Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic β cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.

During host infection, single–celled apicomplexan parasites like Plasmodium and Toxoplasma use a motility mechanism called gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)–actin sandwiched between the plasma membrane and a myosin–coated inner membrane complex. How this surface actin layer drives the diverse apicomplexan gliding modes observed experimentally – helical, circular, and twirling, and patch, pendulum, or rolling – presents a rich biophysical puzzle. Here, we use single–molecule imaging to track individual actin filaments and myosin complexes in live Toxoplasma gondii. Based on these data, we hypothesize that F–actin flows arise by self–organization, rather than following a microtubule-based template as previously believed. We develop a continuum model of emergent F–actin flow within the unusual confines provided by parasite geometry. In the presence of F–actin turnover, our model predicts the emergence of a steady–state mode in which actin transport is largely rearward. Removing actin turnover leads to actin patches that recirculate up and down the cell, a ″cyclosis″ that we observe experimentally for drug–stabilized actin bundles in live parasites. These findings provide a mechanism by which actin turnover governs a transition between distinct self–organized F–actin states, whose properties can account for the diverse gliding modes known to occur. More broadly, we illustrate how different forms of gliding motility can emerge as an intrinsic consequence of the self–organizing properties of F–actin flow in a confined geometry.

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name polycation poisoning. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.