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TRPML Subfamily of Endolysosomal Channels
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Nicholas E. Karagas, Morgan A. Rousseau, Kartik Venkatachalam
To overcome the limitations of LysoTracker, co-labelling with specific-protein markers is also required. For instance, Rab5 and Rab7, which localize to the early- and late-endosomes, respectively, may be used as markers. Other markers of early endosome include HRS and EEA1, and late-endosomes/lysosomes include proteins such as LAMP1 and LAMP3 (Manzoni et al., 2004; Thompson et al., 2007; Venkatachalam et al., 2006). Lysosomes can be visualized using fluorescently tagged substrates for lysosomal enzymes such as cathepsin (e.g. Magic Red cathepsin L substrate) (Johnson et al., 2016). Autophagosomes are detected by labelling with fluorescently tagged Atg proteins or LC3, whereas amphisomes, which are generated by the fusion of autophagosomes and endosomes (Tanida et al., 2008), can be identified by appropriate co-labelling. In addition to these fluorescent techniques, autophagosomes, endosomes, amphisomes, and lysosomes can be effectively discriminated by electron microscopy, and have been shown to accumulate in cells lacking TRPMLs (Wong et al., 2012). Unfortunately, the absence of effective antibodies against TRPML proteins has prevented visualization of natively expressed proteins, which can be circumvented by examining the expression of ectopic expression of tagged TRPMLs.
Gateways of Pathogenic Bacterial Entry into Host Cells—Salmonella
Published in K. Balamurugan, U. Prithika, Pocket Guide to Bacterial Infections, 2019
Balakrishnan Senthilkumar, Duraisamy Senbagam, Chidambaram Prahalathan, Kumarasamy Anbarasu
After host cell invasion, Salmonella are internalized within a membrane-bound compartment known as small colony variants (SCVs). (Many bacterial pathogens have developed different strategies to neutralize the host immune response, either by preventing vacuole–lysosome fusion or by escaping into cytosol.) Salmonella are classified as a vacuolar pathogen; hence upon invasion, this bacterium resides and multiplies within SCV, a specialized vacuole, as the only intracellular niche of Salmonella (Bakowski et al. 2008), and it undergoes different stages of maturation like macrophages. These SCVs consist with markers such as Rab5, EEA1 (early endosome antigen 1), and TfR (transferrin receptor) (Steele-Mortimer et al. 1999; Bakowski et al. 2008). Once the SCV matures and is surrounded by actin, it migrates toward a perinuclear position and initiates formation of Salmonella-induced filaments (SIF), which helps transportation of nutrients to the SCV, thereby facilitating bacterial replication (Knodler and Steele-Mortimer 2003; Salcedo and Holden 2003).
GRP75 as a functional element of cholix transcytosis
Published in Tissue Barriers, 2023
Keyi Liu, Tom Hunter, Alistair Taverner, Kevin Yin, Julia MacKay, Kate Colebrook, Morgan Correia, Amandine Rapp, Randall J. Mrsny
We next examined the relationship of EEA1 with LMAN1 in the context Chx A→B transcytosis. At 5 min post ILI of Chx266-hGH, a subset of the hGH+ vesicles present in the apical region of enterocytes were also EEA1+ (Figure 6a). Simultaneously, LMAN1+ vesicles containing Chx266-hGH vesicles were observed within this same cellular location (Figure 6b). Co-localization of LMAN1 and EEA1 highlighted the extensive distribution of these proteins in the apical vesicular compartment relative to the basal region at this early stage of Chx A→B transcytosis (Figure 6c). Further, apical vesicles that contained EEA1 were observed in a subset of LMAN1+ vesicles and were highly correlated with the distribution of vesicles that were EEA1+/hGH+ (Figure 6c). These results are consistent with a Chx A→B transcytosis process where the contents of EEA1+ vesicles are delivered to LMAN1+ vesicles in an apical vesicular sorting event.
Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis
Published in Tissue Barriers, 2020
Alistair Taverner, Julia MacKay, Floriane Laurent, Tom Hunter, Keyi Liu, Khushdeep Mangat, Lisa Song, Elbert Seto, Sally Postlethwaite, Aatif Alam, Apurva Chandalia, Minji Seung, Mazi Saberi, Weijun Feng, Randall J. Mrsny
Chx utilizes a series of intracellular vesicular compartments to traffic through polarized intestinal epithelial cells using a process that culminates in A→B transcytosis. Engagement of clathrin+ vesicles appears to occur in both the apical and basal vesicular compartments, but there is no strong evidence for a clathrin-mediated endocytosis to begin this A→B transcytosis process. The trafficking mechanism used by Chx overcomes endogenous barriers that restrict vesicular movement within cells in order to maintain systemic homeostasis. Cargos within early endosomes, such as those defined by the presence of EEA1 and Rab7, can be sent to the TGN via retrograde trafficking with ultimate targeting to the transitional ER for refolding and repair or sorted to lysosomes for degradation. Importantly, Chx transcytosis uses trafficking elements that avoid delivery to lysosome-like structures. Co-localizations of Chx with TGN-38, however, suggested limited distribution within the TGN during Chx transcytosis. Instead, Chx appears to access the ERGIC element LMAN1 in the apical vesicular compartment soon after endocytosis and uses this interaction to reach the basal vesicular compartment that culminates in basal exocytosis.
Lithium effects on vesicular trafficking in hepatocellular carcinoma cells
Published in Ultrastructural Pathology, 2019
Iuliia Taskaeva, Nataliya Bgatova, Izabella Gogaeva
Endosomal transport pathways are closely associated with receptor signaling and the Ras-associated binding protein family of small GTPases involved in endocytic trafficking.5 Rab proteins constitute a family of small GTP-binding proteins which localize in distinct intracellular compartments and regulate diverse endocytotic events.6 Endocytotic markers can be detected in different endosomal compartments, such as early, late and recycling endosomes. Early endosome antigen 1 (EEA1) localizes to early endosomes and is associated with endosome fusion7,8 while Ras-related protein 7 (Rab7) has been found in late endosomes and is implicated in transport from early to late endocytic compartments.6 Ras-related protein 11 (Rab11) participates in the endocytic recycling and regulation of transport from early and recycling endosomes to the cell surface and has been detected in recycling endosomes.5,9