Browsing by Subject "Endosome"

Now showing 1 - 2 of 2

Influence of the newly identified Mos10 interaction partner Vps68on ESCRT-III function
(2021) Alsleben, Sören; Kölling, Ralf
The endosomal sorting complex required for transport (ESCRT) is a part of the heteromeric complex machinery consisting of ESCRT-0, -I, -II, and -III ensuring functional protein traffic of endocytic and biosynthetic cargo. Stepwise sorting of labeled cargo material inside the lumen of the endosome by invagination and abscission of the endosomal membrane to form intraluminal vesicles (ILV’s) is mediated by the ESCRT-III complex. The complex consists of eight members of which Vps20, Snf7, Vps2, and Vps24 are considered ESCRT-III essential subunits, and Chm7, Did2, Ist1, and Mos10/Vps60 are commonly labeled as complex associated proteins. The correct interplay between the proteins ensures cargo sorting into the MVB (multivesicular body) pathway and transport from the late endosome into the vacuolar lumen for degradation. Besides the initial function of vacuolar protein sorting (vps), the complex is involved in a multitude of cellular processes like cell abscission, virus budding, autophagy, and remaining nuclear envelope integrity. The step-wise assembly of the ESCRT-III complex is mediated after the cascade-like ESCRT-0 to ESCRT-II complex formation at the membrane budding site, collecting cargo protein for invagination into the endosomal lumen. ESCRT-III Vps20 is recruited to the membrane by the ESCRT-II member Vps25, then nucleating Snf7 association and oligomerization. Additional assembly of ESCRT-III members like Vps24 and Vps2 further drives membrane bending away from the cytosol to the final abscission event, before being recycled back to cytosolic monomers by Vps4. Although Mos10 has been implicated in the recycling step of the ESCRT-III units by interacting with the Vps4/Vta1 complex, the protein’s function remains poorly characterized. This thesis tried to find new insights in Mos10 functionality by finding yet uncharacterized interacting partners, thus connecting the protein to new putative non-endosomal functions or understanding its role in the established ESCRT-III complex. For this purpose, a series of crosslinking experiments with tagged variants of Mos10 were performed. Purification was achieved by IMAC (Immobilized Metal Ion Affinity Chromatography) after adding a poly-his sequence to the protein and by immunoprecipitation of sfGFP tagged Mos10. Both methods revealed a multitude of putative Mos10 interacting partners by MS analysis to be further reduced by applying the SILAC (stable isotope labeling with amino acids in cell culture) technique. After selecting possible Mos10 interacting partners, IP and Co-IP experiments of tagged candidate variants were used to identify an interaction between the two proteins. An interaction between Mos10-6His and Vps68-13myc besides native Mos10 and Vps68-fGFP could be verified by purification of Vps68 and co-precipitating Mos10. The influence of Vps68 on the assembly and composition of the ESCRT-III complex was examined. After Vps68 depletion, an enrichment of the core subunits Snf7, Vps2, and Vps24 in the complex was detected with a reduced number of Did2, Ist1, and Mos10 molecules. Thus, it appears that ESCRT-III disassembly is blocked in ∆vps68 mutant. The influence of VPS68 deletion on the intracellular localization of ESCRT-III proteins was examined by fluorescence microscopy with sfGFP-tagged variants. While the localization of most ESCRT-III proteins was not significantly altered, a marked relocalization was observed for Mos10. In wildtype, Mos10-sfGFP was localized at the vacuolar membrane, while in ∆vps68 it was dispersed into vesicular structures enriched at the cell cortex. Further, the impact of VPS68 deletion on the sorting of the endocytic cargo protein Ste6 was investigated. By cycloheximide chase experiments, it could be shown that Ste6 is strongly stabilized in a ∆vps68 mutant. This indicates that the transport of the protein to the yeast vacuole for degradation is blocked. The ∆vps68 block in endocytic trafficking was compared with other mutants of the vps-pathway, whose site of action has been established. These experiments show that the VPS68 deletion neither leads to a class D phenotype, as in ∆vps21, nor to a class E phenotype, as in ∆snf7. The Ste6-GFP distribution in the ∆vps68 mutant rather resembles wildtype with more pronounced accumulation of endosomal dots. The data taken together suggest that Vps68 acts after the formation of the ESCRT-III complex and is required for cargo delivery from the late endosome to the vacuolar lumen.
Intracellular regulation of Wnt and FGF signal transduction by the late endosomal compartment in Xenopus laevis
(2022) Kreis, Jennifer; Feistel, Kerstin
The endosomal network depicts a vast playground of multiple processing capabilities in terms of signaling. Distinct compartments of the endosomal machinery exert specific functions and thus contribute in signal termination, transduction, attenuation or amplification. Initially, these functions were attributed to early endosomes but recent research likewise considers late endosomes to be just as relevant in mediating such processes. Functionality as well as the molecular identity of these intracellular membranous platforms are orchestrated by a large superfamily of small Ras like GTPases. The collected data of this study particularly highlight the involvement of late endosomes and its associated regulator Rab7 in the early development of the African clawed frog Xenopus laevis. In particular, the first two chapters address the Rab7-dependent specification of the mesodermal germ layer by regulating intracellular pathway activity of Wnt and FGF/MAPK signaling. After fertilization formation of the germ layers is one of the first processes to be initiated. An essential part of mesoderm development comprises subdivision into different mesodermal regions, thus clustering it into ventrolateral and dorsal mesoderm. This patterning is crucial to promote further differentiation into various tissues arising from the mesodermal germ layer. It turned out, Rab7 regulates ventrolateral fates in a Wnt-dependent manner. The small GTPase exerts its function upstream of the Wnt co-transcription factor Ctnnb1 to ensure its nuclear relocalization. In addition to that, Rab7-positive endosomes are likewise required to mediate intracellular FGF/MAPK signal transduction in order to specify dorsal mesoderm. Here, Rab7 regulates proper signaling at the level or downstream of Ras and upstream of Erk/Mapk1. The last chapter then elicits further regulative properties of the late endosomal platform, concerning Cd63 function. The tetraspanin Cd63, which constitutes a transmembrane protein, associates with late endolysosomal compartments and exhibits a similar expression pattern like the small GTPase Rab7 in Xenopus laevis. Contrary to Rab7, function of Cd63 seems to be dispensable whilst gastrulation. However, the presented studies in this chapter suggest a vital function of the tetraspanin Cd63 during axial elongation and correct eye development. Therefore, these investigations regarding Cd63 demonstrated an involvement of the regulative function of late endosomes as signaling platforms for embryonic development beyond mesoderm specification and gastrulation. Overall, the summarized data of this study provides further insights into the determining capacity of Rab7-positive endosomal platforms in intracellular signal transduction of different pathways during early embryonic development.