Establishment of an in vitro implantation model using a newly developed mouse endometrial organoid.

Implantation failure is a major cause of infertility, but its mechanisms remain unclear due to the lack of techniques for constructing organized endometrial structures and recapitulating the implantation process in vitro. Endometrial organoids have recently been developed, but they consist of only epithelial cells, and their apical surface faces inward, preventing blastocyst attachment. We developed an apical-out mouse endometrial organoid incorporating epithelial and stromal cells, and examined its ability to recapitulate implantation with mouse blastocysts.

Protocol for volume correlative light X-ray and electron microscopy of endothelial cells in mouse tissue.

Correlative light and electron microscopy (CLEM) greatly facilitate capturing the ultrastructure of spatially and/or temporally rare events. Here, we present a protocol for targeting regions of interests (ROIs) in tissue endothelial cells (ECs) using X-ray micro-computed tomography (μCT). We describe steps for ROI targeting guided by vasculature patterns and positions of EC nuclei visualized by light and X-ray microscopy.

Crosshair, semi-automated targeting for electron microscopy with a motorised ultramicrotome.

Volume electron microscopy (EM) is a time-consuming process - often requiring weeks or months of continuous acquisition for large samples. In order to compare the ultrastructure of a number of individuals or conditions, acquisition times must therefore be reduced. For resin-embedded samples, one solution is to selectively target smaller regions of interest by trimming with an ultramicrotome.

Branched Actin Maintains Acetylated Microtubule Network in the Early Secretory Pathway.

In the early secretory pathway, the delivery of anterograde cargoes from the endoplasmic reticulum (ER) exit sites (ERES) to the Golgi apparatus is a multi-step transport process occurring via the ER-Golgi intermediate compartment (IC, also called ERGIC). While the role microtubules in ER-to-Golgi transport has been well established, how the actin cytoskeleton contributes to this process remains poorly understood. Here, we report that Arp2/3 inhibition affects the network of acetylated microtubules around the Golgi and induces the accumulation of unusually long RAB1/GM130-positive carriers around the centrosome.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts.

Adipose triglyceride lipase protects renal cell endocytosis in a Drosophila dietary model of chronic kidney disease.

Obesity-related renal lipotoxicity and chronic kidney disease (CKD) are prevalent pathologies with complex aetiologies. One hallmark of renal lipotoxicity is the ectopic accumulation of lipid droplets in kidney podocytes and in proximal tubule cells. Renal lipid droplets are observed in human CKD patients and in high-fat diet (HFD) rodent models, but their precise role remains unclear.

MYO1C stabilizes actin and facilitates the arrival of transport carriers at the Golgi complex.

In this study, we aimed to identify the myosin motor proteins that control trafficking at the Golgi complex. In addition to the known Golgi-associated myosins MYO6, MYO18A and MYH9 (myosin IIA), we identified MYO1C as a novel player at the Golgi in a human cell line. We demonstrate that depletion of induces Golgi complex fragmentation and decompaction.

Myosin 1b promotes axon formation by regulating actin wave propagation and growth cone dynamics.

Single-headed myosin 1 has been identified in neurons, but its function in these cells is still unclear. We demonstrate that depletion of myosin 1b (Myo1b), inhibition of its motor activity, or its binding to phosphoinositides impairs the formation of the axon, whereas overexpression of Myo1b increases the number of axon-like structures. Myo1b is associated with growth cones and actin waves, two major contributors to neuronal symmetry breaking.

Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells.

Mitochondria dynamically change their shape by repeated fission and fusion in response to physiological and pathological conditions. Recent studies have uncovered significant roles of mitochondrial fission and fusion in neuronal functions, such as neurotransmission and spine formation. However, the contribution of mitochondrial fission to the development of dendrites remains controversial.

Synergistic action of dendritic mitochondria and creatine kinase maintains ATP homeostasis and actin dynamics in growing neuronal dendrites.

The distribution of mitochondria within mature, differentiated neurons is clearly adapted to their regional physiological needs and can be perturbed under various pathological conditions, but the function of mitochondria in developing neurons has been less well studied. We have studied mitochondrial distribution within developing mouse cerebellar Purkinje cells and have found that active delivery of mitochondria into their dendrites is a prerequisite for proper dendritic outgrowth. Even when mitochondria in the Purkinje cell bodies are functioning normally, interrupting the transport of mitochondria into their dendrites severely disturbs dendritic growth.

A 3D sphere culture system containing functional polymers for large-scale human pluripotent stem cell production.

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces.

Use of the unroofing technique for atomic force microscopic imaging of the intra-cellular cytoskeleton under aqueous conditions.

Atomic force microscopy (AFM) combined with unroofing techniques enabled clear imaging of the intracellular cytoskeleton and the cytoplasmic surface of the cell membrane under aqueous condition. Many actin filaments were found to form a complex meshwork on the cytoplasmic surface of the membrane, as observed in freeze-etching electron microscopy. Characteristic periodic striations of about 5 nm formed by the assembly of G-actin were detected along actin filaments at higher magnification.

Inhibition by ATP and activation by ADP in the regulation of flagellar movement in sea urchin sperm.

ATP and ADP are known to play inhibitory and activating roles, respectively, in the regulation of dynein motile activity of flagella. To elucidate how these nucleotide functions are related to the regulation of normal flagellar beating, we examined their effects on the motility of reactivated sea urchin sperm flagella at low pH. At pH 7.