Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.

Summary. Background: Recent studies have shown that platelet adhesion and subsequent aggregation can occur in vivo in the absence of the two principal platelets adhesive ligands, von Willebrand factor and fibrinogen. These results highlight a possible role for fibronectin in supporting thrombus formation. Objective and methods: To evaluate the platelet integrins and subsequent activation pathways associated with fibronectindependent platelet adhesion utilizing both human and murine platelets. Results: Platelets can adhere to fibronectin via the integrin a IIb b 3 , leading to formation of lamellipodia. This is mediated through an interaction with the tenth type III domain in fibronectin. Spreading on fibronectin promotes a IIb b 3 -mediated Ca 2+ mobilization and tyrosine phosphorylation of focal adhesion kinase and phospholipase C c2. In contrast, studies with blocking antibodies and a À=À IIb mice demonstrate that a 5 b 1 and a v b 3 support adhesion and promote formation of filopodia but not lamellipodia or tyrosine phosphorylation of these proteins. Further, neither a 5 b 1 nor a v b 3 is able to induce formation of lamellipodia in the presence of platelets agonists, such as collagen-related-peptide (CRP). Conclusions: These observations demonstrate that integrins a 5 b 1 and a v b 3 support platelet adhesion and the generation of filopodia but that, in contrast to the integrin a IIb b 3 , are unable to promote formation of lamellipodia.Keywords: a 5 b 1 , a IIb b 3 , fibronectin, platelet.

The Wiskott-Aldrich syndrome protein (WASP) family activates the Arp2/3 complex leading to the formation of new actin filaments. Here, we study the involvement of Scar1, Scar2, N-WASP, and Arp2/3 complex in dorsal ruffle formation in mouse embryonic fibroblasts (MEFs). Using platelet-derived growth factor to stimulate circular dorsal ruffle assembly in primary E13 and immortalized E9 Scar1 ؉/؉ and Scar1 null MEFs, we establish that Scar1 loss does not impair the formation of dorsal ruffles. Reduction of Scar2 protein levels via small interfering RNA (siRNA) also did not affect dorsal ruffle production. In contrast, wiskostatin, a chemical inhibitor of N-WASP, potently suppressed dorsal ruffle formation in a dose-dependent manner. Furthermore, N-WASP and Arp2 siRNA treatment significantly decreased the formation of dorsal ruffles in MEFs. In addition, the expression of an N-WASP truncation mutant that cannot bind Arp2/3 complex blocked the formation of these structures. Finally, N-WASP ؊/؊ fibroblast-like cells generated aberrant dorsal ruffles. These ruffles were highly unstable, severely depleted of Arp2/3 complex, and diminished in size. We hypothesize that N-WASP and Arp2/3 complex are part of a multiprotein assembly important for the generation of dorsal ruffles and that Scar1 and Scar2 are dispensable for this process.

Complex behaviors are typically associated with animals, but the capacity to integrate information and function as a coordinated individual is also a ubiquitous but poorly understood feature of organisms such as slime molds and fungi. Plasmodial slime molds grow as networks and use flexible, undifferentiated body plans to forage for food. How an individual communicates across its network remains a puzzle, but Physarum polycephalum has emerged as a novel model used to explore emergent dynamics. Within P. polycephalum, cytoplasm is shuttled in a peristaltic wave driven by cross-sectional contractions of tubes. We first track P. polycephalum's response to a localized nutrient stimulus and observe a front of increased contraction. The front propagates with a velocity comparable to the flow-driven dispersion of particles. We build a mathematical model based on these data and in the aggregate experiments and model identify the mechanism of signal propagation across a body: The nutrient stimulus triggers the release of a signaling molecule. The molecule is advected by fluid flows but simultaneously hijacks flow generation by causing local increases in contraction amplitude as it travels. The molecule is initiating a feedback loop to enable its own movement. This mechanism explains previously puzzling phenomena, including the adaptation of the peristaltic wave to organism size and P. polycephalum's ability to find the shortest route between food sources. A simple feedback seems to give rise to P. polycephalum's complex behaviors, and the same mechanism is likely to function in the thousands of additional species with similar behaviors. acellular slime mold | transport network | behavior | Taylor dispersion O ne of the great challenges of unraveling biological complexity is understanding what kind of and how much computational power is required for an organism to generate sophisticated behaviors. Behaviors are typically associated with a nervous system, but many organisms without nervous systems integrate information and function as coordinated individuals (1); examples range from the ability of Escherichia coli to move up chemical gradients (2) to the ability of a multicellular fungus to sense and precisely explore unoccupied space (3). A recently published and striking example of a complex behavior involves bacteria within a biofilm: When a Bacillus subtilis biofilm is deprived of nutrients, bacteria are able to grow networks of channels and evaporatively pump flows, creating intricate structures that benefit the entire community (4).Perhaps the archetypal example of an apparently simple organism able to generate sophisticated behaviors is the slime mold Physarum polycephalum, whose behaviors are repeatedly characterized as "intelligent." This slime mold is able to navigate mazes by finding the shortest route between different food sources (5) and has used its ability to reconstruct the transportation maps of major cities (6). The organism can structure its connections to different nutrient sources to optimize its diet...

How do the topology and geometry of a tubular network affect the spread of particles within fluid flows? We investigate patterns of effective dispersion in the hierarchical, biological transport network formed by Physarum polycephalum. We demonstrate that a change in topology -pruning in the foraging state -causes a large increase in effective dispersion throughout the network. By comparison, changes in the hierarchy of tube radii result in smaller and more localized differences. Pruned networks capitalize on Taylor dispersion to increase the dispersion capability.PACS numbers: 87.18. Vf, 87.16.Wd Transport due to fluid flowing through tubular networks is of great interest, because it has technological applications to biomimetic microfluidic devices [1][2][3], foams [4], fuel cells [5], and other filtration systems [6] and lies at the heart of extended organisms that rely on transport networks to function: animal vasculature [7,8], fungal mycelia [9], and plant tubes [10][11][12]. A big challenge regarding transport networks is to understand how network architecture changes the efficiency of particle spread throughout a network. While it is experimentally tedious to map particle transport in a network, predicting the spread of particles is also a theoretical challenge [13][14][15][16][17][18][19][20]. Attempts to understand how the network topology and geometry affect the transport of particles are scarce [17]. Alternatively, we can study the dynamic changes of tubular network architecture in living beings. Organisms spontaneously reorganize their transport networks, including tube pruning [21][22][23][24]. Examples are vessel development in zebra fish brain development [21], or growth of a large foraging fungal body [22]. Here, we study the slime mold Physarum polycephalum which emerged as an inspiring and yet puzzling model for 'intelligent' living transport networks.P. polycephalum like foraging fungi, actively adapts its network to environmental cues [25][26][27][28][29]. Networks connecting multiple food sources are a good compromise between efficiency, reliability, and cost, comparable to human transport networks [29]. Fluid cytoplasm enclosed in the tubular network exhibits nonstationary shuttle flows [30][31][32] driven by a peristaltic wave of contractions spanning the entire organism [33]. Investigations of transport in these networks are so far limited to estimates based on the minimal distance between tubes [29,34,35]. We tracked a well-reticulated individual trimmed from a larger network (Fig. 1). After several hours, the thin central tubes were abandoned in favor of a few large central tubes and globular structures at the periphery. How does this radical change of topology affect the transport capabilities of the individual? What role do hierarchical tube radii play? We present a method to efficiently map the effective dispersion of particles from any initiation site throughout any network with nonstationary but periodic fluid flows. We use this method to study the change in dispersion patterns ...

Entamoeba histolytica cells, the cause of amoebic dysentery, are highly motile, and this motility is an essential feature of the pathogenesis and morbidity of amoebiasis. However, the control of E. histolytica motility within the gut and during invasion is poorly understood. We have used an improved chemotaxis assay to identify the key extracellular signals mediating Entamoeba chemotaxis. The dominant responses we observe are caused by factors generated by E. histolytica cells themselves. Medium that has been conditioned by E. histolytica growth causes both chemokinesis and negative chemotaxis. The speed of random movement is more than doubled in conditioned compared with fresh medium, and cells move efficiently away from conditioned medium by negative chemotaxis. Ethanol, the product of Entamoeba glucose metabolism, is the principal component of the chemokinetic response. The closely related but nonpathogenic Entamoeba dispar shows no change in motility in response to conditioned medium implying that these responses are central to E. histolytica pathogenesis.invasion ͉ negative chemotaxis ͉ pathogenesis A n estimated 50 million individuals suffer the severe morbidity associated with invasive Entamoeba histolytica infections, with Ϸ100,000 deaths annually (1). Parasite-host interactions that determine the course of infection, in particular asymptomatic colonization vs. symptomatic invasive disease, are largely still a mystery. It is, however, accepted that amoebic adherence to and contactdependent killing of target cells, followed by phagocytosis, are key events (1, 2). The ability to interact with target cell surfaces is therefore a major process underpinning amoebic invasion of the human intestine. Consequently, research centered on E. histolytica cell surface receptors has great clinical promise. In particular, a role of chemotaxis seems likely. Zymosan activated C5a in human serum, lysed red blood cells, whole bacteria, components of the rat colon, N-acetylneuraminic acid (NANA) and NANA-containing compounds, fibronectin and fibronectin-derived fragments, and human TNF have all been shown to provide chemotactic stimuli (3-7).E. histolytica motility and chemotaxis have been studied by using relatively few methods, including in haemocytometers (8), tube migration (9), and Boyden chamber assays (3). Under the first two conditions, cells have almost no resistance to their movement except substrate adhesion. During invasive disease, however, amoebae move in more restrictive conditions, similar to metastasis and extravasation. Under-agarose (under-agar) assays provide such an environment and have been used to study the motility and chemotaxis of a variety of cells, including neutrophils, macrophages (10, 11), and the free-living amoeba Dictyostelium (12), an evolutionary relative of Entamoeba (13). Under-agar assays have the added advantage of allowing moving cells to be visualized and parameters such as cell shape to be studied in detail.To date, there appears to have been only one attempt at establishing an unde...

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