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.

Intravital imaging of cancer xenograft models enables the observation of cells in their tissue microenvironment. Application of molecular activity probes to intravital imaging, however, often poses technical difficulties. Here we sought to visualize Extracellular signal-regulated kinase (ERK) activity in an orthotopic xenograft model of pancreatic cancer. Our custom-made pancreatic imaging window (PIW) enabled long-term tracking (> 2 weeks) of cancer xenografts showing heterogeneous growth potential. Our validation approach of Förster resonance energy transfer (FRET) imaging by a kinase-dead mutant ERK biosensor suggested the local pH may affect the ratiometric interpretation of FRET efficiency. Nevertheless, our FRET analysis allowed to detect pharmacological inhibition of ERK activity, which supports the validity of the experimental model to dissect heterogeneous drug responses of cancer cells in their microenvironment. Of note, we found that collagen signal was negatively correlated with local ERK activity heterogeneity, highlighting the benefit of our imaging system to observe the intricate pharmacodynamics modified by fibrosis. Our intravital live imaging platform paves the way for a novel pre-clinical model that links the molecular dynamics to the living tissue environment of pancreatic cancer.

The endoplasmic reticulum (ER) is the gateway to the eukaryotic protein secretory pathway. Beyond its role in protein biogenesis, it is central to Ca2+ homeostasis and lipid biosynthesis. This organelle, which can constitute more than 50% of the cellular membranes in secretory cells, is highly plastic and must adjust to intracellular or extracellular challenges to ensure proper protein secretion. ER stress, resulting from challenges such as accumulation, misfolding or aggregation of proteins or from disrupted ER lipid composition, can deleteriously affect cell function. Thus, the ER has evolved adaptive mechanisms that lead to cellular reprogramming to adjust its capacity to handle stress, including the unfolded protein response (UPR), which engages various ER quality control systems, protein degradation pathways and ER-organelle contacts. If this succeeds, the cell survives; but if it fails, cell death mechanisms are triggered. In this Cell Science at a Glance article and the accompanying poster, we summarize current knowledge on ER stress and control of the UPR in mammalian cells, highlighting aspects that require further attention. We also discuss recent insights that must be considered to better capture the full understanding of the ability of the ER to adjust to biological variation.

BRD8, a member of the bromodomain-containing protein family, is a chromatin-associated protein that recognizes acetylated histones and recruits regulatory factors to modulate gene expression. Despite extensive studies of BRD8 in tumor biology, its physiological roles in normal tissues remain largely unknown. Given its enriched expression in the testis, we investigated the role of Brd8 in male reproduction using a germ cell-specific Brd8 knockout mouse model. Brd8 deficiency resulted in a marked reduction in testicular size and sperm counts, accompanied by severe defects in germ cell differentiation. Immunofluorescence staining for germ cell markers demonstrated progressive spermatogenic defects, with a reduction in spermatids followed by declines in spermatocytes and spermatogonia at later ages. Single-cell transcriptome analysis of Brd8-knockout testes revealed transcriptional alterations in spermatogonia and spermatocytes, including dysregulation of translation-related pathways and reduced expression of genes involved in meiotic progression. In addition, VEGF-related signaling within the endothelial compartment was reduced in Brd8-knockout testes, suggesting secondary remodeling of the testicular microenvironment associated with progressive germ cell dysfunction. Our findings provide new insights into the role of BRD8, an epigenetic reader, in male fertility.

CLASP2, a microtubule plus-end tracking protein, exhibits changes in phosphorylation as well as localization along the microtubule in response to insulin stimulation. We report that CLASP2 is expressed in insulin target tissues such as skeletal muscle and adipose tissue and that CLASP2 protein abundance is decreased in epididymal fat of insulin resistant mice. Using a commercially available proline-directed phospho-antibody together with site-directed mutagenesis and quantitative proteomics, we establish that mouse CLASP2 (Uniprot ID Q8BRT1) undergoes insulin-stimulated phosphorylation at serines 884/893/1004/1005/1021 in 3T3-L1 adipocytes. Treatment of cells with the ERK inhibitor PD98059 inhibits the insulin-stimulated phosphorylation of CLASP2 at the proline-directed serines 893/1004/1005/1021. In vitro incubation of CLASP2 with ERK2 increases CLASP2 phosphorylation, whereas phosphorylation of a Ser884/893/1004/1005/1021Ala CLASP2 penta-mutant is decreased, indicating ERK2 is capable of directly phosphorylating CLASP2. When expressed in 3T3-L1 adipocytes, the CLASP2 penta-mutant presents significantly diminished insulin-stimulated microtubule plus-end trailing versus wildtype CLASP2. The lack of proline-directed, insulin-stimulated CLASP2 phosphorylation results in reduced insulin-induced microtubule stabilization. We introduce insulin-stimulated, proline-directed CLASP2 phosphorylation under control of the ERK pathway as a contributing factor for insulin-induced microtubule stabilization.

Entry of intracellular pathogenic bacteria is widely considered an actin-driven process, potentially overlooking contributions of the microtubule cytoskeleton. Interphase microtubules exist in distinct states, but whether this heterogeneity influences bacterial entry is unknown. Here, using the obligate intracellular bacterium Chlamydia pneumoniae, we show that microtubules not only contribute to bacterial entry but that their pre-existing state influences host-cell permissiveness. Cells enriched in acetylated microtubules are preferentially infected, whereas detyrosinated microtubules show no such association. Taxol enhances entry, whereas selective elevation of microtubule acetylation with Tubacin does not, indicating that acetylation itself is insufficient and pointing instead to a permissive state associated with stabilized, long-lived microtubules. We next show that this relationship is reciprocal: infection induces a dose-dependent increase in microtubule acetylation that requires bacterial viability and is not reproduced by uptake of Yersinia pseudotuberculosis Invasin-coated beads, indicating that internalization alone is insufficient to trigger this response. Finally, to examine how early chlamydial proteins can alter microtubule behaviour, we assessed the effect of the secreted TarP-family actin-microtubule regulator CPn0572 on microtubule stability. In the Schizosaccharomyces pombe in vivo test system, CPn0572 promotes microtubule persistence and can act together with another chlamydial microtubule modulator to generate a combined microtubule phenotype. Together, these findings identify the pre-existing microtubule state as a determinant of Chlamydia pneumoniae entry, show that infection subsequently remodels the microtubule cytoskeleton, and demonstrate that an early chlamydial protein can directly alter microtubule behaviour.

The cGMP-gated channel β-subunit is required for phototransduction, disc morphogenesis, and structural integrity of rod cells. Its structural role may be mediated by its GARP2 (glutamic acid rich protein) domain. To assess domain functions, we generated transgenic mice expressing a truncated protein lacking the N-terminal region on a Cngb1 null background. In these mice, light microscopy showed normal disk morphology up to 6 months, and cryo-electron tomography revealed disc-to-plasma membrane connections similar to WT. However, the truncated protein was insufficient to prevent progressive degeneration leading to photoreceptor loss by 12-14 months. Truncated protein expression levels resembled WT β-subunit levels. The truncated protein localized both to the outer segment, like WT, but also elsewhere in rods. Electroretinography showed CNG channel function was comparable to WT at 1-month and remained so up to 7.5 months postnatal when marked declines appeared. Thus, the N-terminal extension of the β-subunit is required for long-term maintenance and survival of rod photoreceptors, whereas the major structural defects observed at early ages in the knockouts cannot be attributed to loss of the CNGB1a GARP2 domain or the GARP1 and GARP2 soluble proteins.

Dynamic chromatin behavior, which is related to chromatin accessibility, plays a critical role in various genome functions such as RNA transcription and DNA replication/repair. Previous studies using highly synchronized cells showed that average local chromatin motion, captured by single-nucleosome imaging on a subsecond time scale, remained nearly constant throughout G1, S, and G2 phases in living human cells. Here, we combined single-nucleosome imaging with Fucci cell-cycle probes to test this finding in asynchronous living human cells. Using HeLa and HCT116 cells expressing H2B-HaloTag and Fucci probes, we found that local nucleosome motion remained similar on average throughout interphase. Consistently, H3.3-Halo-labeled nucleosomes, which are enriched in Hi-C A compartments (euchromatin), also showed near-constant motion throughout interphase. Transcription inhibition increased nucleosome motion throughout interphase. Local nucleosome motion also increased following cellular perturbations, such as replication stress or DNA damage. Our findings suggest that near-constant chromatin motion supports housekeeping functions under similar physical conditions during interphase. They also suggest that cells can transiently change chromatin motion to perform ad hoc tasks in response to intra- and extracellular signals, such as DNA damage.

Several cellular processes during morphogenesis, tissue healing or cancer progression involve collective cell migration. To set the cells for motion, there is an initial breaking of organization of the epithelial tissue that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodeled correlating with the buildup of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behavior, but how forces are regulated during the breaking of symmetry at the onset of collective motion remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during collective cell motion remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of epithelial tissue organization. CRB3 interacts with ARP2/3 and controls the remodeling of actin via the modulation of the Rho-/Rac-GTPase balance, and more precisely by acting upstream of the activation of Rac1. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during collective cell motion.

Homologous chromosomes are spatially segregated, or antipaired, in neonatal human endothelial cells at metaphase/anaphase, which may help prevent abnormal recombination. However, it is unclear if this antipairing persists in adult endothelial cells. To test whether the antipairing, or one homolog per nuclear hemisphere motif, is conserved in adult endothelial cells, we examined human aortic endothelial cells at metaphase. Using ImmunoFISH and high-resolution confocal microscopy to visualize the chromosomes and centrosomes, we found that small homologous chromosomes 13, 15, 17, 19, 21, 22, and the sex chromosomes, XY, exhibit a loss of spatial segregation in human adult aortic endothelial cells. In addition, fewer adult endothelial cells showed segregation for the larger chromosomes 1, 4, and XX, as compared to neonatal endothelial cells. Notably, we observed a higher frequency of abnormal pairing in both small and large chromosomes in adult aortic endothelial cells as compared to neonatal umbilical vein endothelial cells. These findings suggest that mechanisms governing chromosome antipairing may decline with aortic endothelial cell age.