Key Points Coordinated contraction of the uterine smooth muscle is essential to parturition.Histologically and physiologically defined pacemaker structures have not been identified in uterine smooth muscle.Here we report combined electrophysiological and histological evidence of zones associated with pacemaker activity in the rat myometrium.Our method relies crucially on the integration of histological and electrophysiological data in an in silico three‐dimensional reconstruction of the rat myometrium at 10 μm resolution.We find that myometrial/placental pacemaking zones are closely related with placental sites and the area of disruptive myometrial remodelling surrounding such sites.If analogues of the myometrial/placental pacemaking zone are present in the human, defining their histology and physiology will be important steps towards treatment of pre‐term birth, pre‐eclampsia, and postpartum haemorrhage. AbstractCoordinated uterine contractions are essential for delivering viable offspring in mammals. In contrast to other visceral smooth muscles, it is not known where excitation within the uterus is initiated, and no defined pacemaking region has hitherto been identified. Using multi‐electrode array recordings and high‐resolution computational reconstruction of the three‐dimensional micro‐structure of late pregnant rat uterus, we demonstrate that electrical potentials are initiated in distinct structures within the placental bed of individual implantation sites. These previously unidentified structures represent modified smooth muscle bundles that are derived from bridges between the longitudinal and circular layers. Coordinated implantation and encapsulation by invading trophoblast give rise to isolated placental/myometrial interface bundles that directly connect to the overlying longitudinal smooth muscle layer. Taken together, these observations imply that the anatomical structure of the uterus, combined with site‐specific implantation, gives rise to emergent patterns of electrical activity that drive effective contractility during parturition.

Generative adversarial networks (GANs) have recently been successfully used to create realistic synthetic microscopy cell images in 2D and predict intermediate cell stages. In the current paper we highlight that GANs can not only be used for creating synthetic cell images optimized for different fluorescent molecular labels, but that by using GANs for augmentation of training data involving scaling or other transformations the inherent length scale of biological structures is retained. In addition, GANs make it possible to create synthetic cells with specific shape features, which can be used, for example, to validate different methods for feature extraction. Here, we apply GANs to create 2D distributions of fluorescent markers for F-actin in the cell cortex of Dictyostelium cells (ABD), a membrane receptor (cAR1), and a cortex-membrane linker protein (TalA). The recent more widespread use of 3D lightsheet microscopy, where obtaining sufficient training data is considerably more difficult than in 2D, creates significant demand for novel approaches to data augmentation. We show that it is possible to directly generate synthetic 3D cell images using GANs, but limitations are excessive training times, dependence on high-quality segmentations of 3D images, and that the number of z-slices cannot be freely adjusted without retraining the network. We demonstrate that in the case of molecular labels that are highly correlated with cell shape, like F-actin in our example, 2D GANs can be used efficiently to create pseudo-3D synthetic cell data from individually generated 2D slices. Because high quality segmented 2D cell data are more readily available, this is an attractive alternative to using less efficient 3D networks.

BackgroundThe fibrous structure of the myometrium has previously been characterised at high resolutions in small tissue samples (< 100 mm3) and at low resolutions (∼500 μm per voxel edge) in whole-organ reconstructions. However, no high-resolution visualisation of the myometrium at the organ level has previously been attained.Methods and resultsWe have developed a technique to reconstruct the whole myometrium from serial histological slides, at a resolution of approximately 50 μm per voxel edge. Reconstructions of samples taken from human and rat uteri are presented here, along with histological verification of the reconstructions and detailed investigation of the fibrous structure of these uteri, using a range of tools specifically developed for this analysis. These reconstruction techniques enable the high-resolution rendering of global structure previously observed at lower resolution. Moreover, structures observed previously in small portions of the myometrium can be observed in the context of the whole organ. The reconstructions are in direct correspondence with the original histological slides, which allows the inspection of the anatomical context of any features identified in the three-dimensional reconstructions.Conclusions and significanceThe methods presented here have been used to generate a faithful representation of myometrial smooth muscle at a resolution of ∼50 μm per voxel edge. Characterisation of the smooth muscle structure of the myometrium by means of this technique revealed a detailed view of previously identified global structures in addition to a global view of the microarchitecture. A suite of visualisation tools allows researchers to interrogate the histological microarchitecture. These methods will be applicable to other smooth muscle tissues to analyse fibrous microarchitecture.

Macropinocytosis is a conserved endocytic process where cells take up medium into micron-sized vesicles. In Dictyostelium, macropinocytic cups form around domains of PIP3 in the plasma membrane and extend by actin polymerization. Using lattice light-sheet microscopy, we describe how cups originate, are supported by an F-actin scaffold and shaped by a ring of actin polymerization, created around PIP3 domains. How cups close is unknown. We find two ways: lip closure, where actin polymerization at the lip is re-directed inwards; and basal closure, where it stretches the cup, eventually causing membrane delamination and vesicle sealing. Cups grow as expanding waves of actin polymerization that travel across the cell surface, capturing new membrane. We propose that cups close when these waves stall. This 'stalled wave' hypothesis is tested through a conceptual model, where the interplay of forces from actin polymerization and membrane tension recreates many of our observations.