Metastatic dissemination requires circulating tumor cells (CTCs) to survive hemodynamic forces, yet the biological traits that enable survival under fluid shear stress (FSS) remain poorly defined. While current paradigms suggest that fluid shear stress (FSS) broadly induces an epithelial-to-mesenchymal transition (EMT), we investigated an alternative mechanism: FSS acts as a mechanical bottleneck that selectively enriches for pre-existing mesenchymal cancer cells by destroying more epithelial-like phenotypes prior to detectable changes in EMT marker expression. Using epithelial and mesenchymal subpopulations isolated from the same cell lines, we exposed single cells in suspension to pulsatile FSS to demonstrate that mesenchymal cells exhibit significantly higher survival rates and lower membrane rupture following pulsatile FSS. Immediate post-shear single-cell analysis revealed stable vimentin-to-cytokeratin (VIM/KRT) ratios in mesenchymal populations, whereas epithelial-like populations showed a selective loss of low-VIM/KRT cells, consistent with immediate mechanical depletion prior to biological induction of EMT. Direct manipulation of EMT states confirmed this causal relationship: TGFβ-induced EMT rescued survival in epithelial cells, while E-cadherin overexpression sensitized mesenchymal cells to mechanical rupture. To validate these findings clinically, we analyzed single-cell RNA-seq data from three patient-derived CTC datasets and found that higher VIM/KRT ratios significantly correlate with increased CTC abundance in the bloodstream. Together, these results demonstrate that hemodynamic forces impose a deterministic mechanical selection pressure on CTCs, identifying epithelial-associated biophysical traits as a primary therapeutic vulnerability in the metastatic cascade.

Lipophagy is an important microautophagic process that degrades lipid droplets (LDs) to mobilize stored lipids as an energy source during nutrient starvation. However, the molecular mechanisms regulating lipophagy in response to nutrient starvation remain poorly understood. We found that budding yeast mutants defective in glycosylphosphatidylinositol (GPI) lipid remodeling exhibited aberrant accumulation of lipid droplets (LDs), neutral lipids, and a reduced degradation of Faa4-GFP, a lipophagy reporter under glucose starvation. Furthermore, we demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) localize to vacuoles in response to glucose depletion, and that a mutant defective in endocytosis has defects in lipophagy. These results imply that GPI lipid remodeling is required for lipophagy upon glucose starvation, and we propose that endocytosis functions to supply the lipid portion of GPI-APs, remodeled to C26 diacylglycerol, to the vacuolar membrane for lipophagy.

The giant scaffold protein obscurin plays a crucial role in anchoring thick filaments at the muscle M-line. Here we show that Drosophila obscurin forms an antiparallel dimer at the M-line with the N-terminal SH3-DH-PH domain facing outwards toward the thin filaments, and the C-terminal kinase region involved in dimerization or oligomerization as well as myosin binding. This arrangement is supported by myosin co-immunoprecipitation, yeast two-hybrid, and super-resolution microscopy data, as well as Obscurin mutants specifically affecting the N- versus the C-terminus. While deletion of the N-terminal domains of obscurin predominantly affects peripheral thin filament length, loss of the C-terminal kinase region of obscurin disrupts early sarcomere assembly leading to various defects during sarcomere maturation, supporting its role in keeping thick filaments in register. In conclusion, we propose that obscurin forms a structural and elastic component organizing thick filaments with its C-terminal kinase region at the M-line, while connecting to thin filaments with its N-terminal domains.

Triple-negative breast cancer is characterized by high rates of chemoresistance and poor clinical outcomes. Although ATP-binding cassette (ABC) transporters are well-established mediators of drug efflux, their role in tumor-immune communication remains poorly defined. Here, we investigated whether the acquisition of doxorubicin resistance alters extracellular vesicle (EV) biogenesis and cargo content. We report that the doxorubicin-resistant MDA-MB-231 cell line exhibits significantly higher rates of EV production, without changes in particle size distribution, compared to the parental cell line. At the cellular level, we observed a significant increase in the number of multivesicular bodies present in doxorubicin-resistant cells. Proteomic and Western blot analysis of isolated EVs derived from the doxorubicin-resistant MDA-MB-231 cell line, compared to the parental MDA-MB-231 cell line, revealed an enrichment of ABCC1, accompanied by enrichment of vesicle-associated pathways. Cellular ABCC1 localized to CD63-positive compartments and was validated to be enriched in resistant cell line-derived EVs. Functionally, EVs from resistant cells promoted M2-like polarization of primary human macrophages, characterized by decreased CD86[+] and increased CD206[+] populations in addition to a decrease in IL-6, IL-8 and TNFα levels. Knocking down ABCC1 in resistant cells significantly attenuated this effect. Together, these findings suggest EV-associated ABCC1 might be a contributor to macrophage polarization and immune modulation in breast cancer.

The Golgi is an essential organelle that serves as a central hub for endomembrane trafficking. In the protozoan parasite Toxoplasma gondii, a single Golgi stack is essential for parasite survival; however, the molecular determinants governing Golgi structure and function remain poorly understood. Here, we characterize a Golgi-associated protein that is required for Golgi integrity and function, which we named Toxoplasma Golgi Integrity Factor (TGIF). Loss of TGIF disrupts parasite replication and natural egress and is lethal to the parasite. To investigate the impact of TGIF depletion on secretory protein trafficking, we adapted a fluorescence-based pulse-chase assay to monitor the synthesis and trafficking of microneme and rhoptry proteins. We found that loss of TGIF significantly impaired the synthesis and trafficking of microneme and rhoptry neck proteins, whereas trafficking of rhoptry bulb proteins was minimally affected. These findings suggest that rhoptry bulb proteins may traffic independently of canonical Golgi-dependent pathways. Collectively, our study provides new insight into the mechanisms of Golgi-mediated trafficking in T. gondii and identifies TGIF as a critical regulator of parasite secretory pathway organization and function.

Cyclic adenosine monophosphate (cAMP) signaling is a major stimulus for lipid and glucose catabolism, yet catabolic processes like these can also coordinate with lysosome-dependent degradation. However, the impact of cAMP signaling on lysosomal dynamics remains unclear. Transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, is regulated by stimulus-dependent nuclear-cytoplasmic shuttling through a variety of phosphorylation events. Here, we find that elevating intracellular cAMP with forskolin and IBMX induces rapid nuclear import of TFEB-GFP within 30 minutes and coincides with a transient upregulation of TFEB target lysosome genes. By 8 hours, TFEB returns to the cytoplasm, accompanied by transcriptional downregulation. Inhibition of cAMP-dependent protein kinase A (PKA) using H89 did not block nuclear import but unexpectedly caused sustained nuclear accumulation, indicating that PKA promotes TFEB nuclear export. Consistent with this, phosphoproteomic profiling revealed increased phosphorylation of a PKA-consensus motif (RRxS) during the export phase. These findings suggest that cAMP-PKA signaling plays a novel role in temporally "tuning" lysosomal gene expression by regulating TFEB nuclear-cytoplasmic shuttling.

Podosomes are actin-rich adhesions that enable cells to probe their environment, migrate across tissue boundaries, and degrade extracellular matrix. They are often organized in clusters, where their spatial organization is governed by complex actin networks, including a vinculin-associated ventral network and a myosin IIA-associated dorsal network. The molecular composition and mechanisms coordinating these networks, however, remain incompletely understood. Here, we identify septins as novel constituents of the podosome network in human immature dendritic cells. Using super-resolution microscopy, we show that septin 2, 7 and 9 are excluded from the actin core and do not colocalize with the canonical podosome ring. Instead, septins localize to the dorsal actin network that interconnects individual podosomes. Disruption of the septin network does not prevent podosome formation but alters the spatiotemporal organization of the podosome actin network. Furthermore, although septins and myosin IIA do not directly colocalize, septin perturbation leads to axial mislocalization and reduced activity of myosin IIA. Together, our findings identify septins as novel regulators of podosome cluster organization, adding an additional layer of complexity to the cytoskeletal coordination of these structures.

Myeloid cell leukemia 1 (MCL-1) is a canonical anti-apoptotic protein crucial for early neurodevelopment. Loss of MCL-1 results in embryonic-lethal neurodevelopmental defects that cannot be rescued by other anti-apoptotic proteins of the B cell lymphoma 2 (BCL-2) family. Here, we pharmacologically inhibit MCL-1 in human neural progenitor cells and find non-apoptotic roles for MCL-1 in sustaining mitochondrial cristae integrity, fatty acid oxidation and neural progenitor identity. MCL-1 inhibition disrupts mitochondrial ultrastructure, leading to a swollen network with disorganized cristae and destabilized OPA1-MICOS machinery that fails to maintain inner mitochondrial membrane architecture. These structural defects are accompanied by displacement of ACSL1 from the outer mitochondrial membrane, impaired fatty acid oxidation, lipid droplet accumulation and reduced mitochondrial oxygen consumption, revealing a tight link between cristae architecture and metabolic competence. Mechanistically, MCL-1 acts through two coordinated functions: maintaining cristae integrity at the inner mitochondrial membrane and retaining ACSL1 at the outer mitochondrial membrane, both independently of caspase activation. Functionally, MCL-1 inhibition selectively depletes intermediate progenitor cells without affecting proliferation, indicating a direct role in lineage progression. Together, our findings position MCL-1 upstream of OPA1, MICOS and ACSL1, and identify it as a critical coordinator of cristae organization, lipid metabolism and neural progenitor fate. This establishes mitochondrial inner membrane architecture as an instructive determinant of human neurogenesis and highlights non-canonical MCL-1 functions as critical regulators of human brain development.

Glioblastoma is the most aggressive and lethal malignancy of the central nervous system and exhibits a complex interplay with endoplasmic reticulum (ER) stress (ERS). The multifunctional ER is the primary site of the unfolded protein response (UPR), which is activated under conditions of ERS to enhance protein-folding capacity and promote cell survival. However, persistent ERS can trigger apoptosis. Despite extensive research over the past decades, the mechanistic insights into the impact of ERS and the UPR on glioblastoma pathogenesis and progression remain unclear. The UPR operates through three canonical signaling pathways: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK, also known as EIF2AK3) and activating transcription factor 6 (ATF6). These pathways regulate glioblastoma development through both independent and synergistic mechanisms. In this Review, we discuss the current state of research on the role of the UPR in glioblastoma and delineate the distinct mechanisms of each signaling pathway. Furthermore, we evaluate the challenges associated with UPR-targeting therapeutic agents and propose possible treatment strategies to leverage the regulatory mechanisms of the different UPR pathways to overcome these limitations.

The cell cycle is controlled in G1-phase by activities of three cyclin-dependent kinases (CDKs), CDK4/6 and CDK2, and their regulatory partners, cyclin D and cyclin E, respectively. p21 (CDKN1A), a member of the KIP/CIP family, is involved in regulation of CDK/cyclin complexes. In the present study, we analysed the impact of p21 on CDK/cyclin complex formation in living cells by fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurements. Depending on the additional expression of p21, both CDK4/cyclin D3 and CDK2/cyclin E1 interactions have been favoured reflected by increased FRET efficiencies (E). Furthermore, post-translational modifications (PTM) as well as the redox status of p21 can regulate the impact on CDK/cyclin complexes. We show that phosphorylation-mimicking mutants of p21 seem to be able to affect the CDK/cyclin protein-protein interactions (PPIs), and that the reduced p21C41S variant decreased binding between CDK4 and cyclin D3. In contrast, p21C41S increased the CDK2/cyclin E1 interaction such as wild-type p21. Hence, wild-type p21 and its specific variants have an impact on the formation of distinct CDK/cyclin complexes under physiological conditions.

Upon meiosis initiation in the fission yeast Schizosaccharomyces pombe, centromeres detach from the spindle pole body (SPB), while telomeres cluster at the SPB, forming the bouquet, which promotes homologous chromosome pairing. Bouquet formation is regulated by mating pheromone signaling and Pat1 kinase, a central regulator of meiosis. Centromere detachment also requires telomere clustering-dependent SPB recruitment of the telomere component Taz1. However, the precise underlying regulatory mechanisms remain unclear. Here, using a haploid cell system that allows synchronous meiosis induction, we show that mating pheromone signaling induces transient bouquet formation, whereas bouquet maintenance additionally requires Pat1 activity reduction, which normally occurs after conjugation. Furthermore, impairment of telomere clustering inhibits centromere detachment regardless of Pat1 activity reduction, whereas SPB-recruited Taz1 restores centromere detachment only when Pat1 activity is reduced. Thus, bouquet formation proceeds in two steps: a transient establishment step dependent on mating pheromone signaling, followed by a maintenance step requiring Pat1 activity reduction and SPB recruitment of Taz1. We propose that this regulation enables rapid cell cycle resumption upon nutrient replenishment in unconjugated cells, while ensuring meiotic commitment in conjugated cells.

Immune cell recruitment into tissues requires cells to migrate on the endothelium while overcoming shear forces. Many extravasating cells, including T lymphocytes and hematopoietic stem cells, migrate upstream against the direction of shear flow on ICAM-1. Upstream migration has been shown to depend on intracellular signaling through Crk family adaptor proteins following engagement of lymphocyte function-associated antigen-1 (LFA-1). Because upstream migration is an important illustration of leukocyte mechanotransduction, we sought to identify key intracellular regulators of upstream migration using CRISPR/Cas9 gene editing and the KG-1a stem cell line. We made targeted deletions of molecules that function downstream of LFA-1 to regulate actin polymerization, link integrin signaling to the actin cytoskeleton, or form scaffolding complexes with Crk proteins. We found that CrkL, CasL (also known as HEF1 or NEDD9), L-plastin (LCP1) and RIAM (APBB1IP) are crucial for dynamic F-actin-rich lamellipodia and upstream migration. Although WASP (WAS), Hem1 (NCKAP1L), EVL and vinculin were found to be dispensable for upstream migration, pan-formin inhibition with SMIFH2 revealed a role for formins in upstream migration. This study illustrates the role of LFA-1 mechanotransduction in driving F-actin assembly at the leading edge to drive upstream migration.

Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluid into giant vesicles known as macropinosomes. This mechanism is used by immune cells to sample the surroundings for antigens and can be exploited by cancer cells for nutrient uptake. What determines macropinosome fate after internalization is largely unknown. Here we investigate the role of the phosphatidylinositol 3-kinase VPS34/PIK3C3 and its product phosphatidylinositol 3-phosphate (PtdIns3P) in macropinosome fate determination. VPS34 inhibition reduced successful macropinosome maturation and fluid-phase uptake and prevented recruitment of early endosomal factors, including the small GTPase RAB5A and its effectors, to forming macropinosomes. Instead, forming macropinosomes under VPS34 inhibition accumulated regulators of endocytic recycling, including RAB8A, RAB10, RAB11A, and PtdIns4P, which led to fusion of macropinosomes with the plasma membrane. Whereas RAB5A was critical for establishment of endosomal identity, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a VPS34-dependent phosphoinositide and RAB transition that determines whether newly formed macropinosomes acquire endosomal identity or recycle to the plasma membrane.