Chemotactic Orientation Research Articles

Nhe1 Is Essential for Potassium but Not Calcium Facilitation of Cell Motility and the Monovalent Cation Requirement for Chemotactic Orientation in Dictyostelium discoideum

In Dictyostelium discoideum, extracellular K+ or Ca2+ at a concentration of 40 or 20 mM, respectively, facilitates motility in the absence or presence of a spatial gradient of chemoattractant. Facilitation results in maximum velocity, cellular elongation, persistent translocation, suppression of lateral pseudopod formation, and myosin II localization in the posterior cortex. A lower threshold concentration of 15 mM K+ or Na or 5 mM Ca2+ is required for chemotactic orientation. Although the common buffer solutions used by D. discoideum researchers to study chemotaxis contain sufficient concentrations of cations for chemotactic orientation, the majority contain insufficient levels to facilitate motility. Here it has been demonstrated that Nhe1, a plasma membrane protein, is required for K+ but not Ca2+ facilitation of cell motility and for the lower K+ but not Ca2+ requirement for chemotactic orientation.

Costars, aDictyosteliumprotein similar to the C-terminal domain of STARS, regulates the actin cytoskeleton and motility

Through analysis of a chemotaxis mutant obtained from a genetic screen in Dictyostelium discoideum, we have identified a new gene involved in regulating cell migration and have named it costars (cosA). The 82 amino acid Costars protein sequence appears highly conserved among diverse species, and significantly resembles the C-terminal region of the striated muscle activator of Rho signaling (STARS), a mammalian protein that regulates the serum response factor transcriptional activity through actin binding and Rho GTPase activation. The cosA-null (cosA(-)) cells formed smooth plaques on bacterial lawns, produced abnormally small fruiting bodies when developed on the non-nutrient agar and displayed reduced migration towards the cAMP source in chemotactic assays. Analysis of cell motion in cAMP gradients revealed decreased speed but wild-type-like directional persistence of cosA(-) cells, suggesting a defect in the cellular machinery for motility rather than for chemotactic orientation. Consistent with this notion, cosA(-) cells exhibited changes in the actin cytoskeleton, showing aberrant distribution of F-actin in fluorescence cell staining and an increased amount of cytoskeleton-associated actin. Excessive pseudopod formation was also noted in cosA(-) cells facing chemoattractant gradients. Expressing cosA or its human counterpart mCostars eliminated abnormalities of cosA(-) cells. Together, our results highlight a role for Costars in modulating actin dynamics and cell motility.

Swimming behaviour of Schistosoma mansoni cercariae: responses to irradiance changes and skin attractants

The swimming behaviour of many cercarial species is governed by sensitive responses to light and dark stimuli. We studied the effect of irradiance changes on swimming behaviour of Schistosoma mansoni cercariae and found only insignificant responses. Decreasing light intensity results in a weak tendency of the cercariae to start swimming movements, and increasing light intensity tends to inhibit the start of swimming. These responses seem not suitable to increase the transmission success. Whether the cercariae show chemo-orientation towards human skin was studied by video-tracking their swimming movements around agar containing human-skin-surface extracts and when immersed into skin extracts. They showed no directed chemotactic orientation, as they did not correct their swimming paths in direction towards the skin-extract substrates, also not when shifting between forward and backward swimming. However, the cercariae shifted more between backward and forward swimming and therewith increased their rate of change of direction. This response may support an accumulation around the skin substrates and could guide the cercariae towards the host's skin surface when they are already in close proximity to it.

Distinct Roles of PI(3,4,5)P3during Chemoattractant Signaling inDictyostelium: A Quantitative In Vivo Analysis by Inhibition of PI3-Kinase

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.

The Shwachman-Bodian-Diamond syndrome gene encodes an RNA-binding protein that localizes to the pseudopod ofDictyosteliumamoebae during chemotaxis

The Shwachman-Bodian-Diamond syndrome (SBDS) is an autosomal disorder with multisystem defects. The Shwachman-Bodian-Diamond syndrome gene (SBDS), which contains mutations in a majority of SBDS patients, encodes a protein of unknown function, although it has been strongly implicated in RNA metabolism. There is also some evidence that it interacts with molecules that regulate cytoskeletal organization. Recently, it has been demonstrated by computer-assisted methods that the single behavioral defect of polymorphonuclear leukocytes (PMNs) of SBDS patients is the incapacity to orient correctly in a spatial gradient of chemoattractant. We considered using the social amoeba Dictyostelium discoideum, a model for PMN chemotaxis, an excellent system for elucidating the function of the SBDS protein. We first identified the homolog of SBDS in D. discoideum and found that the amino acids that are altered in human disease were conserved. Given that several proteins involved in chemotactic orientation localize to the pseudopods of cells undergoing chemotaxis, we tested whether the SBDS gene product did the same. We produced an SBDS-GFP chimeric in-frame fusion gene, and generated transformants either with multiple ectopic insertions of the fusion gene or multiple copies of a non-integrated plasmid carrying the fusion gene. In both cases, the SBDS-GFP protein was dispersed equally through the cytoplasm and pseudopods of cells migrating in buffer. However, we observed differential enrichment of SBDS in the pseudopods of cells treated with the chemoattractant cAMP, suggesting that the SBDS protein may play a role in chemotaxis. In light of these results, we discuss how SBDS might function during chemotaxis.

Acyl‐homoserine lactones modulate the settlement rate of zoospores of the marine alga Ulva intestinalis via a novel chemokinetic mechanism

Bacteria utilize quorum sensing to regulate the expression of cell density-dependant phenotypes such as biofilm formation and virulence. Zoospores of the marine alga Ulva intestinalis exploit the acyl-homoserine lactone (AHL) quorum sensing system to identify bacterial biofilms for preferential settlement. Here, we demonstrate that AHLs act as strong chemoattractants for Ulva zoospores. Chemoattraction does not involve a chemotactic orientation towards the AHL source. Instead, it occurs through a chemokinesis in which zoospore swimming speed is rapidly decreased in the presence of AHLs. The chemoresponse to AHLs was dependant on the nature of the acyl side chain, with N-(3-oxododecanoyl)-homoserine lactone (30-C12-HSL) being the most effective signal molecule. Mean zoospore swimming speed decreased more rapidly over wild-type biofilms of the marine bacteria Vibrio anguillarum relative to biofilms of the vanM mutant, in which AHL synthesis is disrupted. These data implicate a role for AHL-mediated chemokinesis in the location and preferential settlement of Ulva zoospores on marine bacterial assemblages. Exposure to AHLs did not inhibit the negative phototaxis of Ulva zoospores, indicating that chemoattraction to bacterial biofilms does not preclude the response to a light stimulus in substrate location.

Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum

BackgroundStimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.ResultsWe investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae.ConclusionThese results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.

Navigation within host tissues: Schistosoma mansoni and Trichobilharzia ocellata schistosomula respond to chemical gradients

After penetration of human or duck host's skin schistosomula of Schistosoma mansoni and Trichobilharzia ocellata migrate parallel to the surface in the epidermis, then they enter the dermis and venules prior to further migration. This study focuses on potential behavioural mechanisms and host cues which may enable this navigation within host tissues. We stimulated cercariae to penetrate into agar substrates and to transform to schistosomula, and analysed their orientation behaviour within chemical concentration gradients. Both species were chemotactically attracted by low molecular weight fractions of their host's serum (human, duck) and d-glucose and l-arginine were identified as attractive components in serum. They responded to gradients, which established after addition of very low concentrations of d-glucose (1 μM in T. ocellata and 2 μM in S. mansoni) and l-arginine (0.025 μM in T. ocellata and 1.0 μM in S. mansoni). The response to d-glucose was specific as other saccharides had no stimulatory activity. l-Arginine stimulated chemotactic orientation both when free and bound in peptides. However, the two species responded differently to the position of l-arginine within the peptide (terminal or subterminal), and only S. mansoni, not T. ocellata, responded to peptides occurring in serum and endothelial cells: fibronectin (1 μM), bradykinin (25 pM) and its fragment 1–5 (2.5 μM). Both species adjusted their body axis with the ventral side towards the higher concentrations of d-glucose and of l-arginine. We argue that the chemotactic orientation and the alignment of the body axis enable the parasites (i) to orientate towards deeper skin layers and avoid accidental perforation of the covering skin surface layers, (ii) to determine their position during their surface-parallel migration within the epidermis, (iii) to locate blood vessels.

The spatial orientation of Helicobacter pylori in the gastric mucus.

The highly motile human pathogen Helicobacter pylori lives deep in the gastric mucus layer. To identify which chemical gradient guides the bacteria within the mucus layer, combinations of luminal perfusion, dialysis, and ventilation were used to modify or invert transmucus gradients in anaesthetized Helicobacter-infected mice and Mongolian gerbils. Neither changes in lumen or arterial pH nor inversion of bicarbonate/CO2 or urea/ammonium gradients disturbed Helicobacter orientation. However, elimination of the mucus pH gradient by simultaneous reduction of arterial pH and bicarbonate concentration perturbed orientation, causing the bacteria to spread over the entire mucus layer. H. pylori thus uses the gastric mucus pH gradient for chemotactic orientation.

Complex pattern formation by a self-destabilization of established patterns: chemotactic orientation and phyllotaxis as examples

Stable patterns can be generated by molecular interactions involving local self-enhancement and long-range inhibition. In contrast, highly dynamic patterns result if the maxima, generated in this way, become destabilized by a second antagonistic reaction. The latter must act local and must be long-lasting. Maxima either disappear and reappear at displaced positions or they move over the field as travelling waves. The wave can have unusual properties in that they can penetrate each other without annihilation. The resulting pattern corresponds to those observed in diverse biological systems. In the chemotactic orientation of cells, the temporary signals allow the localized extensions of protrusions under control of minute external asymmetries imposed by the chemoattractant. In phyllotaxis, these signals lead to successive leaf initiation, whereby the longer-lasting extinguishing reaction can cause a displacement of the subsequent leaf initiation site by the typical 137.5°, the golden angle. On seashells, this patterns leads either to oblique lines that can cross each other or to oblique rows of dots. For some of the models animated simulations are available at http://www.eb.tuebingen.mpg.de/abt.4/meinhardt/theory.html. To cite this article: H. Meinhardt, C. R. Biologies 326 (2003).

Recognition and invasion of human skin by Schistosoma mansoni cercariae: the key-role of L-arginine.

The attachment of Schistosoma mansoni cercariae to mammalian skin is specifically stimulated by L-arginine. As L-arginine is an unsuitable signal for a specific identification of mammalian skin we examined the following 5 hypotheses to explain the advantage of the cercarial sensitivity to L-arginine. (1) A Schistosoma infection lowered the arginine concentration in the serum of mice, and this could enable the cercariae to avoid attachments to already infected mice. However, the infection did not reduce the arginine concentration in the skin and the cercarial attachment responses to it. (2) Creeping cercariae showed chemotactic orientation specifically along increasing L-arginine gradients. L-arginine could act as a pheromone which could guide cercariae towards common penetration sites. However, the cercarial acetabular gland contents were not attractive and they did not (in contrast to previous reports) contain much arginine. (3) Schistosomula (transformed cercariae) could use L-arginine to produce nitric oxide (NO) for blood vessel dilation during their migration in the host. However, in vitro the transformed cercariae did not convert L-arginine into citrulline and NO. (4) Schistosomula could bind L-arginine from the surrounding tissues and so escape the cellular immune attack (which needs L-arginine as the precursor of NO). However, transformed cercariae bound no more L-arginine than L-serine and L-lysine. (5) Schistosomula, migrating parallel to the surface in the mammalian epidermis, are dependent on information on their position between the inner and the surface layers of the skin. In the mouse skin, they adjusted their body axis with the ventral side toward the deeper (arginine-residue rich) epidermis layers. When migrating in agar, they showed chemo-orientation toward serum, and D-glucose and L-arginine were the stimulating compounds therein. The burrowing schistosomula adjusted their body axis (as in the epidermis) with the ventral side toward the higher concentration of L-arginine and not of glucose. We argue that the sensitivity for L-arginine has its primary function in orientation within mammalian skin and in location of blood vessels.

Stereochemical specificity of lamoxirene, the sperm-releasing pheromone in kelp (Laminariales, Phaeophyceae).

Next article No AccessResearch NotesStereochemical Specificity of Lamoxirene, the Sperm-Releasing Pheromone in Kelp (Laminariales, Phaeophyceae)Ingo Maier, Christian Hertweck, and Wilhelm BolandIngo Maier1. Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany Search for more articles by this author , Christian Hertweck2. Max-Planck-Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, 07745 Jena, Germany Search for more articles by this author , and Wilhelm Boland2. Max-Planck-Institute for Chemical Ecology, Carl-Zeiss-Promenade 10, 07745 Jena, Germany Search for more articles by this author Received 18 December 2000; accepted 28 June 2001.PDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 201, Number 2October 2001 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.2307/1543327 Views: 99Total views on this site PDF download Crossref reports no articles citing this article.

Chemoattractant-controlled accumulation of coronin at the leading edge of Dictyostelium cells monitored using a green fluorescent protein–coronin fusion protein

The highly motile cells of Dictyostelium discoideum rapidly remodel their actin filament system when they change their direction of locomotion either spontaneously or in response to chemoattractant. Coronin is a cytoplasmic actin-associated protein that accumulates at the coritcal sites of moving cells and contributes to the dynamics of the actin system. It is a member of the WD-repeat family of proteins and is known to interact with actin-myosin complexes. In coronin null mutants, cell locomotion is slowed down and cytokinesis is impaired. We have visualized the redistribution of coronin by fluorescence imaging of motile cells that have been transfected with an expression plasmid containing the coding sequence of coronin fused to the sequence encoding the green fluorescent protein (GFP). This coronin-GFP fusion protein (GFP). This coronin-GFP fusion protein transiently accumulates in the front regions of growth-phase cells, reflecting the changing positions of leading edges and the competition between them. During the aggregation stage, local accumulation of coronin-GFP is biased by chemotactic orientation of the cells in gradients of cAMP. The impairment of cell motility in coronin null mutants shows that coronin has an important function at the front region of the cells. The mutant cells are distinguished by the formation of extended particle-free zones at their front regions, from where pseudopods often break out as blebs. Cytochalasin A reduces the size of these zones, indicating that actin filaments prevent entry of the particles. These data demonstrate that coronin is reversibly recruited from the cytoplasm and is incorporated into the actin network of a nascent leading edge, where it participates in the reorganization of the cytoskeleton. Monitoring the dynamics of protein assembly using GFP fusion proteins and fluorescence microscopy promises to be a generally applicable method for studying the dynamics of cytoskeletal proteins in moving and dividing cells.

Finding and recognition of the snail intermediate hosts by 3 species of echinostome cercariae

Finding and recognition of snail second intermediate hosts was studied in cercariae of 3 echinostome species. The cercariae of the 3 species accumulated in snail-conditioned water (SCW) with 2 types of orientation mechanisms and responded to different small molecular weight ( < 500 Da) components of SCW. Pseudechinoparyphium echinatum and Echinostoma revolutum cercariae returned by swimming an arc, when swimming in decreasing concentration gradients of SCW (turn-back swimming). The stimulating cues of SCW were identified as hydrophilic organic molecules, probably possessing amino groups. Amino acids contributed to the attractivity of SCW, at least in P. echinatum, but they could not account for the complete attractivity of SCW. Hypoderaeum conoideum were directed chemotactically and swam along increasing concentration gradients of small peptides within SCW, but in decreasing SCW gradients they showed no turn-back swimming. Chemotactic orientation in H. conoideum only started 1 h after emission, which may assist the cercariae to leave the immediate area of their first intermediate host snails and to disperse. Attachments occurred specifically to snail hosts in the 3 species and were stimulated by macromolecular mucus compounds, probably mainly by viscoelastic properties of the mucus. The results of this study show, that host-finding mechanisms and the stimulating host cues of snail invading echinostome cercariae differ considerably from those of schistosome miracidia.

Behavioral responses of streamer F mutants of Dictyostelium discoideum: effects of cyclic GMP on cell motility.

Streamer F (stmF) mutants have a prolonged increase in intracellular cGMP in response to addition of the chemoattractant cAMP. The speed of movement and area of stmF cells were quantitated as the cells were stimulated with a rapid, uniform increase in extracellular cAMP. The speed of stmF cells rapidly drops as does that of the wild-type, but then requires about 300 seconds to recover. In contrast, the speed of the parental strain, XP55, recovers within 60-70 seconds. This prolonged drop in speed correlates with the time during which intracellular cGMP remains high, suggesting that intracellular cGMP induces this prolonged reduction in speed. Mutants from other streamer complementation groups do not show this altered response. Area measurements indicate that stmF cells do not cringe or round up as XP55 does, but spread with the same kinetics as XP55. Chemotactic orientation of the stmF cells in stable spatial gradients is similar to or slightly greater than that of the wild-type. Tracking of cells moving during aggregation indicates that the stmF cells show large drops in speed between pulses, resulting in the banding pattern seen in streams. The cells can still respond to new pulses, resulting in an aggregation time that is similar to that of XP55.

Orientation of Aplysia californien to distant food sources

The behavior of the marine mollusc Aplysia was examined under different experimental conditions designed to determine the food searching strategy of the animals. In a small, open field tank with still water, the animals took an average of 42 min to find a piece of seaweed, even though the stimulus was never located more than 30 cm away from the animal. Observations of the animals indicated that their search was not directed, without a clear tendency towards the food, and during the course of a search, they often crawled through most of the area of the tank. The search time, the distance travelled, and the strategy of the search of the animals was similar for different types of seaweed. If animals were aroused into activity by the presence of seaweed extract, the time for them to contact a piece of odorless glass fiber paper in the open field was not significantly different than that for a piece of seaweed. The probability at which the animals contacted the seaweed, as a function of the distance travelled, resembled the detection probability determined according to a theory of random search. We thus propose that the aroused animals move in a random pattern until they are very close to the food. This strategy can be advantageous in still water since chemicals do not provide distinct gradients that can serve as cues for chemotactic orientation from distances greater than a few centimeters from the source. In a Y-maze in still water, Aplysia did not perform above chance in selecting the arm that contained the seaweed.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.

A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Consequences of chemosensory phenomena for leukocyte chemotactic orientation

The stochastic nature of cell surface receptor-ligand binding is known to limit the accuracy of detection of chemoattractant gradients by leukocytes, thus limiting the orientation ability that is crucial to the chemotactic response in host defense. The probabilistic cell orientation model of Lauffenburger is extended here to assess the consequences of recently discovered receptor phenomena: "down-regulation" of total surface receptor number, spatial asymmetry of surface receptors, and existence of a higher-affinity receptor subpopulation. In general, a reduction in orientation accuracy is predicted by inclusion of these phenomena. An orientation signal based on a simple model of chemosensory adaptation (i.e., a spatial difference in relative receptor occupancy) is found to be functionally different from the signal suggested by an experimental correlation (i.e., a spatial difference in absolute receptor occupancy). However, in the context of receptor "signal noise," the signal based on adaptation yields predictions in better qualitative agreement with the experimental orientation data of Zigmond. From this cell orientation model we can estimate the effective time-averaging period required for noise diminution to a level allowing orientation predictions to match observed levels. This time-averaging period presumably reflects the time constant for receptor signal transduction and locomotory response.