This article has an associated First Person interview with the first author of the paper.The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single-cell RNA-sequencing and identify Amalgam (Ama), which encodes a cell adhesion protein of the immunoglobulin IgLON family, as regulating the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB), which leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of the RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPROUTY2 in glioblastoma cell lines, suggesting that the relationship between Ama and Sty is conserved.The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. selleck compound Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.Cellular polarization underlies many facets of cell behavior, including cell growth. The rod-shaped fission yeast Schizosaccharomyces pombe is a well-established, genetically tractable system for studying growth polarity regulation. S. pombe cells elongate at their two cell tips in a cell cycle-controlled manner, transitioning from monopolar to bipolar growth in interphase when new ends established by the most recent cell division begin to extend. We previously identified cytokinesis as a critical regulator of new end growth and demonstrated that Fic1, a cytokinetic factor, is required for normal polarized growth at new ends. Here, we report that Fic1 is phosphorylated on two C-terminal residues, which are each targeted by multiple protein kinases. Endogenously expressed Fic1 phosphomutants cannot support proper bipolar growth, and the resultant defects facilitate the switch into an invasive pseudohyphal state. Thus, phosphoregulation of Fic1 links the completion of cytokinesis to the re-establishment of polarized growth in the next cell cycle. These findings broaden the scope of signaling events that contribute to regulating S. pombe growth polarity, underscoring that cytokinetic factors constitute relevant targets of kinases affecting new end growth.This article has an associated First Person interview with Anthony M. Rossi, joint first author of the paper.Intimal stiffening upregulates endothelial cell contractility, disrupting barrier integrity; however, intimal stiffening is non-uniform. The impact of local changes in intimal stiffness on proximal and distal cell-cell interactions is unknown. To investigate the range at which matrix stiffness heterogeneities impact neighboring endothelial cells within a monolayer, we built a micropillar system with adjacent regions of stiff and compliant matrix. The stiffness interface results in an oscillatory pattern of neutrophil transendothelial migration, symmetrical about the interface and well-fit by a sinusoid function. ‘Peaks’ of the sinusoid were found to have increased cellular contractility and decreased barrier function relative to ‘troughs’ of the sinusoid. Pharmacological modulation of contractility was observed to break symmetry, altering the amplitude and wavelength of the sinusoid, indicating that contractility may regulate this effect. This work illuminates a novel biophysical phenomenon of the role of stiffness-mediated cell-matrix interactions on cell-cell interactions at a distance.