- Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity. [Journal Article]
- PPPLoS Pathog 2019 Jun 26; 15(6):e1007851
- Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion syste…
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling machineries to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
- Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI). [Journal Article]
- ATArterioscler Thromb Vasc Biol 2019; 39(7):1390-1401
- Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generatio…
Objective- Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results- Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa-PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa-PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions- PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa-PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview- An online visual overview is available for this article.
- Size-dependent cellular uptake and localization profiles of silver nanoparticles. [Journal Article]
- IJInt J Nanomedicine 2019; 14:4247-4259
- CONCLUSIONS: The results suggest that the size of AgNPs can not only affect the efficiency of cellular uptake, but also the type of endocytosis. The clathrin-mediated endocytosis may be the most common endocytic pathway for AgNPs in B16 cells, and AgNPs at each size were likely to enter cells by a major internalization pathway.
- Recycling endosomes in mature epithelia restrain tumorigenic signaling. [Journal Article]
- CRCancer Res 2019 Jun 25
- The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and…
The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, while suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis.
- Uptake of Ranibizumab but Not Bevacizumab into Uveal Melanoma Cells Correlates with a Sustained Decline in VEGF-A Levels and Metastatic Activities. [Journal Article]
- CCancers (Basel) 2019 Jun 21; 11(6)
- Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. …
Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. Here, we evaluated whether bevacizumab and ranibizumab, a humanized Fab-fragment against VEGF-A, can enter UM cells and induce a sustained physiological response. The primary and metastatic UM cell lines Mel-270 and OMM-2.5 were exposed to bevacizumab or ranibizumab for one day and were maintained further in untreated medium for a total of three days. Both antibodies significantly reduced the levels of extracellular VEGF-A and the angiogenic potential of the conditioned medium after one day. These inhibitory effects of bevacizumab diminished by day three. Ranibizumab suppressed the metabolic activity, proliferation, and intracellular VEGF-A levels in a cell-type and concentration-dependent manner, whereas bevacizumab exerted no effect. Both drugs were detected inside early endosomes within the UM cells, with the stronger and sustained colocalization of ranibizumab. Our results therefore demonstrated the more potent and persistent suppressive activity of ranibizumab on the UM cells, possibly due to its higher level of uptake and prolonged intracellular retention. Further research on the endosome dynamics in UM cells might provide valuable insight into the response of these heterogenous tumors to therapeutic antibodies.
- Lysosome-related organelles as functional adaptations of the endolysosomal system. [Review]
- COCurr Opin Cell Biol 2019 Jun 21; 59:147-158
- Unique functions of specialised cells such as those of the immune and haemostasis systems, skin, blood vessels, lung, and bone require specialised compartments, collectively referred to as lysosome-r…
Unique functions of specialised cells such as those of the immune and haemostasis systems, skin, blood vessels, lung, and bone require specialised compartments, collectively referred to as lysosome-related organelles (LROs), that share features of endosomes and lysosomes. LROs harbour unique morphological features and cell type-specific contents, and most if not all undergo regulated secretion for diverse functions. Ongoing research, largely driven by analyses of inherited diseases and their model systems, is unravelling the mechanisms involved in LRO generation, maturation, transport and secretion. A molecular understanding of these features will provide targets and markers that can be exploited for diagnosis and therapy of a myriad of diseases.
- Tumor-derived extracellular vesicles in breast cancer: from bench to bedside. [Journal Article]
- CLCancer Lett 2019 Jun 21
- Tumor-derived extracellular vesicles (TEVs) released from various tumor cell types comprise endosome-derived exosomes and microvesicles (MVs), which originate from plasma membrane budding. TEVs incor…
Tumor-derived extracellular vesicles (TEVs) released from various tumor cell types comprise endosome-derived exosomes and microvesicles (MVs), which originate from plasma membrane budding. TEVs incorporate a myriad of biomolecules such as proteins, DNAs, metabolites and microRNAs, which can be transferred from cell-to-cell. Besides their role in the disposal of biomolecules, TEVs serve to orchestrate fundamental processes of normal and malignant development, including breast cancer (BC). As such, TEVs are important constituents of the tumor microenvironment (TME) that act as communication shuttles through transduction of encapsulated molecular cargos from a parent to a recipient cell and through direct interaction with target cells. Emerging evidence suggests that TEVs support BC development and disease progression by fostering invasion, angiogenesis, pre-metastatic niche preparation, escape from immune surveillance, and induction of resistance to treatment. Although there is a long way to go in order to translate the current knowledge into actual clinical applications, TEVs represent promising candidates for diagnostic biomarkers, therapeutic carriers and targets. In the present review, we will summarize the current knowledge on TEVs in BC.
- Protein kinase C regulates ErbB3 turnover. [Journal Article]
- ECExp Cell Res 2019 Jun 21
- ErbB3, which belongs to the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases, is involved in progression of several human cancers and a tight regulation of its expr…
ErbB3, which belongs to the epidermal growth factor receptor (EGFR) or ErbB family of receptor tyrosine kinases, is involved in progression of several human cancers and a tight regulation of its expression is crucial. An important mechanism for regulation of ErbB proteins is endocytosis and we recently showed that ErbB3, contrary to other ErbB proteins, like EGFR and ErbB2, is constitutively internalized and degraded. Several studies show that protein kinase C (PKC) can regulate the activation, localization and stability of EGFR and ErbB2. Activation of PKC causes their down-regulation from the plasma membrane, but instead of being degraded the receptors accumulate in an endosomal recycling compartment. Since little is known about possible connections between ErbB3 and PKC, we have in the present study investigated effects PKC activity has on ErbB3 stability and intracellular trafficking. While PKC inhibition tends to increase ErbB3 degradation, activation of PKC causes ErbB3 stabilization. The stabilization was not due to inhibited internalization, on the contrary we find that expression of ErbB3 at the plasma membrane is reduced upon PMA-induced PKC activation. However, while endocytosed ErbB3 under normal conditions and upon PKC inhibition is found in early endosomal antigen 1 (EEA1) positive early endosomes and lysosomal-associated membrane protein 1 (LAMP1) positive late endosomes/lysosomes, indicating that it follows the classic degradative pathway, ErbB3 localizes to EEA1 and LAMP1 negative compartments upon PMA-induced activation of PKC. Altogether this shows that PKC regulates the stability of ErbB3, and knockdown experiments show that PKCδ is essential in this process. A likely explanation is that PKC regulates endosomal sorting of ErbB3 and that activated PKC sorts ErbB3 away from the degradative pathway.
- Selective Cell Penetrating Peptide-Functionalized Envelope-Type Chimeric Lipopepsomes Boost Systemic RNAi Therapy for Lung Tumors. [Journal Article]
- AHAdv Healthc Mater 2019 Jun 24; :e1900500
- Small interfering RNA (siRNA) is considered a highly specific and potent biotherapeutic that holds tremendous potential for the treatment of various diseases. The clinical translation of siRNA is, ho…
Small interfering RNA (siRNA) is considered a highly specific and potent biotherapeutic that holds tremendous potential for the treatment of various diseases. The clinical translation of siRNA is, however, greatly impeded by the lack of safe and efficient delivery vehicles in vivo. Here, the development of selective cell penetrating peptide (CPP33)-functionalized chimeric lipopepsomes (CPP33-CLP) for efficient encapsulation and selective delivery of polo-like kinase 1 specific siRNA (siPLK1) to orthotopic A549 human lung tumor in vivo is reported. Interestingly, siRNA is tightly encapsulated into CPP33-CLP with a superb encapsulation efficiency of over 95% owing to the thick strong electrostatic interactions. Notably, siPLK1-loaded CPP33-CLP (siPLK1-CPP33-CLP) is selectively internalized by A549 human lung cancer cells, efficiently escapes from endosomes, and swiftly releases siRNA into the cytoplasm, affording a significant sequence-specific gene silencing in vitro. Moreover, siPLK1-CPP33-CLP exhibits prolonged blood circulation, enhanced tumor accumulation, effective suppression of tumor growth, and considerably elevated survival time of orthotopic A549 human lung tumor-bearing nude mice. These chimeric lipopepsomes appear as an attractive and potent nanoplatform for safe and targeted siRNA delivery.
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- The chloride anion as a signalling effector. [Journal Article]
- BRBiol Rev Camb Philos Soc 2019 Jun 23
- The specific role of the chloride anion (Cl-) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellu…
The specific role of the chloride anion (Cl-) as a signalling effector or second messenger has been increasingly recognized in recent years. It could represent a key factor in the regulation of cellular homeostasis. Changes in intracellular Cl- concentration affect diverse cellular functions such as gene and protein expression and activities, post-translational modifications of proteins, cellular volume, cell cycle, cell proliferation and differentiation, membrane potential, reactive oxygen species levels, and intracellular/extracellular pH. Cl- also modulates functions in different organelles, including endosomes, phagosomes, lysosomes, endoplasmic reticulum, and mitochondria. A better knowledge of Cl- signalling could help in understanding the molecular and metabolic changes seen in pathologies with altered Cl- transport or under physiological conditions. Here we review relevant evidence supporting the role of Cl- as a signalling effector.