequence and then transfected into cells. After addition of either FlAsH-EDT(2) or ReAsH-EDT(2), the fluorescence intensity of cells increased in a time-dependent manner and reached a plateau after 3 h of incubation. ReAsH had a much stronger and more specifically localized fluorescent signal compared with FlAsH. After removal of the ReAsH-EDT(2) reagent, the fluorescence signal remained detectable for at least 24 h. The localization of filamin labelled by ReAsH was similar to that of an FLN-mRFP fusion protein, but the fluorescence signal from the ReAsH-labelled protein was stronger. Our findings suggest that the ReAsH-tetracysteine tagging system can be a useful alternative for in vivo protein tagging in Dictyostelium.

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells.

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca2+]i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca2+]i. We observed small, brief, Ca2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.

Automated real-time measurement of chemotactic cell motility.

We have developed a novel method, (ECIS/taxis), for monitoring cell movement in response to chemotactic and chemokinetic factors. In this system, cells migrate in an under-agarose environment, and their positions are monitored using the electric cell-substrate impedance sensor technology to measure the impedance change at a target electrode, that is lithographed onto the substrate, as the cells arrive at the target. In the studies reported here, Dictyostelium discoideum was used as a prototypical, motile eukaryotic cell. Using the ECIS/taxis system, the arrival of cells at the target electrode was proportional to the dose offolate used to stimulate the cells and could be assessed by changes in resistance at the electrode. ECIS/taxis was readily able to distinguish between wild-type cells and a mutant that is deficient in its chemotactic response. Finally, we have shown that an agent that interferes with chemotactic motility leads to the delayed arrival of cells at the target electrode. The multi-well assay configuration allows for simultaneous automated screening of many samples for chemotactic or anti-chemotactic activity. This assay system is compatible with measurements of mammalian cell movement and should be valuable in the assessment of both agonists and antagonists of cell movement.

Under-agarose folate chemotaxis of Dictyostelium discoideum amoebae in permissive and mechanically inhibited conditions.

Under-agarose chemotaxis has been used previously to assess the ability of neutrophils to respond to gradients of chemoattractant. We have adapted this assay to the chemotactic movement of Dictyostelium amoebae in response to folic acid. Troughs are used instead of wells to increase the area along which the cells can be visualized and to create a uniform front of moving cells. Imaging the transition zone where the cells first encounter the agarose, we find that the cells move perpendicular to the gradient and periodically manage to squeeze under the agarose and move up the gradient. As cells exit the troughs, their cross-sectional area increases as the cells become flattened. Three-dimensional reconstruction of confocal optical sections through GFP-labeled cells demonstrates that the increase in cross-sectional area is due to the flattening of the cells. Since the cells locally deform the agarose and become deformed by it, the concentration of the agarose, and therefore its stiffness, should affect the ability of the cells to migrate. Consistent with this hypothesis, cells in 0.5% agarose move faster and are less flat than cells under 2% agarose. Cells do not exit the troughs and move under 3% agarose at all. Therefore, this assay can be used to compare and quantify the ability of different cell types or mutant cell lines to move in a restrictive environment.

Regulated expression of myosin II heavy chain and RacB using an inducible tRNA suppressor gene.

An inducible expression system that indirectly regulates gene expression through the use of an inducible suppressor tRNA has been used to express both endogenous and exogenous genes in Dictyostelium. The tetracycline repressor and tRNA suppressor (Glu) are expressed from a single G418 selectable vector, while a gene engineered to contain a stop codon is expressed from a separate hygromycin selectable vector. beta-Galactosidase could be induced over 300 fold with this system, and the extent of induction could be varied depending upon the amount of tetracycline added. It took 3 days to fully induce expression, and about 3 days for expression to decrease to baseline after removal of the tetracycline. Dictyostelium myosin II heavy chain could also be expressed in an inducible manner, although the induction ratio was not as high as beta-galactosidase and the maximum expression level was not as high as wild-type levels. A significant accumulation of the truncated peptide indicates that complete suppression of the stop codon was not achieved. Partial phenotypic reversion was observed in null mutants inducibly expressing myosin II. RacB could also be inducibly expressed, whereas the protein could not be expressed from a constitutive promoter, presumably because expression at high levels is lethal. Therefore, the inducible tRNA system can be used to control expression of endogenous Dictyostelium genes.

Sequential activities of phosphoinositide 3-kinase, PKB/Aakt, and Rab7 during macropinosome formation in Dictyostelium.

Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes.

Cytoskeletal alterations in Dictyostelium induced by expression of human cdc42.

The rho family of small G proteins has been shown to be involved in controlling actin filament dynamics in cells. To evaluate the functional overlap between human and Dictyostelium G proteins, we conditionally expressed constitutively active human cdc42 (V12-cdc42) in Dictyostelium cells. Upon induction, cells adopted a unique morphology: a flattened shape with wrinkles running from the cell edge toward the center. The appearance of these wrinkles is highly dynamic so that the cells cycle between the wrinkled and relatively normal morphologies. Phalloidin staining indicates that the stellate wrinkles contain dense actin structures and also that numerous filopods project vertically from the center of these cells. Consistent with the hypothesis that cdc42 induces actin polymerization in vivo, cells expressing V12-cdc42 show an increase in the amount of F-actin associated with the cytoskeleton. This is accompanied by an increase in the association of the actin-binding proteins 34-kDa bundler, ABP-120 and alpha-actinin with the cytoskeleton. In conclusion, human cdc42 has various effects on the Dictyostelium actin cytoskeleton consistent with a conserved role of small GTPases in control of the cytoskeleton.

During multicellular migration, myosin ii serves a structural role independent of its motor function.

We have shown previously that cells lacking myosin II are impaired in multicellular motility. We now extend these results by determining whether myosin contractile function is necessary for normal multicellular motility and shape control. Myosin from mutants lacking the essential (mlcE(-)) myosin light chain retains the ability to form bipolar filaments that bind actin, but shows no measurable in vitro or in vivo contractile function. The contractile function is necessary for cell shape control since mlcE(-) cells, like myosin heavy-chain null mutants (mhcA(-)), were defective in their ability to control their three-dimensional shape. When mixed with wild-type cells in chimeric aggregation streams, the mlcE(-) cells were able to move normally, unlike mhcA(-) cells which accumulated at the edges of the stream and became distorted by their interactions with wild-type cells. When mhcA(-) cells were mixed with mlcE(-) streams, the mhcA(-) cells were excluded. The normal behavior of the mlcE(-) cells in this assay suggests that myosin II, in the absence of motor function, is sufficient to allow movement in this constrained, multicellular environment. We hypothesize that myosin II is a major contributor to cortical integrity even in the absence of contractile function.

The regulation of actin polymerization and cross-linking in Dictyostelium.

It is clear that the polymerization and organization of actin filament networks plays a critical role in numerous cellular processes. Inhibition of actin polymerization by pharmacological agents will completely prevent chemotactic motility, macropinocytosis, endocytosis, and phagocytosis. Recently there has been great progress in understanding the mechanisms that control the assembly and structure of the actin cytoskeleton. Members of the Rho family of GTPases have been identified as major players in the signal transduction pathway leading from a cell surface signal to actin polymerization. The Arp2/3 complex has been added to the list of means by which new actin filaments can be nucleated. However, it is clear that actin polymerization by Arp2/3 complex is not the whole story. In principle, the final structures formed by actin filaments will depend on factors such as: the length of actin filaments, the degree of branching, how they are cross-linked and the tensions imparted on them. In addition, the means by which actin polymerization generates protrusion of membranes is still controversial. A phagosome, filopodium and a lamellipodium all require polymerization of new actin filaments, but each has a characteristic morphology and cytoskeletal structure. In the following chapter, we will discuss actin polymerization and filament organization, especially as it relates to the machinery of phagocytosis in Dictyostelium.

Mutant Rac1B expression in Dictyostelium: effects on morphology, growth, endocytosis, development, and the actin cytoskeleton.

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.

Actin filaments are severed by both native and recombinant dictyostelium cofilin but to different extents.

Cofilin has been reported to depolymerize F-actin alternately by either severing filaments to increase the number of depolymerizing ends or by increasing the off-rate of monomers from F-actin without increasing the number of filament ends. We have compared directly the ability of native and recombinant cofilins from Dictyostelium to sever F-actin. Our results demonstrate that native cofilin has a higher level of severing activity than recombinant cofilin. Significantly, the measurement of cofilin's severing activity by two independent methods, direct visualization with an improved light microscope assay and by scoring of the number of pointed ends by DNase I binding, clearly shows that both native and recombinant cofilins sever F-actin but to different extents. The severing activity in preparations of recombinant cofilin is variable depending on the method of preparation and, in some cases, is difficult to detect by microscopy assays. This latter point is particularly significant because it may lead to the conclusion that cofilin severs weakly or not at all depending on its method of isolation.

Variables controlling the expression level of exogenous genes in Dictyostelium.

Ectopic expression of genes from recombinant plasmids is commonly used to study gene function. In Dictyostelium, three drug resistance cassettes are commonly used as selectable markers in vectors. We report here a comparative study of the expression of green fluorescent protein (GFP) gene from vectors containing each of the drug-resistant cassettes. The expression was highest in cells transformed with the vectors containing the neomycin-resistant cassette (pDNeoGFP), followed by the hygromycin-resistant cassette (pDHygGFP) and the blasticidin-resistant cassette (pDBsrGFP). The level of GFP expression was directly related to the copy number of the vector in transformants. In turn, the copy number of the vector depended on the drug resistance cassette as well as the concentration of the drug used in selection. In general, cells with higher copy numbers could be selected by a higher drug concentration. The expression of GFP was also affected by the method of transformation. For pDHygGFP, expression of GFP was much higher in cells transformed by electroporation than those transformed by calcium phosphate coprecipitation. However, only a slight difference was observed for pDNeoGFP or pDBsrGFP.

Overexpression of a novel rho family GTPase, RacC, induces unusual actin-based structures and positively affects phagocytosis in Dictyostelium discoideum.

Rho family proteins have been implicated in regulating various cellular processes, including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel Dictyostelium discoideum Rho family protein (RacC). A cell line was generated that conditionally overexpressed wild-type RacC three- to fourfold relative to endogenous RacC. Light and scanning electron microscopy indicated that the morphology of the RacC-overexpressing cells [RacC WT(+) cells] was significantly altered compared with control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin-rich surface blebs (petalopodia, for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced threefold relative to control Ax2 cells, whereas fluid-phase pinocytosis was reduced threefold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid-phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate postinternalization steps along the endolysosomal pathway. Treatment of cells with Wortmannin and LY294002 (phosphatidylinositol 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol-binding motif-containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium.

Reconstruction and display of curvilinear objects from optical section data using 3-D curve fitting algorithms.

Biological objects resembling filaments are often highly elongated while presenting a small cross-sectional area. Examination of such objects requires acquisition of images from regions large enough to contain entire objects, but at sufficiently high resolution to resolve individual filaments. These requirements complicate the application of conventional optical sectioning and volume reconstruction techniques. For example, objective lenses used to acquire images of entire filaments or filament networks may lack sufficient depth (Z) resolution to localize filament cross-sections along the optical axis. Because volume reconstruction techniques consider only the information represented by a single volume element (voxel), views of filament networks reconstructed from images obtained at low Z-resolution will not accurately represent filament morphology. A possible solution to these problems is to simultaneously utilize all available information on the path of an object by fitting 3-D curves through data points localized in 2-D images. Here, we present an application of this approach to the reconstruction of microtubule networks from 2-D optical sections obtained using confocal microscopy, and to synthesized curves which have been distorted using a simple mathematical model of optical sectioning artefacts. Our results demonstrate that this strategy can produce high resolution 3-D views of filamentous objects from a small number of optical sections.

Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures.

Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1] [2] [3]. We have been able to visualize these changes in live cells using a fusion protein (GFP-ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP-ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP-ABD fluorescence in these cells coincided with that of rhodamine-phalloidin, indicating that GFP-ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP-ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

Formation of F-actin aggregates in cells treated with actin stabilizing drugs.

We have electroporated Dictyostelium amoebae with fluorescent phalloidin in order to visualize the localization and behavior of F-actin filaments in living cells. Immediately after electroporation with phalloidin, cells became round and showed bright staining in the cortical region. Over time, the cortical staining disappeared and was replaced by a large aggregate of actin filaments. The aggregates were predominantly localized to the apical posterior of actively moving cells and in the middle of dividing cells or stationary AX4 cells. Mutants lacking myosin II or ABP-120 also formed actin aggregates; however, the rate of formation of aggregates was slower in myosin II mutant cells. In order to investigate this phenomenon further, we have used jasplakinolide, a membrane-permeable drug that also stabilizes F-actin filaments. Cells treated with jasplakinolide formed actin aggregates in a concentration-dependent manner. Drug treatment led to an increase in the proportion of actin associated with the cytoskeleton. Jasplakinolide-treated cells were still motile; however, their rate of movement was less than that of untreated cells. Cytochalasin B and nocodazole had inhibitory effects on aggregate formation, while azide blocked the process completely. We hypothesize that aggregates are formed from the cortical flow of F-actin filaments. These filaments would normally be depolymerized but are artificially stabilized by phalloidin or jasplakinolide binding. The localization of the aggregate is likely to be an indication of the direction of cortical flow.