Single mutations frequently alter several aspects of cell behavior but it is often not clear whether a particular statistically significant change is biologically significant. To determine which behavioral changes are most important for multicellular self-organization, we devised a new methodology using Myxococcus xanthus as a model system. During development, myxobacteria coordinate their movement to aggregate into spore-filled fruiting bodies. We investigate how aggregation is restored in two mutants, csgA and pilC, that cannot aggregate unless mixed with wild type (WT) cells. To this end, we use cell tracking to follow movement of fluorescently labeled cells in combination with data-driven agent-based modeling. The results indicate that just like WT cells, both mutants bias their movement toward aggregates and reduce motility inside aggregates. However, several aspects of mutant behavior remain uncorrected by WT demonstrating that perfect recreation of WT behavior is unnecessary. In fact, synergies between errant behaviors can make aggregation robust. 12 partial rescue of the mutants during multicellular development of Myxococcus xanthus 13 biofilms. 14 Myxococcus xanthus is a rod-shaped member of the delta-Protobacteria with a 15 lifecycle centered around surface motility of cells in a biofilm. M. xanthus has evolved 16 1/20multiple social mechanisms such as S-motility [11] and C-signaling [5, 17, 26] to achieve 17 coordinated group behaviors such as predation [25], rippling [3,12,28] and 18 development [28,35]. Upon amino acid limitation, M. xanthus cells move into 19 three-dimensional aggregates called fruiting bodies where they sporulate [14,18,23]. 20 Recent studies based on cell tracking have provided unprecedented detail of cell 21 movement during development [7]. In combination with mathematical modeling, these 22 datasets unambiguously identified individual cell behaviors that are essential for 23 42 help of A-motility system that uses a novel molecular motor and focal adhesion 43 complexes [9,21]. However, most S-system mutants fail to develop because they cannot 44 produce an extracellular matrix (ECM) that is both essential for S-motility and vital for 45 development. The ECM is required for some types of chemotaxis [15,16] as well as for 46 cell cohesion, which could play a role in the inhibition of motility inside the 47 65 2/20 1 Results 66 1.1 Quantifying aggregation dynamics in mixtures of wild-type 67 and mutant strains 68 Fluorescence microscopy was used to quantify the behavior of mutant cells at both 69 single cell and population levels. A small fraction of cells expressing the fluorescent 70 protein tdTomato were mixed with cells expressing eYFP. Each cell expressing 71 tdTomato is bright enough to be segmented and tracked, allowing quantification of their 72 behaviors, whereas the weaker eYFP signal was used to quantify cell density during 73 aggregate growth [7]. 74 When either pilC or csgA cells are mixed with differentially labeled cells of their 75 own genotype, no aggregates were o...
