10 research outputs found
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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
The potential of bioorthogonal chemistry for correlative light and electron microscopy: a call to arms
Bio-organic Synthesi
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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 chemistryî—¸chemistry
performed inside a cell or organismî—¸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