Kazrin C entraps early endosomes at the pericentriolar region and facilitates endocytic recycling

We identified kazrin C as a human protein that inhibits clathrin-mediated endocytosis when overexpressed. We now generated kazrin knock out and GFP-kazrin C expressing MEF lines to investigate in detail its function in endocytic traffic. We find that kazrin depletion delays recycling of internalized material and causes accumulation and dispersal of early endosomes (EE), indicating a role in transport from the early to the perinuclear recycling endosomes (RE). Consistently, we found that the C-terminal domain of kazrin C, predicted to be an intrinsically disordered region (IDR), specifically interacts with several endosomal components, including Epsin Homology Domain (EHD) proteins, γ-adaptin, and phosphatidyl-inositol-3 phosphate. Further, kazrin C shares homology with dynein/dynactin adaptors, it directly interacts with the dynactin complex and the dynein light intermediate chain LIC1, and overexpressed GFP-kazrin C forms condensates that entrap EE in the vicinity of the centrosome, in a microtubule-dependent manner. Altogether, the data indicates that kazrin C facilitates cargo recycling by trapping EE or EE-derived transport intermediates at the perinuclear region, where transfer of cargo to the RE might occur.

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Exocyst Requirement for Endocytic Traffic Directed Toward the Apical and Basolateral Poles of Polarized MDCK Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e07-02-0097 ◽

2007 ◽

Vol 18 (10) ◽

pp. 3978-3992 ◽

Cited By ~ 81

Author(s):

Asli Oztan ◽

Mark Silvis ◽

Ora A. Weisz ◽

Neil A. Bradbury ◽

Shu-Chan Hsu ◽

...

Keyword(s):

Mdck Cells ◽

Small Gtpase ◽

Junctional Complex ◽

Recycling Endosomes ◽

Permeabilized Cells ◽

Exocyst Complex ◽

C Terminus ◽

Streptolysin O ◽

Early Endosomes ◽

Endocytic Traffic

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

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Co-translational targeting of transcripts to endosomes

10.1101/2020.07.17.208652 ◽

2020 ◽

Author(s):

Doris Popovic ◽

Wilco Nijenhuis ◽

Lukas C. Kapitein ◽

Lucas Pelkmans

Keyword(s):

Cellular Response ◽

Single Cells ◽

Translational Efficiency ◽

Dependent Manner ◽

Coding Region ◽

Independent Manner ◽

Knock Out ◽

Multiple Transcripts ◽

Extrinsic Cues ◽

Early Endosomes

AbstractAsymmetric localization and translation of mRNAs is used by single cells to sense their environment and integrate extrinsic cues with the appropriate cellular response. Here we investigate the extent to which endosomes impact subcellular patterning of transcripts and provide a platform for localized translation. Using image-based transcriptomics, indirect immunofluorescence, and RNAseq of isolated organelles, we discover mRNAs that associate with early endosomes in a translation-dependent and -independent manner. We explore this in more detail for the mRNA of a major endosomal tethering factor and fusogen, Early Endosomal Antigen 1, EEA1, which localizes to early endosomes in a puromycin-sensitive manner. By reconstituting EEA1 knock-out cells with either the coding sequence or 3’UTR of EEA1, we show that the coding region is sufficient for endosomal localization of mRNA. Finally, we use quantitative proteomics to discover proteins associated with EEA1 mRNA and identify CSRP1 as a factor that controls EEA1 translational efficiency. Our findings reveal that multiple transcripts associate with early endosomes in a translation-dependent manner and identify mRNA-binding proteins that may participate in controlling endosome-localized translation.

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Convergence of Non-clathrin- and Clathrin-derived Endosomes Involves Arf6 Inactivation and Changes in Phosphoinositides

Molecular Biology of the Cell ◽

10.1091/mbc.02-04-0053 ◽

2003 ◽

Vol 14 (2) ◽

pp. 417-431 ◽

Cited By ~ 195

Author(s):

Naava Naslavsky ◽

Roberto Weigert ◽

Julie G. Donaldson

Keyword(s):

Major Histocompatibility Complex ◽

Kinase Inhibitor ◽

Interleukin 2 ◽

Dependent Manner ◽

Complex Class ◽

Α Subunit ◽

Recycling Endosomes ◽

Histocompatibility Complex ◽

Late Endosomes ◽

Early Endosomes

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor α subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the “classical” early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP2-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.

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TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0604 ◽

2014 ◽

Vol 25 (23) ◽

pp. 3779-3797 ◽

Cited By ~ 20

Author(s):

Luciana I. Gallo ◽

Yong Liao ◽

Wily G. Ruiz ◽

Dennis R. Clayton ◽

Min Li ◽

...

Keyword(s):

Mdck Cells ◽

Growth Factor Receptor ◽

Dependent Manner ◽

Limited Information ◽

Nucleotide Exchange ◽

Recycling Endosomes ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Gtpase Activating Proteins ◽

Early Endosomes

Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the guanine nucleotide-exchange factors and GTPase-activating proteins (GAPs) that regulate its GTP-GDP cycle. We observed that in the presence of Mg2+(2.5 mM), TBC1D9B interacted via its Tre2-Bub2-Cdc16 (TBC) domain with Rab11a, Rab11b, and Rab4a in a nucleotide-dependent manner. However, only Rab11a was a substrate for TBC1D9B-stimulated GTP hydrolysis. At limiting Mg2+concentrations (<0.5 mM), Rab8a was an additional substrate for this GAP. In polarized Madin–Darby canine kidney cells, endogenous TBC1D9B colocalized with Rab11a-positive recycling endosomes but less so with EEA1-positive early endosomes, transferrin-positive recycling endosomes, or late endosomes. Overexpression of TBC1D9B, but not an inactive mutant, decreased the rate of basolateral-to-apical IgA transcytosis—a Rab11a-dependent pathway—and shRNA-mediated depletion of TBC1D9B increased the rate of this process. In contrast, TBC1D9B had no effect on two Rab11a-independent pathways—basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor. Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A. We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

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JRAB/MICAL-L2 undergoes liquid–liquid phase separation to form tubular recycling endosomes

Communications Biology ◽

10.1038/s42003-021-02080-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ayuko Sakane ◽

Taka-aki Yano ◽

Takayuki Uchihashi ◽

Kazuki Horikawa ◽

Yusuke Hara ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Closed Form ◽

Liquid Phase Separation ◽

Cellular Functions ◽

Recycling Endosomes ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Multiple Molecules ◽

Liquid Liquid Phase Separation

AbstractElongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid–liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

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Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles

Molecular Biology of the Cell ◽

10.1091/mbc.01-07-0380 ◽

2002 ◽

Vol 13 (1) ◽

pp. 169-182 ◽

Cited By ~ 152

Author(s):

Ellen M. van Dam ◽

Willem Stoorvogel

Keyword(s):

Plasma Membrane ◽

Brefeldin A ◽

Fluid Phase ◽

Coated Vesicles ◽

Temperature Sensitive ◽

Dependent Manner ◽

Coated Pits ◽

Recycling Endosomes ◽

Early Endosomes ◽

Golgi Network

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1G273D) or, as a control, dynamin-1 wild type. In dynamin-1G273D–expressing cells, 29–36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1G273D associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1G273D–expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.

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Tensin3 interaction with talin drives formation of fibronectin-associated fibrillar adhesions

10.1101/2021.07.16.452612 ◽

2021 ◽

Author(s):

Paul Atherton ◽

Rafaella Konstantinou ◽

Suat Peng Neo ◽

Emily Wang ◽

Eleonora Balloi ◽

...

Keyword(s):

Mass Spectrometry ◽

Extracellular Matrix ◽

Healthy Tissue ◽

Mitochondrial Targeting ◽

Knock Out ◽

Intrinsically Disordered ◽

Adhesion Sites ◽

Intrinsically Disordered Region ◽

Underlying Mechanisms ◽

Α5β1 Integrin

The formation of healthy tissue involves continuous remodelling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here we examine how tensin3 contributes to formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for formation of α5β1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.

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Association/dissociation Mechanisms of Intrinsically Disordered Region of Protein Beyond Conformational Selection and Induced Fit

10.26434/chemrxiv.9874379 ◽

2019 ◽

Author(s):

Duy Phuoc Tran ◽

Akio Kitao

Keyword(s):

Free Energy ◽

Transcriptional Activation ◽

Binding Free Energy ◽

Energy Structure ◽

Induced Fit ◽

Hydrogen Bond Formation ◽

Conformational Selection ◽

Intrinsically Disordered ◽

Intrinsically Disordered Region ◽

Dissociation Mechanisms

<p>We investigate association and dissociation mechanisms of a typical intrinsically disordered region (IDR), transcriptional activation subdomain of tumor repressor protein p53 (TAD-p53) with murine double-minute clone 2 protein (MDM2). Using the combination of cycles of association and dissociation parallel cascade molecular dynamics, multiple standard MD, and Markov state model, we are successful in obtaining the lowest free energy structure of MDM2/TAD-p53 complex as the structure very close to that in crystal without prior knowledge. This method also reproduces the experimentally measured standard binding free energy, and association and dissociation rate constants solely with the accumulated MD simulation cost of 11.675 μs, in spite of the fact that actual dissociation occurs in the order of a second. Although there exist a few complex intermediates with similar free energies, TAD-p53 first binds MDM2 as the second lowest free energy intermediate dominantly (> 90% in flux), taking a form similar to one of the intermediate structures in its monomeric state. The mechanism of this step has a feature of conformational selection. In the second step, dehydration of the interface, formation of π-π stackings of the side-chains, and main-chain relaxation/hydrogen bond formation to complete α-helix take place, showing features of induced fit. In addition, dehydration (dewetting) is a key process for the final relaxation around the complex interface. These results demonstrate a more fine-grained view of the IDR association/dissociation beyond classical views of protein conformational change upon binding.</p>

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