Biology subjects

Stenmark, H.

Publications and source records attributed to Stenmark, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Structure and function of the DFCP1 membrane barrier responsible for ER microdomain organization in lysosome repair

The endoplasmic reticulum (ER) forms specialized microdomains that organize autophagy, lipid droplet formation, and the repair of damaged lysosomes. The PI3P-binding protein DFCP1/ZFYVE1 marks these microdomains, raising the question as to whether and how one protein could define such a subcompartment. Here we determine the structure of full-length human DFCP1 bound to membranes by cryo-electron tomography and single-particle cryo-electron microscopy. DFCP1 assembles into a helical coat, built from tetramers that stack around a constricted membrane tube. Its ATPase domains form the organizing core of the helix, while its ER-targeting domains insert deeply into the outer leaflet, and its FYVE domains grip PI3P. Coarse-grained molecular dynamics simulations show that the coat does not simply follow the curvature of the membrane but acts as a barrier to lipid diffusion, almost completely blocking the movement of its cognate lipid PI3P past the filament and concentrating it beneath the coat. Point mutations that break the assembly, membrane-insertion or PI3P-binding interfaces each impair lysosomal repair in cells, and the assembly is also required for the focal recruitment of the lipid transporter VPS13C. We propose that DFCP1 collars fence off a specialized ER microdomain, so that locally made lipids can reach sites of repair rather than escaping into bulk ER.

cell biology↗

bioRxiv2026

The kinesin-4 family member KIF27 regulates mitotic progression, cytokinesis and genome stability

Kinesins are microtubule-dependent motors, yet the functions of the kinesin-4 family member KIF27 remain poorly understood. Here, we demonstrate a dynamic and cell-cycle-dependent localization of KIF27, consistent with its functional roles in mitotic progression. Upon mitotic entry, KIF27 relocates to condensed chromosomes. During anaphase, a fraction of KIF27 accumulates at the spindle midzone, and in telophase and late stages of cytokinesis it localizes at the midbody, colocalizing with key cytokinetic regulators at both structures. Recruitment of KIF27 to the midbody depends on KIF23 and CEP55. KIF27 depletion results in profound cell division defects, altered midbody and microtubule morphology, delayed cytokinesis and cytokinesis failure. Beyond cell division, KIF27 depletion directly compromises nuclear morphology, and pan-cancer transcriptomic analyses correlate low KIF27 expression with aneuploidy and poor patient survival in several cancer types. Together, our results identify KIF27 as a novel regulator of mitotic fidelity and genome stability.

Cell Biology↗

bioRxiv2026

ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

The ATG8 conjugation machinery supports both canonical autophagy, in which ATG8 proteins are lipidated on forming autophagosomes, and CASM, in which ATG8 proteins are conjugated to stressed single-membrane compartments. How cells allocate this shared machinery between these competing membrane programs remains unclear. Here, we identify ATG16L2 as a specialized regulator of V-ATPase-responsive ATG8 lipidation (atg8ylation). ATG16L2 does not form stable homodimers and is inactive in the absence of ATG16L1, but it becomes functional through heterodimerization with ATG16L1. The ATG16L1-ATG16L2 heterodimer constitutes a VAIL-competent E3-like complex that is excluded from WIPI2-positive autophagic membranes but readily recruited to stressed lysosomes. ATG16L2 also confers VAIL activity onto the otherwise VAIL-deficient ATG16L1 isoform. We propose that ATG16L1 homodimers and ATG16L1-ATG16L2 heterodimers represent alternative assemblies of the ATG8 conjugation machinery: ATG16L1 homodimers are dedicated to WIPI2-positive autophagic membranes, whereas ATG16L1-ATG16L2 heterodimers containing either ATG16L1 isoform form a VAIL-selective assembly.

molecular biology↗

bioRxiv2026

Structural basis for the ATP-dependence of omegasome biogenesis by DFCP1

Autophagosome formation begins at phosphatidylinositol 3-phosphate-enriched endoplasmic reticulum (ER) subdomains termed omegasomes. DFCP1/ZFYVE1 is recruited to omegasomes through its FYVE domains and has recently been shown to function as an ATPase involved in omegasome constriction and autophagosome biogenesis. However, the structural basis of DFCP1 ATPase activity and how nucleotide-dependent conformational states regulate omegasome dynamics remain unknown. Here, we determined the crystal structures of the DFCP1 ATPase domain in complex with either ADP or the non-hydrolyzable ATP analogue, AppNHp at near atomic resolution. We employed structure-guided mutagenesis to define residues required for DFCP1 function in cells. The structures reveal that the active site contains a trans-acting Arg271 finger coordinating the {psi}-phosphate and trans-acting His323 that stacks on the ATP adenine ring. ATP hydrolysis results in a conformational switch in the vicinity of Arg271, while contacts with His323 persist in the presence of ADP. Biochemical analyses of DFCP1 and its mutants show that DFCP1 forms ATP-dependent microdomains on membranes. Disruption of His323 and Arg271 uncouples nucleotide binding from ATP hydrolysis and alters the oligomeric state of the protein. Live-cell imaging of DFCP1 knockout cells reconstituted with wild-type or mutant DFCP1 further demonstrates that these biochemical defects translate into distinct omegasome phenotypes. Together, our data provides a structural-function framework for DFCP1 ATPase activity and reveals how distinct catalytic elements control omegasome dynamics in vivo. We propose that nucleotide-dependent assembly and hydrolysis-driven conformational changes enable DFCP1 to regulate omegasome formation and its progression toward autophagosome closure. Significance StatementDFCP1 organizes the ER subdomains known as omegasomes, which are sites of autophagosome biogenesis. DFCP1 is an ATPase whose catalytic activity is required for function, but the precise role of its ATPase activity is unknown. Crystal structures of the DFCP1 ATPase domain in the presence of a non-hydrolyzable ATP analogue and ADP reveal an Arg finger that operates in trans, such that dimerization is required for ATP hydrolysis, and ATP hydrolysis destabilizes dimers. Dimerization is also promoted by an adenine base-stacking interaction in trans with H323. These residues are important for DFCP1 clustering on membranes and omegasome constriction, clarifying that the role of ATP is to promote the dimerization of the ATPase domain and thereby control the organization of DFCP1 on membranes.

biochemistry↗

bioRxiv2022

Vesicle-mediated transport of ALIX and ESCRT-III to the intercellular bridge during cytokinesis

Cellular abscission is the final step of cytokinesis that leads to the physical separation of the two daughter cells. The scaffold protein ALIX and the ESCRT-I protein TSG101 contribute to recruiting ESCRT-III to the midbody, which orchestrates the final membrane scission of the intercellular bridge. Here, we addressed by which mechanisms ALIX and the ESCRT-III subunit CHMP4B are transported to the midbody. Structured illumination microscopy revealed gradual accumulation of ALIX at the midbody, resulting in the formation of spiral-like structures extending from the midbody to the abscission site, which strongly co-localized with CHMP4B. Live-cell microscopy uncovered that ALIX appeared together with CHMP4B in vesicular structures, whose motility was microtubule-dependent. Depletion of ALIX led to structural alterations of the midbody and delayed recruitment of CHMP4B, resulting in delayed abscission. Likewise, depletion of the kinesin-1 motor KIF5B reduced the motility of ALIX-positive vesicles and caused delayed recruitment of ALIX, TSG101 and CHMP4B to the midbody, accompanied by impeded abscission. We propose that ALIX, TSG101 and CHMP4B are associated with endosomal vesicles transported along microtubules by kinesin-1, leading to their directional transport to the cytokinetic bridge and midbody, thereby contributing to their function in abscission.

cell biology↗

bioRxiv2022

"Double-Effect" strategy against Triple-Negative Breast Cancer via a synergistic therapy of Magneto-mechanical Force enhancing NIR-II Hypothermal Ablation

Triple-negative breast cancer (TNBC) is a form of breast cancer that is more aggressive and harder to treat than others, with a higher probability of relapse. Its highly efficient capabilities for migrating and invading other parts of the body together with the current lack of clinically established effective therapies account for a low survival rate. Thus, we propose the in-tandem use of two complementary therapeutic routes to effectively combat TNBC. Herein, a versatile magnetic-photothermal converter (MPC) is elaborately designed via integrating zinc-doped ferrite nanoparticles and polyethene glycol, synergistically enhanced through magneto-mechanical force (MMF) and near-infrared-II (NIR-II) hypothermal ablation, thereby displaying excellent therapeutic efficiency. Their combined use, which is less aggressive to the human body compared to conventional chemotherapeutic approaches, results in the splendid suppression of TNBC migration and invasion. Remotely controlling the MPCs by an external magnetic stimulus, results in cellular MMF effects that cause direct mechanical destruction to the cancer cell membrane, leading to its necrosis. Furthermore, the MMF disrupts intracellular lysosomes, thereby triggering the release of large amounts of protein hydrolases, which induce intracellular oxidative stress, and accelerate the induction of apoptosis. Complementing the therapeutic approach based on MMF, the excellent photothermal performance of the MPC in the NIR-II region (1064nm) is exploited to enable effective hypothermal ablation of the tumours, which can be achieved in deep tissue layers. The proposed multifunctional nanocomposites, together with the demonstrated "double effect" therapeutic approach, hold significant potential to pave the way for future cutting-edge weapons against the dreadful TNBC.

cancer biology↗

bioRxiv2022

Lysosome repair by ER-mediated cholesterol transfer

Lysosome integrity is essential for cell viability, and lesions in lysosome membranes are repaired by the ESCRT machinery. Here we describe an additional mechanism for lysosome repair that is activated independently of ESCRT recruitment. Lipidomic analyses showed increases in lysosomal phosphatidylserine and cholesterol after damage. Electron microscopy demonstrated that lysosomal membrane damage is rapidly followed by formation of contacts with the endoplasmic reticulum (ER), which depend on the ER proteins VAPA/B. The cholesterol-binding protein ORP1L was recruited to damaged lysosomes, accompanied by cholesterol accumulation by a mechanism that required VAP-ORP1L interactions. The PtdIns 4-kinase PI4K2A rapidly produced PtdIns4P on lysosomes upon damage, and knockout of PI4K2A inhibited damage-induced accumulation of ORP1L and cholesterol and led to failure of lysosomal membrane repair. The cholesterol-PtdIns4P transporter OSBP was also recruited upon damage, and its depletion caused lysosomal accumulation of PtdIns4P and resulted in cell death. We conclude that ER contacts are activated on damaged lysosomes in parallel to ESCRTs to provide lipids for membrane repair, and that PtdIns4P generation and removal are central in this response.

cell biology↗

bioRxiv2022

ATPase activity of DFCP1 controls selective autophagy

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic flux, DFCP1 was required to maintain the autophagic flux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.

cell biology↗

bioRxiv2021

A phosphoinositide and RAB switch controls early macropinocytosis

Macropinocytosis is a non-selective endocytic process by which cells take up large amounts of extracellular fluids 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 the fate of macropinosomes after they have been internalized 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. Inhibition of VPS34 led to a decrease in macropinosome survival and fluid phase uptake as well as preventing recruitment of early endosomal factors, including the small GTPase RAB5 and its effectors, to the 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 RAB5 was critical for macropinosome formation, macropinosome fusion with the plasma membrane depended on RAB8A. Thus, macropinosome maturation is regulated by a PtdIns3P-controlled switch that balances macropinosome fate between the default, endolysosomal maturation and an alternative, secretory route.

cell biology↗

bioRxiv2021

Divalent ligand-monovalent molecule binding

Simultaneous binding of divalent ligands to two identical molecules is a widespread phenomenon in biology and chemistry. Here, we describe this binding event as a divalent ligand AA that can bind to two identical monovalent molecules B to form the complex AA {middle dot} B2. Cases where the total concentration [AA]T is either much larger or much smaller than the total concentration [B]T have been studied earlier, but a description of intermediate concentrations is missing. In this paper, we describe the general case of any ratio of{xi} {equiv} [B]T /[AA]T. We show that the concentration of the intermediate complex AA {middle dot} B is governed by a cubic equation and discuss several scenarios in which this cubic equation simplifies. Our numerical results, which cover the entire range of 0 <{xi} < {infty}, are relevant to processes wherein the concentrations of free ligands and proteins both decrease upon binding. Such ligand and protein depletion is expected to be important in cellular contexts, e.g., in antigen detection and in coincidence detection of proteins or lipids.

biochemistry↗

bioRxiv2020

JIP4 is recruited by the phosphoinositide-binding protein Phafin2 to promote recycling tubules on macropinosomes

Macropinocytosis allows cells to take up extracellular material in a non-selective manner. The molecular mechanisms that mediate recycling of membranes and transmembrane proteins from macropinosomes still need to be defined. Here we report that JIP4, a coiled-coil containing protein previously described to bind to microtubule motors, is recruited to retromer- and actin-containing tubulating subdomains on macropinosomes by binding to the PH domain of the phosphatidylinositol 3-phosphate (PtdIns3P)-binding protein Phafin2. This recruitment is not shared by the closely related isoforms JIP3 and Phafin1. Disruption of Phafin2 or PtdIns3P impairs JIP4 recruitment to macropinosomes whereas forced localization of Phafin2 to mitochondria causes mitochondrial targeting of JIP4. While knockout of JIP4 suppresses tubulation, overexpression enhances tubulation from macropinosomes. JIP4 knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes, consistent with a recycling defect. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes.

cell biology↗

bioRxiv2020

The phosphatidylinositol 3-phosphate binding protein SNX4 controls ATG9A recycling and autophagy

Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Whereas these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P) binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the transmembrane autophagy machinery protein ATG9A from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. Altogether, we propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.

cell biology↗

bioRxiv2020

GAS6-AXL signaling triggers actin remodeling and macropinocytosis that drive cancer cell invasion

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand GAS6 are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion (FA) turnover, cell spreading and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL but not TYRO3, and downstream activation of PI3K. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements and macropinocytosis that jointly contribute to cancer cell invasion.

cell biology↗

bioRxiv2019

Protein Crowding mediates Passive Membrane Remodeling in ESCRT-induced Formation of Intraluminal Vesicles

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0-III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0-II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding on the membrane induces membrane deformation and facilitates ILV formation: upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.Significance Statement Intraluminal vesicle (ILV) formation plays a crucial role in the attenuation of growth factor receptor signaling, which is mediated by the endosomal sorting complex required for transport (ESCRT-0-III/VPS4). The general dogma has been that the upstream ESCRTs (0-II) sequester the receptors at the surface of endosomes and the downstream ESCRTs (III/VPS4) remodel the endosome membrane leading to the abscission and formation of receptor-containing ILVs. We now show that the upstream ESCRTs not only sequester cargo, but in addition play an essential role for the initiation of membrane shape remodeling in ILV budding. Through a combination of mathematical modeling and experimental measurements we show that upstream ESCRTs facilitate ILV budding by crowding with a high density in the membrane neck region.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2019

Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) is a major global health problem and causes extensive cytotoxicity in patient cells and tissues. Here we define an NLRP3, caspase-1 and gasdermin D-mediated pathway to pyroptosis in human monocytes following exposure to Mtb. We demonstrate an ESX-1 mediated, contact-induced plasma membrane (PM) damage response that occurs during phagocytosis or from the cytosolic side of the PM after phagosomal rupture in Mtb infected cells. This PM injury in turn causes K+ efflux and activation of NLRP3 dependent IL-1{beta} release and pyroptosis, facilitating the spread of Mtb to neighbouring cells. Further we reveal a dynamic interplay of pyroptosis with ESCRT-mediated PM repair. Collectively, these findings reveal a novel mechanism for pyroptosis and spread of infection acting through dual PM disturbances both during and after phagocytosis. We also highlight dual PM damage as a common mechanism utilized by other NLRP3 activators that have previously been shown to act through lysosomal damage.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC=\"FIGDIR/small/747014v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@1911356org.highwire.dtl.DTLVardef@18c99dcorg.highwire.dtl.DTLVardef@1c94bb9org.highwire.dtl.DTLVardef@8a69db_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Unrestrained ESCRT-III drives chromosome fragmentation and micronuclear catastrophe

The ESCRT-III membrane fission machinery1,2 restores nuclear envelope integrity during mitotic exit3,4 and interphase5,6. Whereas primary nuclei resealing takes minutes, micronuclear envelope ruptures appear irreversible and result in catastrophic collapse associated with chromosome fragmentation and rearrangements (chromothripsis), thought to be a major driving force in cancer development7-10. Despite its importance11-13, the mechanistic underpinnings of nuclear envelope sealing in primary nuclei and the defects observed in micronuclei remain largely unknown. Here we show that CHMP7, the nucleator of ESCRT-III filaments at the nuclear envelope3,14, and the inner nuclear membrane protein LEMD215 act as a compartmentalization sensor detecting the loss of nuclear integrity. In cells with intact nuclear envelope, CHMP7 is actively excluded from the nucleus to preclude its binding to LEMD2. Nuclear influx of CHMP7 results in stable association with LEMD2 at the inner nuclear membrane that licenses local polymerization of ESCRT-III. Tight control of nuclear CHMP7 levels is critical, as induction of nuclear CHMP7 mutants is sufficient to induce unrestrained growth of ESCRT-III foci at the nuclear envelope, causing dramatic membrane deformation, local DNA torsional stress, single-stranded DNA formation and fragmentation of the underlying chromosomes. At micronuclei, membrane rupture is not associated with repair despite timely recruitment of ESCRT-III. Instead, micronuclei inherently lack the capacity to restrict accumulation of CHMP7 and LEMD2. This drives unrestrained ESCRT-III recruitment, membrane deformation and DNA defects that strikingly resemble those at primary nuclei upon induction of nuclear CHMP7 mutants. Preventing ESCRT-III recruitment suppresses membrane deformation and DNA damage, without restoring nucleocytoplasmic compartmentalization. We propose that the ESCRT-III nuclear integrity surveillance machinery is a double-edged sword, as its exquisite sensitivity ensures rapid repair at primary nuclei while causing unrestrained polymerization at micronuclei, with catastrophic consequences for genome stability16-18.

cell biology↗

bioRxiv2018

ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival

Although lysosomes perform a number of essential cellular functions, damaged lysosomes represent a potential hazard to the cell. Such lysosomes are therefore engulfed by autophagic membranes in the process known as lysophagy, which is initiated by recognition of luminal glycoprotein domains by cytosolic lectins such as Galectin-3. Here we show that, under various conditions that cause injury to the lysosome membrane, components of the endosomal sorting complex required for transport (ESCRT) machinery are recruited. This recruitment occurs before that of Galectin-3 and the lysophagy machinery. Subunits of the ESCRT-III complex show a particularly prominent recruitment, which depends on the ESCRT-I component TSG101 and the TSG101- and ESCRT-III-binding protein ALIX. Interference with ESCRT recruitment abolishes lysosome repair and causes otherwise reversible lysosome damage to become cell lethal. Vacuoles containing the intracellular pathogen Coxiella burnetii show reversible ESCRT recruitment, and interference with this recruitment reduces intravacuolar bacterial replication. We conclude that the cell is equipped with an endogenous mechanism for lysosome repair which protects against lysosomal damage-induced cell death but which also provides a potential advantage for intracellular pathogens.

cell biology↗

bioRxiv2018

TRPML1-mediated Modulation of Dense-core Granules Tunes Functional Potential in NK Cells

Inhibitory signaling during natural killer (NK) cell education translates into increased responsiveness to activation; however the intracellular mechanism for functional tuning by inhibitory receptors remains unclear. We found that educated NK cells expressing self-MHC specific inhibitory killer cell immunoglobulin-like receptors (KIR) show accumulation of granzyme B, localized in dense-core secretory lysosomes, converged close to the centrosome. This discrete morphological phenotype persists in self-KIR+ NK cells independently of transcriptional programs that regulate effector function, metabolism and lysosomal biogenesis. The granzymeB dense, large secretory lysosomes in self-KIR+ NK cells were efficiently released upon target cell recognition, contributing to their enhanced cytotoxic capacity. Secretory lysosomes are part of the acidic lysosomal compartment, which has been shown to channel calcium and mediate intracellular signalling in several cell types. Interference of signaling from acidic Ca2+ stores in primary NK cells reduced both target-specific Ca2+-flux, degranulation and cytokine production. Furthermore, inhibition of PI(3,5)P2 synthesis or genetic silencing of the PI(3,5)P2-regulated lysosomal Ca2+-channel TRPML1 led to increased levels of granzyme B and enhanced functional potential. These results indicate an intrinsic role for lysosomal homeostasis in NK cell education.

immunology↗