Summary
This brief overview of endocytic trafficking is written in honor of Renate Fuchs, who retires this year. In the mid-1980s, Renate pioneered studies on the ion-conducting properties of the ...recently discovered early and late endosomes and the mechanisms governing endosomal acidification. As described in this review, after uptake through one of many mechanistically distinct endocytic pathways, internalized proteins merge into a common early/sorting endosome. From there they again diverge along distinct sorting pathways, back to the cell surface, on to the trans-Golgi network or across polarized cells. Other transmembrane receptors are packaged into intraluminal vesicles of late endosomes/multivesicular bodies that eventually fuse with and deliver their content to lysosomes for degradation. Endosomal acidification, in part, determines sorting along this pathway. We describe other sorting machinery and mechanisms, as well as the rab proteins and phosphatidylinositol lipids that serve to dynamically define membrane compartments along the endocytic pathway.
Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low‐density lipoproteins in macrophages. Furthermore, several groups have previously ...used IKA to inhibit clathrin‐mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 μm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long‐term incubation with IKA has cytotoxic effects, the short‐term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.
The authors have characterized the natural product, ikarugamycin, to demonstrate its utility as a potent and selective acute inhibitor of clathrin‐mediated endocytosis.
Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by ...endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics.
Rhinovirus infection is associated with the majority of asthma exacerbations. The role of fractalkine in anti-viral (type 1) and pathogenic (type 2) responses to rhinovirus infection in allergic ...asthma is unknown. To determine whether (1) fractalkine is produced in airway cells and in peripheral blood leucocytes, (2) rhinovirus infection increases production of fractalkine and (3) levels of fractalkine differ in asthmatic compared to non-asthmatic subjects. Fractalkine protein and mRNA levels were measured in bronchoalveolar lavage (BAL) cells and peripheral blood mononuclear cells (PBMCs) from non-asthmatic controls (n = 15) and mild allergic asthmatic (n = 15) subjects. Protein levels of fractalkine were also measured in macrophages polarised ex vivo to give M1 (type 1) and M2 (type 2) macrophages and in BAL fluid obtained from mild (n = 11) and moderate (n = 14) allergic asthmatic and non-asthmatic control (n = 10) subjects pre and post in vivo rhinovirus infection. BAL cells produced significantly greater levels of fractalkine than PBMCs. Rhinovirus infection increased production of fractalkine by BAL cells from non-asthmatic controls (P<0.01) and in M1-polarised macrophages (P<0.05), but not in BAL cells from mild asthmatics or in M2 polarised macrophages. Rhinovirus induced fractalkine in PBMCs from asthmatic (P<0.001) and healthy control subjects (P<0.05). Trends towards induction of fractalkine in moderate asthmatic subjects during in vivo rhinovirus infection failed to reach statistical significance. Fractalkine may be involved in both immunopathological and anti-viral immune responses to rhinovirus infection. Further investigation into how fractalkine is regulated across different cell types and into the effect of stimulation including rhinovirus infection is warranted to better understand the precise role of this unique dual adhesion factor and chemokine in immune cell recruitment.
Germline cancer genetic testing has become a standard evidence-based practice, with established risk reduction and screening guidelines for genetic carriers. Access to genetic services is limited in ...many places, which leaves many genetic carriers unidentified and at risk for late diagnosis of cancers and poor outcomes. This poses a problem for childhood cancer survivors, as this is a population with an increased risk for subsequent malignant neoplasms (SMN) due to cancer therapy or inherited cancer predisposition. The ENGaging and Activating cancer survivors in Genetic services (ENGAGE) study evaluates the effectiveness of an in-home, collaborative PCP model of remote telegenetic services to increase uptake of cancer genetic testing in childhood cancer survivors compared to usual care options for genetic testing.
The ENGAGE study is a 3-arm randomized hybrid type 1 effectiveness and implementation study within the Childhood Cancer Survivor Study population which tests a clinical intervention while gathering information on its delivery during the effectiveness trial and its potential for future implementation among 360 participants. Participants are randomized into three arms. Those randomized to Arm A receive genetic services via videoconferencing, those in Arm B receive these services by phone, and those randomized to Arm C will receive usual care services.
With many barriers to accessing genetic services, innovative delivery models are needed to address this gap and increase uptake of genetic services. The ENGAGE study evaluates the effectiveness of an adapted model of remote delivery of genetic services to increase the uptake of recommended genetic testing in childhood cancer survivors. This study assesses the uptake in remote genetic services and identify barriers to uptake to inform future recommendations and a theoretically-informed process evaluation which can inform modifications to enhance dissemination beyond this study population and to realize the benefits of precision medicine.
This protocol was registered at clinicaltrials.gov (NCT04455698) on July 2, 2020.
Increased use of neonicotinoid‐coated crop seeds introduces greater amounts of insecticides into the environment, where they are vulnerable to transport. To understand the transport of neonicotinoids ...from agricultural fields, we planted maize (Zea mays L.) seeds coated with thiamethoxam in lysimeter plots in central Pennsylvania. Over the next year, we sampled water generated by rainfall and snowmelt and analyzed these samples with mass spectrometry for the neonicotinoids thiamethoxam and clothianidin (metabolite), which originated from the coated seeds. For surface and subsurface transport, thiamethoxam exhibited “first‐flush” dynamics, with concentrations highest during the first events following planting and generally decreasing for the remainder of the study. The metabolite clothianidin, however, persisted throughout the study. The mass of thiamethoxam and clothianidin exported during the study period accounted for 1.09% of the mass applied, with more than 90% of the mass transported in subsurface flow and less than 10% in surface runoff. These results suggest that surface runoff, at least for our site, is a relatively small contributor to the overall fate and transport of these insecticides and that the delivery ratio (i.e., mass exported/mass applied) observed for these compounds is similar to those of other trace‐level emerging contaminants known to negatively influence aquatic ecosystems.
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