Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

Mitochondria-ER-PM contacts regulate mitochondrial division and PI(4)P distribution.

The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.

MTM1-mediated production of phosphatidylinositol 5-phosphate fuels the formation of podosome-like protrusions regulating myoblast fusion.

Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.

A connection between phosphatidylinositol 5-phosphate and the Hippo pathway to prevent epithelial-mesenchymal transition in cancer.

The Hippo pathway blocks epithelial-mesenchymal transition and metastasis in cancer mediated by the transcriptional coactivator YAP. In this issue of Science Signaling, Palamiuc et al. demonstrate that phosphatidylinositol 5-phosphate (PI5P) enhances Hippo pathway activation and that simultaneously the Hippo pathway initiates a positive feedback loop by inhibiting the conversion of PI5P into PIP2.

Coxiella burnetii effector CvpE maintains biogenesis of Coxiella-containing vacuoles by suppressing lysosome tubulation through binding PI(3)P and perturbing PIKfyve activity on lysosomes.

Coxiella burnetii (C. burnetii) is the causative agent of Q fever, a zoonotic disease. Intracellular replication of C. burnetii requires the maturation of a phagolysosome-like compartment known as the replication permissive Coxiella-containing vacuole (CCV). Effector proteins secreted by the Dot/Icm secretion system are indispensable for maturation of a single large CCV by facilitating the fusion of promiscuous vesicles. However, the mechanisms of CCV maintenance and evasion of host cell clearance remain to be defined. Here, we show that C. burnetii secreted Coxiella vacuolar protein E (CvpE) contributes to CCV biogenesis by inducing lysosome-like vacuole (LLV) enlargement. LLV fission by tubulation and autolysosome degradation is impaired in CvpE-expressing cells. Subsequently, we found that CvpE suppresses lysosomal Ca2+ channel transient receptor potential channel mucolipin 1 (TRPML1) activity in an indirect manner, in which CvpE binds phosphatidylinositol 3-phosphate [PI(3)P] and perturbs PIKfyve activity in lysosomes. Finally, the agonist of TRPML1, ML-SA5, inhibits CCV biogenesis and C. burnetii replication. These results provide insight into the mechanisms of CCV maintenance by CvpE and suggest that the agonist of TRPML1 can be a novel potential treatment that does not rely on antibiotics for Q fever by enhancing Coxiella-containing vacuoles (CCVs) fission.

Omegasomes control formation, expansion, and closure of autophagosomes.

Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double-membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER-associated omegasomes, characterized by their distinctive omega-shaped structure and accumulation of phosphatidylinositol 3-phosphate (PI3P), play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation.

Recombinant biosensors for multiplex and super-resolution imaging of phosphoinositides.

Phosphoinositides are a small family of phospholipids that act as signaling hubs and key regulators of cellular function. Detecting their subcellular distribution is crucial to gain insights into membrane organization and is commonly done by the overexpression of biosensors. However, this leads to cellular perturbations and is challenging in systems that cannot be transfected. Here, we present a toolkit for the reliable, fast, multiplex, and super-resolution detection of phosphoinositides in fixed cells and tissue, based on recombinant biosensors with self-labeling SNAP tags. These are highly specific and reliably visualize the subcellular distributions of phosphoinositides across scales, from 2D or 3D cell culture to Drosophila tissue. Further, these probes enable super-resolution approaches, and using STED microscopy, we reveal the nanoscale organization of PI(3)P on endosomes and PI(4)P on the Golgi. Finally, multiplex staining reveals an unexpected presence of PI(3,5)P2-positive membranes in swollen lysosomes following PIKfyve inhibition. This approach enables the versatile, high-resolution visualization of multiple phosphoinositide species in an unprecedented manner.

Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL.

Metabolic crosstalk of the major nutrients glucose, amino acids and fatty acids (FAs) ensures systemic metabolic homeostasis. The coordination between the supply of glucose and FAs to meet various physiological demands is especially important as improper nutrient levels lead to metabolic disorders, such as diabetes and metabolic dysfunction-associated steatohepatitis (MASH). In response to the oscillations in blood glucose levels, lipolysis is thought to be mainly regulated hormonally to control FA liberation from lipid droplets by insulin, catecholamine and glucagon. However, whether general cell-intrinsic mechanisms exist to directly modulate lipolysis via glucose sensing remains largely unknown. Here we report the identification of such an intrinsic mechanism, which involves Golgi PtdIns4P-mediated regulation of adipose triglyceride lipase (ATGL)-driven lipolysis via intracellular glucose sensing. Mechanistically, depletion of intracellular glucose results in lower Golgi PtdIns4P levels, and thus reduced assembly of the E3 ligase complex CUL7FBXW8 in the Golgi apparatus. Decreased levels of the E3 ligase complex lead to reduced polyubiquitylation of ATGL in the Golgi and enhancement of ATGL-driven lipolysis. This cell-intrinsic mechanism regulates both the pool of intracellular FAs and their extracellular release to meet physiological demands during fasting and glucose deprivation. Moreover, genetic and pharmacological manipulation of the Golgi PtdIns4P-CUL7FBXW8-ATGL axis in mouse models of simple hepatic steatosis and MASH, as well as during ex vivo perfusion of a human steatotic liver graft leads to the amelioration of steatosis, suggesting that this pathway might be a promising target for metabolic dysfunction-associated steatotic liver disease and possibly MASH.

Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

A p85 isoform switch enhances PI3K activation on endosomes by a MAP4- and PI3P-dependent mechanism.

Phosphatidylinositol 3-kinase α (PI3Kα) is a heterodimer of p110α catalytic and p85 adaptor subunits that is activated by agonist-stimulated receptor tyrosine kinases. Although p85α recruits p110α to activated receptors on membranes, p85α loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. p110α localizes to microtubules via microtubule-associated protein 4 (MAP4), facilitating its interaction with activated receptor kinases on endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85α knockdown, the residual p110α, coupled predominantly to p85ß, exhibits enhanced recruitment with receptor tyrosine kinases to endosomes. Moreover, the p110α C2 domain binds PI3-phosphate, and this interaction is also required to recruit p110α to endosomes and for PI3K/Akt signaling. Stable knockdown of p85α, which mimics the reduced p85α levels observed in cancer, enhances cell growth and tumorsphere formation, and these effects are abrogated by MAP4 or p85ß knockdown, underscoring their role in the tumor-promoting activity of p85α loss.

Inositol Hexaphosphate as an Inhibitor and Potential Regulator of p47phox Membrane Anchoring.

As a key component for NADPH oxidase 2 (NOX2) activation, the peripheral membrane protein p47phox translocates a cytosolic activating complex to the membrane through its PX domain. This study elucidates a potential regulatory mechanism of p47phox recruitment and NOX2 activation by inositol hexaphosphate (IP6). Through NMR, fluorescence polarization, and FRET experimental results, IP6 is shown to be capable of breaking the lipid binding and membrane anchoring events of p47phox-PX with low micromolar potency. Other phosphorylated inositol species such as IP5(1,3,4,5,6), IP4(1,3,4,5), and IP3(1,3,4) show weaker binding and no ability to inhibit lipid interactions in physiological concentration ranges. The low micromolar potency of IP6 inhibition of the p47phox membrane anchoring suggests that physiologically relevant concentrations of IP6 serve as regulators, as seen in other membrane anchoring domains. The PX domain of p47phox is known to be promiscuous to a variety of phosphatidylinositol phosphate (PIP) lipids, and this regulation may help target the domain only to the membranes most highly enriched with the highest affinity PIPs, such as the phagosomal membrane, while preventing aberrant binding to other membranes with high and heterogeneous PIP content, such as the plasma membrane. This study provides insight into a potential novel regulatory mechanism behind NOX2 activation and reveals a role for small-molecule regulation in this important NOX2 activator.

Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

Rim aperture of yeast autophagic membranes balances cargo inclusion with vesicle maturation.

Autophagy eliminates cytoplasmic material by engulfment in membranous vesicles targeted for lysosome degradation. Nonselective autophagy coordinates sequestration of bulk cargo with the growth of the isolation membrane (IM) in a yet-unknown manner. Here, we show that in the budding yeast Saccharomyces cerevisiae, IMs expand while maintaining a rim sufficiently wide for sequestration of large cargo but tight enough to mature in due time. An obligate complex of Atg24/Snx4 with Atg20 or Snx41 assembles locally at the rim in a spatially extended manner that specifically depends on autophagic PI(3)P. This assembly stabilizes the open rim to promote autophagic sequestration of large cargo in correlation with vesicle expansion. Moreover, constriction of the rim by the PI(3)P-dependent Atg2-Atg18 complex and clearance of PI(3)P by Ymr1 antagonize rim opening to promote autophagic maturation and consumption of small cargo. Tight regulation of membrane rim aperture by PI(3)P thus couples the mechanism and physiology of nonselective autophagy.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.

BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function. METHODS: The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ. RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP. CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.

Self-organization of PIP3 waves is controlled by the topology and curvature of cell membranes.

Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results.

GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

OSBP-mediated PI(4)P-cholesterol exchange at endoplasmic reticulum-secretory granule contact sites controls insulin secretion.

Insulin is packaged into secretory granules that depart the Golgi and undergo a maturation process that involves changes in the protein and lipid composition of the granules. Here, we show that insulin secretory granules form physical contacts with the endoplasmic reticulum and that the lipid exchange protein oxysterol-binding protein (OSBP) is recruited to these sites in a Ca2+-dependent manner. OSBP binding to insulin granules is positively regulated by phosphatidylinositol-4 (PI4)-kinases and negatively regulated by the PI4 phosphate (PI(4)P) phosphatase Sac2. Loss of Sac2 results in excess accumulation of cholesterol on insulin granules that is normalized when OSBP expression is reduced, and both acute inhibition and small interfering RNA (siRNA)-mediated knockdown of OSBP suppress glucose-stimulated insulin secretion without affecting insulin production or intracellular Ca2+ signaling. In conclusion, we show that lipid exchange at endoplasmic reticulum (ER)-granule contact sites is involved in the exocytic process and propose that these contacts act as reaction centers with multimodal functions during insulin granule maturation.

Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

The temporal association of CapZ with early endosomes regulates endosomal trafficking and viral entry into host cells.

BACKGROUND: Many viruses enter host cells by hijacking endosomal trafficking. CapZ, a canonical actin capping protein, participates in endosomal trafficking, yet its precise role in endocytosis and virus infection remains elusive. RESULTS: Here, we showed that CapZ was transiently associated with early endosomes (EEs) and was subsequently released from the matured EEs after the fusion of two EEs, which was facilitated by PI(3)P to PI(3,5)P2 conversion. Vacuolin-1 (a triazine compound) stabilized CapZ at EEs and thus blocked the transition of EEs to late endosomes (LEs). Likewise, artificially tethering CapZ to EEs via a rapamycin-induced protein-protein interaction system blocked the early-to-late endosome transition. Remarkably, CapZ knockout or artificially tethering CapZ to EEs via rapamycin significantly inhibited flaviviruses, e.g., Zika virus (ZIKV) and dengue virus (DENV), or beta-coronavirus, e.g., murine hepatitis virus (MHV), infection by preventing the escape of RNA genome from endocytic vesicles. CONCLUSIONS: These results indicate that the temporal association of CapZ with EEs facilitates early-to-late endosome transition (physiologically) and the release of the viral genome from endocytic vesicles (pathologically).

Shigella generates distinct IAM subpopulations during epithelial cell invasion to promote efficient intracellular niche formation.

The facultative intracellular pathogen Shigella flexneri invades non-phagocytic epithelial gut cells. Through a syringe-like apparatus called type 3 secretion system, it injects effector proteins into the host cell triggering actin rearrangements leading to its uptake within a tight vacuole, termed the bacterial-containing vacuole (BCV). Simultaneously, Shigella induces the formation of large vesicles around the entry site, which we refer to as infection-associated macropinosomes (IAMs). After entry, Shigella ruptures the BCV and escapes into the host cytosol by disassembling the BCV remnants. Previously, IAM formation has been shown to be required for efficient BCV escape, but the molecular events associated with BCV disassembly have remained unclear. To identify host components required for BCV disassembly, we performed a microscopy-based screen to monitor the recruitment of BAR domain-containing proteins, which are a family of host proteins involved in membrane shaping and sensing (e.g. endocytosis and recycling) during Shigella epithelial cell invasion. We identified endosomal recycling BAR protein Sorting Nexin-8 (SNX8) localized to IAMs in a PI(3)P-dependent manner before BCV disassembly. At least two distinct IAM subpopulations around the BCV were found, either being recycled back to cellular compartments such as the plasma membrane or transitioning to become RAB11A positive "contact-IAMs" involved in promoting BCV rupture. The IAM subpopulation duality was marked by the exclusive recruitment of either SNX8 or RAB11A. Hindering PI(3)P production at the IAMs led to an inhibition of SNX8 recruitment at these compartments and delayed both, the step of BCV rupture time and successful BCV disassembly. Finally, siRNA depletion of SNX8 accelerated BCV rupture and unpeeling of BCV remnants, indicating that SNX8 is involved in controlling the timing of the cytosolic release. Overall, our work sheds light on how Shigella establishes its intracellular niche through the subversion of a specific set of IAMs.