Evolution of an ancient protein function involved in organized multicellularity in animals.

To form and maintain organized tissues, multicellular organisms orient their mitotic spindles relative to neighboring cells. A molecular complex scaffolded by the GK protein-interaction domain (GKPID) mediates spindle orientation in diverse animal taxa by linking microtubule motor proteins to a marker protein on the cell cortex localized by external cues. Here we illuminate how this complex evolved and commandeered control of spindle orientation from a more ancient mechanism. The complex was assembled through a series of molecular exploitation events, one of which - the evolution of GKPID's capacity to bind the cortical marker protein - can be recapitulated by reintroducing a single historical substitution into the reconstructed ancestral GKPID. This change revealed and repurposed an ancient molecular surface that previously had a radically different function. We show how the physical simplicity of this binding interface enabled the evolution of a new protein function now essential to the biological complexity of many animals

Bacterial cues regulate multicellular development and mating in the choanoflagellate, S. rosetta

Animals first diverged from their unicellular ancestors in oceans dominated by bacteria, and have lived in close association with bacteria ever since. Interactions with bacteria critically shape diverse aspects of animal biology today, including developmental processes that were long thought to be autonomous. Yet, the multicellularity of animals and the often-complex communities of bacteria with which they are associated make it challenging to characterize the mechanisms underlying many bacterial-animal interactions. Thus, developing experimentally tractable host-microbe model systems will be essential for revealing the molecules and mechanisms by which bacteria influence animal development. The choanoflagellate Salpingoeca rosetta, one of the closest living relatives of animals, has emerged as an attractive model for studying host-microbe interactions. Like all choanoflagellates, S. rosetta feeds on bacteria; however, we have found that interactions between S. rosetta and bacteria extend beyond those of predator and prey. In fact, two key transitions in the life history of S. rosetta, multicellular “rosette” development and sexual reproduction, are regulated by environmental bacteria. The experimental tractability of S. rosetta allowed us to characterize the molecules and regulatory logic underpinning the bacterial regulation of rosette development (Chapters 2 and 3). We found that the bacterium Algoriphagus machipongonensis produces three classes of structurally distinct lipids that are interpreted by S. rosetta as activators, synergistic enhancers, and inhibitors of rosette development. Although activating sulfonolipid RIFs (Rosette Inducing Factors) elicited relatively low levels of rosette development, the combined activity of the RIFs and synergizing lysophosphatidylethanolamines (LPEs; which alone had no detectable activity) was sufficient to fully recapitulate the rosette-inducing activity of Algoriphagus bacteria. Moreover, we identified a potent antagonist of the RIFs, IOR-1 (Inhibitor of Rosettes), but found that the synergistic activities of the RIFs and the LPEs overcame the inhibitory activities of IOR-1. We hypothesize that the integration of multiple activating, enhancing, and inhibitory bacterial cues act to ensure that rosette development is not initiated under the wrong environmental conditions. Until recently, bacteria were not known to influence any life history transition in S. rosetta other than rosette development. We serendipitously discovered that the bacterium Vibrio fischeri produces an “aphrodisiac” that regulates sexual reproduction in S. rosetta (Chapter 4). To our knowledge, the interaction between Vibrio and S. rosetta is the first known example of bacteria regulating mating in a eukaryote. After observing that S. rosetta cells aggregate into large swarms in response to Vibrio bacteria, we demonstrated that swarming, a behavior that had not been previously observed in choanoflagellates, was a prelude to sexual fusion. We next found that Vibrio secreted a chondroitinase aphrodisiac (EroS) that depolymerized chondroitin sulfate, a glycosaminoglycan previously thought to be restricted to animals, in the S. rosetta extracellular matrix. Finally, we determined mating in S. rosetta was triggered by low cell densities of Vibrio bacteria, and picomolar concentrations of EroS (as well as other bacterial chondroitinases), indicating that bacteria could plausibly trigger S. rosetta swarming and mating in the environment. We predict that the presence of chondroitinase-producing bacteria may indicate environmental factors that favor mating in S. rosetta

Lessons from simple marine models on the bacterial regulation of eukaryotic development

Molecular cues from environmental bacteria influence important developmental decisions in diverse marine eukaryotes. Yet, relatively little is understood about the mechanisms underlying these interactions, in part because marine ecosystems are dynamic and complex. With the help of simple model systems, including the choanoflagellate Salpingoeca rosetta, we have begun to uncover the bacterial cues that shape eukaryotic development in the ocean. Here, we review how diverse bacterial cues-from lipids to macromolecules-regulate development in marine eukaryotes. It is becoming clear that there are networks of chemical information circulating in the ocean, with both eukaryotes and bacteria acting as nodes; one eukaryote can precisely respond to cues from several diverse environmental bacteria, and a single environmental bacterium can regulate the development of different eukaryotes

Synergistic Cues from Diverse Bacteria Enhance Multicellular Development in a Choanoflagellate.

Bacteria regulate the life histories of diverse eukaryotes, but relatively little is known about how eukaryotes interpret and respond to multiple bacterial cues encountered simultaneously. To explore how a eukaryote might respond to a combination of bioactive molecules from multiple bacteria, we treated the choanoflagellate Salpingoeca rosetta with two sets of bacterial cues, one that induces mating and another that induces multicellular development. We found that simultaneous exposure to both sets of cues enhanced multicellular development in S. rosetta, eliciting both larger multicellular colonies and an increase in the number of colonies. Thus, rather than conveying conflicting sets of information, these distinct bacterial cues synergize to augment multicellular development. This study demonstrates how a eukaryote can integrate and modulate its response to cues from diverse bacteria, underscoring the potential impact of complex microbial communities on eukaryotic life histories.IMPORTANCE Eukaryotic biology is profoundly influenced by interactions with diverse environmental and host-associated bacteria. However, it is not well understood how eukaryotes interpret multiple bacterial cues encountered simultaneously. This question has been challenging to address because of the complexity of many eukaryotic model systems and their associated bacterial communities. Here, we studied a close relative of animals, the choanoflagellate Salpingoeca rosetta, to explore how eukaryotes respond to diverse bacterial cues. We found that a bacterial chondroitinase that induces mating on its own can also synergize with bacterial lipids that induce multicellular "rosette" development. When encountered together, these cues enhance rosette development, resulting in both the formation of larger rosettes and an increase in the number of rosettes compared to rosette development in the absence of the chondroitinase. These findings highlight how synergistic interactions among bacterial cues can influence the biology of eukaryotes

Mating in the Closest Living Relatives of Animals Is Induced by a Bacterial Chondroitinase

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here, we show that a single V. fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V. fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S. rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V. fischeri, purified EroS, and other bacterial chondroitin lyases induce S. rosetta mating at environmentally relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature