Cooperatively generated stresslet flows supply fresh fluid to multicellular choanoflagellate colonies.

The flagellated protozoan Salpingoeca rosetta is one of the closest relatives of multicellular animals. Unicellular S. rosetta can be induced to form multicellular colonies, but colonies swim more slowly than individual cells so the advantages conferred by colony formation are uncertain. Here we use theoretical models to show that hydrodynamic cooperation between cells can increase the fluid supply to the colony, an important predictor of feeding rate. Our results suggest that hydrodynamic benefits may have been an important selective factor in the evolution of early multicellular animals.

Cell differentiation and morphogenesis in the colony-forming choanoflagellate Salpingoeca rosetta.

It has been posited that animal development evolved from pre-existing mechanisms for regulating cell differentiation in the single celled and colonial ancestors of animals. Although the progenitors of animals cannot be studied directly, insights into their cell biology may be gleaned from comparisons between animals and their closest living relatives, the choanoflagellates. We report here on the life history, cell differentiation and intercellular interactions in the colony-forming choanoflagellate Salpingoeca rosetta. In response to diverse environmental cues, S. rosetta differentiates into at least five distinct cell types, including three solitary cell types (slow swimmers, fast swimmers, and thecate cells) and two colonial forms (rosettes and chains). Electron microscopy reveals that cells within colonies are held together by a combination of fine intercellular bridges, a shared extracellular matrix, and filopodia. In addition, we have discovered that the carbohydrate-binding protein wheat germ agglutinin specifically stains colonies and the slow swimmers from which they form, showing that molecular differentiation precedes multicellular development. Together, these results help establish S. rosetta as a model system for studying simple multicellularity in choanoflagellates and provide an experimental framework for investigating the origin of animal multicellularity and development.

In silico reconstitution of actin-based symmetry breaking and motility.

Eukaryotic cells assemble viscoelastic networks of crosslinked actin filaments to control their shape, mechanical properties, and motility. One important class of actin network is nucleated by the Arp2/3 complex and drives both membrane protrusion at the leading edge of motile cells and intracellular motility of pathogens such as Listeria monocytogenes. These networks can be reconstituted in vitro from purified components to drive the motility of spherical micron-sized beads. An Elastic Gel model has been successful in explaining how these networks break symmetry, but how they produce directed motile force has been less clear. We have combined numerical simulations with in vitro experiments to reconstitute the behavior of these motile actin networks in silico using an Accumulative Particle-Spring (APS) model that builds on the Elastic Gel model, and demonstrates simple intuitive mechanisms for both symmetry breaking and sustained motility. The APS model explains observed transitions between smooth and pulsatile motion as well as subtle variations in network architecture caused by differences in geometry and conditions. Our findings also explain sideways symmetry breaking and motility of elongated beads, and show that elastic recoil, though important for symmetry breaking and pulsatile motion, is not necessary for smooth directional motility. The APS model demonstrates how a small number of viscoelastic network parameters and construction rules suffice to recapture the complex behavior of motile actin networks. The fact that the model not only mirrors our in vitro observations, but also makes novel predictions that we confirm by experiment, suggests that the model captures much of the essence of actin-based motility in this system.

Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2.

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly-with no detectable lag-upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2-Arp3-actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.

Prey capture and phagocytosis in the choanoflagellate Salpingoeca rosetta.

Choanoflagellates are unicellular and colonial aquatic microeukaryotes that capture bacteria using an apical flagellum surrounded by a feeding collar composed of actin-filled microvilli. Flow produced by the apical flagellum drives prey bacteria to the feeding collar for phagocytosis. We report here on the cell biology of prey capture in rosette-shaped colonies and unicellular "thecate" or substrate attached cells from the choanoflagellate S. rosetta. In thecate cells and rosette colonies, phagocytosis initially involves fusion of multiple microvilli, followed by remodeling of the collar membrane to engulf the prey, and transport of engulfed bacteria into the cell. Although both thecate cells and rosette colony cells produce ∼ 70 nm "collar links" that connect and potentially stabilize adjacent microvilli, only thecate cells were observed to produce a lamellipod-like "collar skirt" that encircles the base of the collar. This study offers insight into the process of prey ingestion by S. rosetta, and provides a context within which to consider potential ecological differences between solitary cells and colonies in choanoflagellates.