The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7

Trofimenko, Evgeniya; Homma, Yuta; Fukuda, Mitsunori; Widmann, Christian

doi:10.1016/j.celrep.2021.109945

Cited by 22 publications

(20 citation statements)

References 138 publications

(178 reference statements)

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“…Following cellular uptake/internalization, the intracellular trafficking pathway mapped for PGua 4 is identical to that reported for TAT and R 8 (at low concentrations (1 μM)), which colocalized with both early (Rab5+) and late (Rab7+) endosomes after 30 min of internalization . Recently, an unconventional endocytic route fully independent of Rab5 and Rab7 but dependent on Rab14 has been reported to target cationic CPPs (including R 9 and Tat, at high concentrations (40 μM)) to the nonacidic LAMP1-positive compartment . While we cannot exclude the role of Rab14 in the intracellular transport of PGua 4 , at the concentrations used in our study (5 μM), PGua 4 was observed to traffic mainly through the classical endocytic pathway and to primarily remain trapped within it, a major limitation for many Arg-rich CPPs, including TAT, R 8 , and R 9 . ,, Based on qualitative assessment (detection of fluorescence in the cytoplasm, which is a limiting method for the detection of low level of cytoplasmic delivery), the release of PGua 4 from endosomes occurs at a concentration ≥ 10 μM, with the maximal cells showing cytosolic labeling at a concentration of 20 μM.…”

Section: Discussionsupporting

confidence: 64%

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

et al. 2023

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To investigate the structure-cellular penetration relationship of guanidinium-rich transporters (GRTs), we previously designed PGua4, a five-amino acid peptoid containing a conformationally restricted pattern of eight guanidines, which showed high cell-penetrating abilities and low cell toxicity. Herein, we characterized the cellular uptake selectivity, internalization pathway, and intracellular distribution of PGua4, as well as its capacity to deliver cargo. PGua4 exhibits higher penetration efficiency in HeLa cells than in six other cell lines (A549, Caco-2, fibroblast, HEK293, Mia-PaCa2, and MCF7) and is mainly internalized by clathrin-mediated endocytosis and macropinocytosis. Confocal microscopy showed that it remained trapped in endosomes at low concentrations but induced pH-dependent endosomal membrane destabilization at concentrations ≥10 μM, allowing its diffusion into the cytoplasm. Importantly, PGua4 significantly enhanced macropinocytosis and the cellular uptake and cytosolic delivery of large IgGs following noncovalent complexation. Therefore, in addition to its peptoid nature conferring high resistance to proteolysis, PGua4 presents characteristics of a promising tool for IgG delivery and therapeutic applications.

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Section: Discussionsupporting

confidence: 64%

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

et al. 2023

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“…S5h ). GII.4-induced membrane wounding was further confirmed by staining the VLP-treated and untreated cells for (1) LAMP-1 and (2) regulators of endo-lysosomal processes and wound healing mechanisms, Rab11 and Rab14 30 , 31 . GII.4 VP1 localized with LAMP-1 (Fig.…”

Section: Resultsmentioning

confidence: 90%

CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

et al. 2023

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Globally, most cases of gastroenteritis are caused by pandemic GII.4 human norovirus (HuNoV) strains with no approved therapies or vaccines available. The cellular pathways that these strains exploit for cell entry and internalization are unknown. Here, using nontransformed human jejunal enteroids (HIEs) that recapitulate the physiology of the gastrointestinal tract, we show that infectious GII.4 virions and virus-like particles are endocytosed using a unique combination of endosomal acidification-dependent clathrin-independent carriers (CLIC), acid sphingomyelinase (ASM)-mediated lysosomal exocytosis, and membrane wound repair pathways. We found that besides the known interaction of the viral capsid Protruding (P) domain with host glycans, the Shell (S) domain interacts with both galectin-3 (gal-3) and apoptosis-linked gene 2-interacting protein X (ALIX), to orchestrate GII.4 cell entry. Recognition of the viral and cellular determinants regulating HuNoV entry provides insight into the infection process of a non-enveloped virus highlighting unique pathways and targets for developing effective therapeutics.

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“…It is now widely believed that several endocytic pathways other than the clathrin-dependent pathway are present in single cells (reviewed in Sigismund et al, 2021), and some of them presumably function in a Rab5-independent manner. It has recently been reported that endo-lysosomal maturation for internalized cell-penetrating peptides, homeoproteins and polyamines depends mainly on Rab14 rather than on Rab5 (Trofimenko et al, 2021). Thus, the Rab5-CKO cells established in our study should serve as an ideal tool for analyzing such Rab5-independent endocytic pathways.…”

Section: Rab1 Functions Under Rab1-depleted Conditionsmentioning

confidence: 77%

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system

Hatoyama

Homma

Hiragi

et al. 2021

Journal of Cell Science

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Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. Since they have recently been shown to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, it has been extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed conditional KO (CKO) cells for Rab1 (Rab1A/1B) and Rab5 (Rab5A/5B/5C) by using the auxin-inducible protein degradation system. Rab1-CKO and Rab5-CKO led to eventual cell death >18 h and >48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and ER-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

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The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7

Cited by 22 publications

References 138 publications

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system

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The endocytic pathway taken by cationic substances requires Rab14 but not Rab5 and Rab7

Cited by 22 publications

References 138 publications

Characterization of PGua4, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

Characterization of PGua4, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system

Contact Info

Product

Resources

About

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis

Characterization of PGua₄, a Guanidinium-Rich Peptoid that Delivers IgGs to the Cytosol via Macropinocytosis