[Transdisciplinary Expert Statement: care guide for people severely affected by ME/CFS in home-based care].

is a widely studied eukaryote and an NIH-recognized model organism. Some significant features include its robust growth and similarities with key cellular processes that are observed in major biological systems such as wound healing, cancer metastasis, and embryogenesis []. Dictyostelium cells undergo a life cycle after starvation that involves transitioning from single cells to multi-cellular aggregates. These aggregates then cluster together to form mounds, which transform into motile slugs before their final transition into a fruiting body. All these transitions are initiated by a chemoattractant, cyclic adenosine 3‘,5’-monophosphate (cAMP), that acts as a signal between cells and aggregates []. This chemical signaling regulates gene expression and furthers development through chemotactic aggregation and, eventually, cell differentiation []. When sufficient extracellular cAMP is bound to a cell’s receptors, it triggers an intracellular cascade of events that stimulates the synthesis and secretion of additional cAMP and phosphodiesterase (PDE), an enzyme that breaks down cAMP. The secreted cAMP molecules will then diffuse to the neighboring cells, triggering further cAMP secretion, and thus relaying the signal throughout the entire population. Additionally, as part of the intracellular cascade of events, cells also polarize in response to the external cAMP gradient by redistributing proteins to create a ‘front’ and ‘back’ of the cell, allowing for directed migration in the direction opposite to the signal propagation. This entire process creates a chemical wave that propagates through a population of cells and can be visualized using intracellular fluorescent reporters (Flamindo 2). These fluorescent reporters undergo conformational changes when bound to cAMP, resulting in a decrease in fluorescence intensity [, ]. This method allows for visualization of the signal in a single cell and any propagation through a population of cells. Many experimental studies have sought to understand the cAMP signaling in Dictyostelium systems, both in a single cell [–] and at the population level [–]. There have also been attempts to understand these dynamics through theoretical studies [–]. Most of these studies, however, are limited to either the single cell, aggregation, or the slug stage. A detailed discussion on both the chemical signaling and the cell motility in the pre-mound formation stage is lacking. One key component of the pre-mound formation stage is the collective rotation of cells. As cells continue to signal to each other, they merge together to form small clusters, which subsequently coalesce into larger clusters. These clusters, containing anywhere from a few cells to a few thousands of cells depending on cluster size, collectively rotate in either clockwise or counterclockwise direction. As reported in earlier work, these aggregates also exhibit cAMP waves that rotate like spiral waves in the direction opposite to the cell rotation. The number of spiral arms within each cluster is not fixed and can vary [–]. A quantitative investigation of the rotational properties of cAMP waves and cells in rotating clusters remains unclear. This study aims to characterize the rotational properties of cell motion and cAMP signaling in the pre-mound stage clusters in more detail, using a combination of experiments and numerical simulations. We focus on cluster size as the key player in determining a number of signaling properties within a cluster, including the angular velocity of the cAMP wave (), the cell angular velocity (), and the number of spiral arms. We investigate this experimentally with GFP tagged cell aggregates (AX4 Flamindo2) under agar confinement to analyze the rotations and cAMP signaling properties in clusters of various sizes. The agar confinement method is commonly used to improve visualization of cells and their cAMP signaling by restricting motion in the third dimension, allowing dynamics to be viewed in an effectively 2D slice []. Our experimental results show that in larger aggregates both and decrease. Furthermore, we demonstrate that and are linearly correlated. Finally, we also show that spirals with multiple arms are more probable within larger aggregates compared to smaller aggregates.