A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Abstract: Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export

Repetitive non-typhoidal Salmonella exposure is an environmental risk factor for colon cancer and tumor growth

During infection, Salmonella hijacks essential host signaling pathways. These molecular manipulations disrupt cellular integrity and may induce oncogenic transformation. Systemic S. Typhi infections are linked to gallbladder cancer, whereas severe non-typhoidal Salmonella (NTS) infections are associated with colon cancer (CC). These diagnosed infections, however, represent only a small fraction of all NTS infections as many infections are mild and go unnoticed. To assess the overall impact of NTS infections, we performed a retrospective serological study on NTS exposure in patients with CC. The magnitude of exposure to NTS, as measured by serum antibody titer, is significantly positively associated with CC. Repetitively infecting mice with low NTS exposure showed similar accelerated tumor growth to that observed after high NTS exposure. At the cellular level, NTS preferably infects (pre-)transformed cells, and each infection round exponentially increases the rate of transformed cells. Thus, repetitive exposure to NTS associates with CC risk and accelerates tumor growth

A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Abstract: Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export

Quantification of Bioorthogonal Stability in Immune Phagocytes Using Flow Cytometry Reveals Rapid Degradation of Strained Alkynes

One of the areas in which bioorthogonal chemistrychemistry performed inside a cell or organismhas become of pivotal importance is in the study of host–pathogen interactions. The incorporation of bioorthogonal groups into the cell wall or proteome of intracellular pathogens has allowed study within the endolysosomal system. However, for the approach to be successful, the incorporated bioorthogonal groups must be stable to chemical conditions found within these organelles, which are some of the harshest found in metazoans: the groups are exposed to oxidizing species, acidic conditions, and reactive thiols. Here we present an assay that allows the assessment of the stability of bioorthogonal groups within host cell phagosomes. Using a flow cytometry-based assay, we have quantified the relative label stability inside dendritic cell phagosomes of strained and unstrained alkynes. We show that groups that were shown to be stable in other systems were degraded by as much as 79% after maturation of the phagosome

Ultrastructural imaging of Salmonella-Host interactions using super-resolution correlative light-electron microscopy of bioorthogonal pathogens

\u3cp\u3eThe imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20nm. This STORM-CLEM approach thus presents a new approach to understand these pathogens during infection.\u3c/p\u3

Repetitive non-typhoidal Salmonella exposure is an environmental risk factor for colon cancer and tumor growth

During infection, Salmonella hijacks essential host signaling pathways. These molecular manipulations disrupt cellular integrity and may induce oncogenic transformation. Systemic S. Typhi infections are linked to gallbladder cancer, whereas severe non-typhoidal Salmonella (NTS) infections are associated with colon cancer (CC). These diagnosed infections, however, represent only a small fraction of all NTS infections as many infections are mild and go unnoticed. To assess the overall impact of NTS infections, we performed a retrospective serological study on NTS exposure in patients with CC. The magnitude of exposure to NTS, as measured by serum antibody titer, is significantly positively associated with CC. Repetitively infecting mice with low NTS exposure showed similar accelerated tumor growth to that observed after high NTS exposure. At the cellular level, NTS preferably infects (pre-)transformed cells, and each infection round exponentially increases the rate of transformed cells. Thus, repetitive exposure to NTS associates with CC risk and accelerates tumor growth

Negatively Charged Lipid Membranes Catalyze Supramolecular Hydrogel Formation

In this contribution we show that biological membranes can catalyze the formation of supramolecular hydrogel networks. Negatively charged lipid membranes can generate a local proton gradient, accelerating the acid-catalyzed formation of hydrazone-based supramolecular gelators near the membrane. Synthetic lipid membranes can be used to tune the physical properties of the resulting multicomponent gels as a function of lipid concentration. Moreover, the catalytic activity of lipid membranes and the formation of gel networks around these supramolecular structures are controlled by the charge and phase behavior of the lipid molecules. Finally, we show that the insights obtained from synthetic membranes can be translated to biological membranes, enabling the formation of gel fibers on living HeLa cells

Negatively Charged Lipid Membranes Catalyze Supramolecular Hydrogel Formation

In this contribution we show that biological membranes can catalyze the formation of supramolecular hydrogel networks. Negatively charged lipid membranes can generate a local proton gradient, accelerating the acid-catalyzed formation of hydrazone-based supramolecular gelators near the membrane. Synthetic lipid membranes can be used to tune the physical properties of the resulting multicomponent gels as a function of lipid concentration. Moreover, the catalytic activity of lipid membranes and the formation of gel networks around these supramolecular structures are controlled by the charge and phase behavior of the lipid molecules. Finally, we show that the insights obtained from synthetic membranes can be translated to biological membranes, enabling the formation of gel fibers on living HeLa cells