Endocytosis In Cells Research Articles

Branched actin networks are organized for asymmetric force production during clathrin-mediated endocytosis in mammalian cells

Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood. Here, branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches. When endocytic dynamics of unperturbed CME sites are compared, sites with actin assembly show a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. In addition, N-WASP and the Arp2/3 complex are recruited to one side of CME sites, where they are positioned to stimulate asymmetric actin assembly and force production. We propose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetrically, much as a bottle opener pulls off a bottle cap.

Sequential compound fusion and kiss-and-run mediate exo- and endocytosis in excitable cells.

Vesicle fusion at preestablished plasma membrane release sites releases transmitters and hormones to mediate fundamental functions like neuronal network activities and fight-or-flight responses. This half-a-century-old concept—fusion at well-established release sites in excitable cells—needs to be modified to include the sequential compound fusion reported here—vesicle fusion at previously fused Ω-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we real-time visualized sequential compound fusion pore openings and content releases in generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic ranges in excitable cells. We also visualized subsequent compound fusion pore closure, a new mode of endocytosis termed compound kiss-and-run that enhances vesicle recycling capacity. These results suggest modifying current exo-endocytosis concepts by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion and kiss-and-run take place to enhance exo-endocytosis capacity and dynamic ranges.

A mathematical model of in vitro hepatocellular cholesterol and lipoprotein metabolism for hyperlipidemia therapy.

Cardiovascular diseases associated with high cholesterol (hypercholesterolemia) and low-density lipoproteins (LDL) levels are significant contributors to total mortality in developing and developed countries. Mathematical modeling of LDL metabolism is an important step in the development of drugs for hypercholesterolemia. The aim of this work was to develop and to analyze an integrated mathematical model of cholesterol metabolism in liver cells and its interaction with two types of drugs, statins and PCSK9 inhibitors. The model consisted of 21 ordinary differential equations (ODE) describing cholesterol biosynthesis and lipoprotein endocytosis in liver cells in vitro. The model was tested for its ability to mimic known biochemical effects of familial hypercholesterolemia, statin therapy, and PCSK9 inhibitors. The model qualitatively reproduced the well-known biology of cholesterol regulation, which confirms its potential for minimizing cellular research in initial testing of new drugs for cardiology.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

The morphology of hydroxyapatite nanoparticles regulates clathrin-mediated endocytosis in melanoma cells and resultant anti-tumor efficiency

The morphology of hydroxyapatite nanoparticles regulates clathrin-mediated endocytosis in melanoma cells and resultant anti-tumor efficiency

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

Targeting AKT and CK2 represents a novel therapeutic strategy for SMO constitutive activation-driven medulloblastoma.

AimsSonic hedgehog subtype medulloblastoma is featured with overactivation of hedgehog pathway and can be targeted by SMO‐specific inhibitors. However, the resistance is frequently developed leading to treatment failure of SMO inhibitors. W535L mutation of SMO (SMOW535L) is thought to be an oncogenic driver for Sonic hedgehog subtype MB and confer resistance to SMO inhibitors. The regulation network of SMOW535L remains to be explored in comparison with wild‐type SMO (SMOWT).MethodsIn this study, we profiled transcriptomes, methylomes, and interactomes of MB cells expression SMOWT or SMOW535L in the treatment of DMSO or SMO inhibitor, respectively.ResultsAnalysis of transcriptomic data indicated that SMO inhibitor disrupted processes of endocytosis and cilium organization in MB cells with SMOWT, which are necessary for SMO activation. In MB cells with SMOW535L, however, SMO inhibitor did not affect the two processes‐related genes, implying resistance of SMOW535L toward SMO inhibitor. Moreover, we noticed that SMO inhibitor significantly inhibited metabolism‐related pathways. Our metabolic analysis indicated that nicotinate and nicotinamide metabolism, glycerolipid metabolism, beta‐alanine metabolism, and synthesis and degradation of ketone bodies might be involved in SMOW535L function maintenance. Interactomic analysis revealed casein kinase II (CK2) as an important SMO‐associated protein. Finally, we linked CK2 and AKT together and found combination of inhibitors targeting CK2 and AKT showed synergetic effects to inhibit the growth of MB cells with SMO constitutive activation mutation.ConclusionsTaken together, our work described SMO‐related transcriptomes, metabolomes, and interactomes under different SMO status and treatment conditions, identifying CK2 and AKT as therapeutic targets for SHH‐subtype MB cells with SMO inhibitor resistance.

Fusion-inhibition peptide broadly inhibits influenza virus and SARS-CoV-2, including Delta and Omicron variants

ABSTRACT Pandemic influenza virus and SARS-CoV-2 vaiants have posed major global threats to public health. Broad-spectrum antivirals blocking viral entry can be an effective strategy for combating these viruses. Here, we demonstrate a frog-defensin-derived basic peptide (FBP), which broadly inhibits the influenza virus by binding to haemagglutinin so as to block low pH-induced HA-mediated fusion and antagonizes endosomal acidification to inhibit the influenza virus. Moreover, FBP can bind to the SARS-CoV-2 spike to block spike-mediated cell–cell fusion in 293T/ACE2 cells endocytosis. Omicron spike shows a weak cell–cell fusion mediated by TMPRSS2 in Calu3 cells, making the Omicron variant sensitive to endosomal inhibitors. In vivo studies show that FBP broadly inhibits the A(H1N1)pdm09 virus in mice and SARS-CoV-2 (HKU001a and Delta)in hamsters. Notably, FBP shows significant inhibition of Omicron variant replication even though it has a high number of mutations in spike. In conclusion, these results suggest that virus-targeting FBP with a high barrier to drug resistance can be an effective entry-fusion inhibitor against influenza virus and SARS-CoV-2 in vivo.

On-demand assembly of polymeric nanoparticles for longer-blood-circulation and disassembly in tumor for boosting sonodynamic therapy

Sonodynamic therapy (SDT) is one of the promising strategies for tumor therapy, but its application is usually hindered by fast clearance in blood-circulation, abnormal tumor microenvironment, and inefficient generation of reactive oxygen species. To solve these problems, we proposed an on-demand assembly-disassembly strategy, where the assembly is favorable for longer-blood-circulation and then the disassembly in tumor is favorable for boosting SDT. Hematoporphyrin monomethyl ether (HMME) as the model of organic sonosensitizers were conjugated with hyaluronic acid (HA). Then HA-HMME was mixed with catalase (CAT) and assembled into polymeric nanoparticles (CAT@HA-HMME NPs) with size of ∼80 nm. CAT@HA-HMME NPs exhibit good biocompatibility and a longer blood half-time (t1/2 = 4.17 h) which is obviously longer than that (∼0.82 h) of HMME molecules. After HA receptor-mediated endocytosis of cancer cells, CAT@HA-HMME NPs can be cleaved by endogenous hyaluronidase, resulting in the on-demand disassembly in tumor to release HA-HMME molecules and CAT. The CAT catalyzes the endogenous H2O2 into O2 to relieve the hypoxic microenvironment, and the released HA-HMME exhibits a higher ROS generation ability, greatly boosting SDT for the inhibition of tumor growth. Therefore, the on-demand assembly-disassembly strategy may provide some insight in the design and development of nanoagents for tumor therapy.

DT7 peptide-modified lecithin nanoparticles co-loaded with γ-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemia and reduce gastrointestinal toxicity

T-cell acute lymphoblastic leukemia (T-ALL) is a serious hematologic malignancy and glucocorticoid resistance is the main recurrent cause for a high relapsed and death rate. Here, we proposed an effective therapeutic regimen of combining gamma-secretase inhibitors (GSIs) with dexamethasone (DEX) to overcome glucocorticoid resistance. Moreover, the bone marrow targeting DT7 peptide-modified lecithin nanoparticles co-loaded with DEX and GSI (TLnp/D&G) were developed to enhance T-ALL cells recognition and endocytosis. In vitro cytotoxicity studies showed that TLnp/D&G significantly inhibited cell survival and promoted apoptosis of T-ALL cells. Mechanically, we found that GSIs promoted DEX-induced cell apoptosis by two main synergetic mechanisms: 1) GSIs significantly upregulated glucocorticoid receptor (GR) expression in T-ALL and restored the glucocorticoid-induced pro-apoptotic response. 2) Both DEX and GSI synergistically inhibited BCL2 and suppressed the survival of T-ALL cells. Furthermore, in vivo studies demonstrated that TLnp/D&G showed high bone marrow accumulation and better antileukemic efficacy both in leukemia bearing models and in systemic Notch1-induced T-ALL models, with excellent biosafety and reduced gastrointestinal toxicity. Overall, our study provides new strategies for the treatment of T-ALL and promising bone marrow targeting systems with high transformation potential.

Flurbiprofen inhibits cell proliferation in thyroid cancer through interrupting HIP1R-induced endocytosis of PTEN

BackgroundThe incidence of thyroid cancer, a most common tumor in the endocrine system, has increased in recent years. A growing number of studies have focused on the molecular mechanisms of thyroid cancer subtypes, aiming to identify effective therapeutic targets. Endocytosis is of vital significance in the malignant development of tumors, although its involvement in thyroid cancer has been rarely reported.MethodsHIP1R expressions in thyroid cancer from the TCGA database were analyzed by UALCAN software. Thyroid epithelial and cancer cell lines were cultured in vitro. Western blotting and quantitative PCR were used to analyze protein and mRNA levels, respectively. Cell viability was measured by CCK-8 assay. Immunofluorescence staining indicated protein distribution in cell. Co-immunoprecipitation was used to study protein–protein interaction. Immunohistochemical staining was used to analyze protein expression in clinical tissues. Differences between groups were compared using the two-tailed Student’s t test, and those among three or more groups were compared by one-way or two-way ANOVA.ResultsIn the present study, HIP1R (Huntingtin Interacting Protein 1 Related) was found upregulated in thyroid cancer tissues and cell lines compared with that in the controls, while knockdown of HIP1R significantly inhibited the proliferation of thyroid cancer cells. Since HIP1R is essential for the clathrin-dependent endocytic process, we thereafter explored the effect of HIP1R on the endocytosis of thyroid cancer cells. Interestingly, knockdown of HIP1R significantly reduced the number of clathrin-coated pits (CCPs) in thyroid cancer cells. In addition, the interaction between HIP1R and PTEN (phosphatase and tensin homolog) was identified in thyroid cancer cells. Knockdown of HIP1R downregulated intracellular PTEN in thyroid cancer cells, but upregulated membrane-binding PTEN. Notably, flurbiprofen, a commonly used analgesic, significantly inhibited the proliferation of thyroid cancer cells and interfered with the interaction between HIP1R and PTEN, thereby enhancing the binding of PTEN to cell membrane. However, the proliferation inhibitory effect of flurbiprofen was attenuated when knocking down HIP1R or PTEN.ConclusionsUpregulated HIP1R in thyroid cancer cells promotes cell proliferation and mediates the endocytosis of PTEN. Flurbiprofen may exert an anti-tumor effect on thyroid cancer by blocking the interaction between HIP1R and PTEN.

Dual-responsive drug release and fluorescence imaging based on disulfide-pillar[4]arene aggregate in cancer cells

The construction of multistimuli-responsive nanoaggregate has become one of the increasingly significant research topics in supramolecular chemistry. We herein reported the pH- and glutathione dual-responsive supramolecular assemblies fabricated by the disulfide-containing pillar[4]arene and tetraphenylethylene derivatives possessing different alkyl chains in length. Morphological characterization experiments showed the binary supramolecular assemblies formed well-defined nanoparticles, which could facilitate their endocytosis in cells. More remarkably, due to the compact nanostructures and the existence of acidifiable carboxyl group and bioreducible disulfide linkage in pillar[4]arene, the obtained nanoaggregates presented high drug-loading efficiency and sustained drug release behaviors, as well as the targeted fluorescence imaging ability in cancer cells. Thus, it can be envisioned that such microenvironment-adaptable supramolecular nanoassemblies featuring dual stimuli-responsiveness and fluorescence-imaging abilities may be developed as more appealing nanosystems for the therapy of refractory disease.

Medicated Scaffolds Prepared with Hydroxyapatite/Streptomycin Nanoparticles Encapsulated into Polylactide Microfibers.

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanoparticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaffolds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concentrations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles

SummaryClathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.

Exosome-based rare earth nanoparticles for targeted in situ and metastatic tumor imaging with chemo-assisted immunotherapy.

In this research, a tumor exosome system DOX/2DG@E-RENPs with good biocompatibility, low immunogenicity, and a high targeting effect was proposed for theranostics with high chemo-/starvation/immunotherapy efficiency. DOX and 2-deoxy-D-glucose (DOX/2DG) together with rare earth nanoparticles (RENPs) can be simultaneously carried on the exosome by endocytosis of tumor cells and then exocytosis in vitro. This platform has a good monodispersity with an average size of 70 nm, and the system can emit upconversion luminescence and NIR II luminescence under a single NIR laser. In particular, this exosome can target homing cancer cells and kill the origin tumor cells. The strong targeting effect was proved by different cell lines with exosomes from different orthogonal cells (normal/cancer cells and human/mouse sources, respectively), and the in vivo NIR II imaging guided targeted cancer imaging and liver metastases can be realized by intravenous injection of E-RENPs. Furthermore, the good targeted therapeutic effect and in vivo NIR II imaging and metastases of this platform can be proved. The chemotherapy, starvation therapy, and immunotherapy (immune checkpoint inhibitors of an anti-PD-L1 antibody) could achieve effective synergistic therapy for lung adenocarcinoma, and the immunotherapy can be further proved by the clinical data. This will provide a new strategy for the precise targeting and treatment of tumors.

Phospholipase D1-generated phosphatidic acid modulates secretory granule trafficking from biogenesis to compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis is a multi-step process that allows specialized secretory cells to release informative molecules such as neurotransmitters, neuropeptides, and hormones for intercellular communication. The biogenesis of secretory vesicles from the Golgi cisternae is followed by their transport towards the cell periphery and their docking and fusion to the exocytic sites of the plasma membrane allowing release of vesicular content. Subsequent compensatory endocytosis of the protein and lipidic constituents of the vesicles maintains cell homeostasis. Despite the fact that lipids represent the majority of membrane constituents, little is known about their contribution to these processes. Using a combination of electrochemical measurement of single chromaffin cell catecholamine secretion and electron microscopy of roof-top membrane sheets associated with genetic, silencing and pharmacological approaches, we recently reported that diverse phosphatidic acid (PA) species regulates catecholamine release efficiency by controlling granule docking and fusion kinetics. The enzyme phospholipase D1 (PLD1), producing PA from phosphatidylcholine, seems to be the major responsible of these effects in this model. Here, we extended this work using spinning disk confocal microscopy showing that inhibition of PLD activity also reduced the velocity of granules undergoing a directed motion. Furthermore, a dopamine β-hydroxylase (DβH) internalization assay revealed that PA produced by PLD is required for an optimal recovery of vesicular membrane content by compensatory endocytosis. Thus, among numerous roles that have been attributed to PA our work gives core to the key regulatory role in secretion that has been proposed in different cell models. Few leads to explain these multiple functions of PA along the secretory pathway are discussed.

Evaluation of inhibitor activity of bacterial sialidase from Clostridium perfringens against Newcastle disease virus in the cell culture model using chicken embryo fibroblast.

Objective:The Newcastle disease virus (NDV) is an infectious disease that causes very high economic losses due to decreased livestock production and poultry deaths. The vaccine’s ineffectiveness due to mutation of the genetic structure of the virus impacts obstacles in controlling the disease, especially in some endemic areas. This study aimed to provide an alternative treatment for NDV infection by observing the viral replication inhibitor activity of Clostridium perfringens sialidase in primary chicken embryo fibroblast (CEF) cells.Materials and Methods:The virus was adapted in CEF monolayer cells, then collected thrice using the freeze–thaw method and stored at −20°C for the next step in the challenge procedure. C. perfringens crude sialidase was obtained, but it was further purified via stepwise elution in ion exchange using Q Sepharose® Fast Flow and affinity chromatography with oxamic acid agarose. The purified sialidase was tested for its toxicity, ability to breakdown sialic acid, stopping viral replication, and how treated cells expressed their genes.Results:According to this study, purified C. perfringens sialidase at dosages of 187.5, 93.75, and 46.87 mU effectively hydrolyzes CEF cells’ sialic acid and significantly inhibits viral replication on the treated cells. However, sialidase dosages of 375 and 750 mU affected the viability of monolayer CEF cells. Interestingly, downregulation of toll-like receptor (TLR)3 and TLR7 (p < 0.05) in the sialidase-treated group indicates viral endocytosis failure.Conclusions:By stopping endocytosis and viral replication in host cells, sialidase from C. perfringens can be used as an alternative preventive treatment for NDV infection.

Using BODIPY FL-Sphingolipid Analogs to Study Sphingolipid Metabolism in Mouse Embryonic Stem Cells.

Sphingolipids are important structural components of cellular membranes. They also function as prominent signaling molecules to control a variety of cellular events, such as cell growth, differentiation, and apoptosis. Impaired sphingolipid metabolism, particularly defects in sphingolipid degradation, has been associated with many human diseases. Fluorescence sphingolipid analogs have been widely used as efficient probes to study sphingolipid metabolism and intracellular trafficking in living mammalian cells. Compared with nitrobenzoxadiazole fluorophores (NBD FL), the boron dipyrromethene difluoride fluorophores (BODIPY FL) have much higher absorptivity and fluorescence quantum. These features allow more intensive labeling of cells for fluorescence microscopy imaging and flow cytometry analysis. Here, we describe a protocol employing BODIPY FL-labeled sphingolipid analogs to elucidate sphingolipid internalization, trafficking, and endocytosis in mouse embryonic stem cells. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.67452 Graphical abstract.

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

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found 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, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (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.