The adhesive interactions of neutrophils with postcapillary venules during inflammation have been well studied. However, how neutrophils trigger molecular changes in endothelial cells (EC) during their extravasation requires further exploration. The endothelial actin-binding protein cortactin regulates endothelial contacts and neutrophil-endothelial interactions, but the associated mechanisms remain elusive. Hypothesizing that endothelial cortactin dynamics change during inflammation, using super-resolution confocal microscopy of inflamed mouse cremasteric venules and HUVEC, we report that neutrophil interaction with EC induces reduction in EC cortactin levels. This response was specifically mediated by neutrophil serine proteases, including cathepsin G, that were detected inside EC. The observed cortactin degradation was abolished after inhibition of serine proteases or blockade of neutrophil exocytosis. Finally, the endogenous serine protease inhibitor α1-antitrypsin suppressed cortactin degradation in vivo and reduced neutrophil adhesion and extravasation. Collectively, our data unveil a new mechanism by which neutrophils manipulate proteins inside EC to facilitate their extravasation.

Depletion of TRF2 from chromosome ends causes telomeric fusions and genome instability in mammals, but in mouse neural stem cells (mNSCs), Trf2's role is non-telomeric. Although essential for mNSC proliferation and survival, Trf2 does not protect telomeres, aligning with findings that Trf2 is dispensable for telomere protection in pluripotent stem cells. In Trf2-deficient adult mNSCs (Trf2fl/fl; Nestin-Cre), proliferation decreased and neuronal differentiation was impaired, yet no telomere dysregulation or DNA damage response was observed. Similarly, TRF2 depletion in SH-SY5Y cells induced differentiation without telomere dysfunction. Mechanistically, non-telomeric TRF2 directly binds to the promoters of key genes that regulate differentiation, recruiting the polycomb repressor complex (PRC2) for H3K27 trimethylation, repressing differentiation genes to maintain NSC identity. G-quadruplex (G4) motifs are crucial for TRF2 binding; disrupting this interaction via G4-binding ligands or the G4-specific helicase DHX36 induces differentiation genes, promoting neurogenesis. These findings highlight TRF2's non-telomeric role in NSC survival, offering insights into neurogenesis and aging-related neurodegeneration.

Tetraspanins are integral membrane proteins that play a crucial role in organizing and regulating cellular signaling by serving as scaffolds that compartmentalize receptors and other signaling molecules within membrane microdomains. Here, we report how the tetraspanin CD82 modulates the molecular organization and signaling of the EGF receptor (EGFR), a key molecule involved in cellular proliferation, differentiation, and survival. Combining multicolor super-resolution microscopy with advanced image reconstruction and analysis techniques, we demonstrate that CD82 selectively associates with EGFR, promotes receptor oligomerization, and acts as a regulator of ligand-independent receptor phosphorylation in a palmitoylation-dependent manner. Additionally, CD82 promotes a more compact molecular organization of EGFR, which correlates with altered endocytosis and downstream signaling outcomes. These findings underscore the importance of tetraspanins in the spatial and functional regulation of cell surface receptors, with implications for controlling aberrant signaling in disease and positions CD82 as a potential target for developing therapeutic strategies aimed at modulating EGFR signaling by influencing receptor organization.

Secreted proteins are essential for processes like immune responses, cellular communication, and extracellular matrix remodeling. Once synthesized and processed at the Golgi, some of these proteins are packaged for delivery to the plasma membrane. While this transport and sorting rely on complex molecular machinery, the precise mechanisms remain unclear. In this study, we affinity-isolated and analyzed post-Golgi carriers by mass spectrometry. Candidate machinery was subsequently assessed in a pooled CRISPR-KO screen. This led to the identification of a rich set of new genes functionally important for Golgi-to-plasma membrane delivery including PTPN23, a component of the endosomal sorting complex required for transport (ESCRT) complex. Depletion of PTPN23, as well as the ESCRT subunits CHMP1 and VPS4, disrupts tubule fission from the trans-Golgi, impairing cargo delivery to the surface. Furthermore, the loss of PTPN23 also prevents the constitutive secretion of soluble cargoes, and of endogenous hormones and antibodies in specialized cells. We propose that PTPN23 is essential for secretion from the trans-Golgi.

Activation of WNT signaling in human pluripotent stem cells efficiently drives lateral mesoderm specification and subsequent cardiomyocyte differentiation. Stabilization of the WNT effector β-catenin induces mesodermal genes such as TBXT (Brachyury) and triggers an epithelial-mesenchymal transition (EMT). Although mechanical forces are essential for embryonic development, the role of actomyosin contractility during human mesoderm specification remains unclear. We show that increasing contractility through constitutively active Rho kinase or myosin light-chain kinase unexpectedly blocks β-catenin-dependent mesoderm induction and prevents EMT. In contrast, pharmacological or genetic suppression of contractility enhances Brachyury expression and advances EMT onset by 24 h. While β-catenin signaling alone promotes colony-level contractility, we find that contractility must be reduced prior to WNT activation to promote mesoderm specification, indicating a sensitization effect at the pluripotent state. Mechanistically, reduced tension decreases junctional β-catenin and increases nuclear active β-catenin, identifying actomyosin contractility as a key regulator of lineage commitment following WNT pathway activation.

Cells in metabolically active tissues with high biosynthetic and secretory demands often use robust stress-responsive mechanisms to maintain homeostasis. Coordinating such stress response mechanisms requires intercellular communication and coordination. Such modalities of intercellular communication have been relatively understudied in the context of stress tolerance. Here, we use the Drosophila melanogaster third instar fat body to demonstrate that adipocytes communicate with each other through intercellular bridges called ring canals to buffer endoplasmic reticulum (ER) stress. The fat body supports the exponential growth from embryo to late larval stage over a short period of time through its energy storage and secretory functions, enduring a high basal level of stress in the process. We discovered that individual cells in the fat body are paired to one neighboring cell through ring canals. We further demonstrate that ring canals mediate rapid and highly specific intercellular cargo and organellar trafficking, and allow the transport of cytoplasmic, ER-bound, and Golgi vesicular proteins. Disrupting fat body ring canals resulted in higher levels of stress response markers, aberrant cell size, and increased cell sensitivity and lethality in response to various exogenous stressors. We also find that animals with disrupted fat body ring canals display an overall delay in larval development, likely due to reduced secretion of larval serum proteins from the fat body. In sum, our work reveals a novel feature of intercellular communication in adipose tissue that serves to buffer stress across cells, which is required for both homeostatic secretory function and maintaining tissue viability under exogenous stress.

Cilia, despite sharing a conserved axonemal structure, display diverse structural and molecular features, particularly at their tips. This study uncovers a mechanism that shapes the molecular features of fly mechanosensory cilia tips through localized active transport mediated by Kif19A, a kinesin-8 family member. Kif19A is predominantly expressed in peripheral neurons and is essential for enriching mechanosensory molecules at the ciliary tip, though it does not affect overall ciliary structure. In vitro studies show that Kif19A motors bind transiently to microtubules and exhibit non-processive movement individually, while multiple motors work together to drive plus end-directed movement of the cargoes, essential for precise localization and function of Kif19A in vivo. Building on the experimental observations, we propose a theoretical model in which nanoscale molecular polarity is established by local transport and binding sites that counteract rapid diffusion. This model highlights key principles by which molecular motors shape nanoscale cellular structures, thereby contributing to the diversity of cell architecture.

Enteroendocrine cells (EEs) are secretory cells in the gut epithelium that exist in two morphologically distinct forms: "open" and "closed." However, the mechanisms governing this morphological divergence remain unclear. Here, we show that in the adult Drosophila midgut, open-type EEs are the predominant form throughout most regions, whereas the closed type is found exclusively in the middle gastric region (R3). We identify Ptx1, a region-specific transcription factor, as a key determinant of the closed EE morphology. Ptx1 maintains the expression of the Snail-family transcription factor escargot (esg) in newly formed EEs, which in turn suppresses the expression of smooth septate junction (sSJ) genes. This repression prevents apical integration into the epithelium, resulting in the formation of closed-type EEs. Ectopic expression of ptx1 or esg in non-R3 regions induces the formation of closed-type EEs, which exhibit impaired sensing of dietary amino acids. Together, our findings reveal a regional transcriptional regulatory axis that controls EE morphology and its associated luminal sensing function.

Bridge-like lipid-transport protein 2 (BLTP2) transfers lipids at membrane contact sites, but its precise localization is unclear. Dziurdzik et al. and Dai et al. identify and characterize a conserved contact site between the endoplasmic reticulum and the plasma membrane mediated by BLTP2.

Bridge-like lipid transfer proteins (LTPs) contain a repeating β-groove domain and long hydrophobic grooves that act as bridges at membrane contact sites (MCSs) to efficiently transfer lipids. Atg2 is one such bridge-like LTP essential for autophagosome formation, during which a newly synthesized isolation membrane (IM) emerges and expands through lipid supply. However, studies on Atg2-mediated lipid transfer are limited to in vitro studies due to the lack of a suitable probe for monitoring phospholipid dynamics in vivo. Here, we characterized the lipophilic dye octadecyl rhodamine B (R18), which internalizes and labels the endoplasmic reticulum (ER) in a manner that requires flippases and oxysterol-binding protein-related proteins. Using R18, we demonstrated phospholipid transfer from the ER to the IM during autophagy in vivo. Upon autophagy termination, our data suggested the reversible phospholipid flow from the IM to the ER in response to environmental changes. Our findings highlight the critical role of bridge-like LTPs in MCS-mediated phospholipid homeostasis.