RESUMEN
Human papillomavirus (HPV) infects epithelial basal cells in the mucosa and either proliferates with the differentiation of the basal cells or persists in them. Multiple host factors are required to support the HPV life cycle; however, the molecular mechanisms involved in cell entry are not yet fully understood. In this study, we performed a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) knockout (KO) screen in HeLa cells and identified folliculin (FLCN), a GTPase-activating protein for Rag GTPases, as an important host factor for HPV infection. The introduction of single guide RNAs for the FLCN gene into HeLa, HaCaT, and ectocervical Ect1 cells reduced infection by HPV18 pseudovirions (18PsVs) and 16PsVs. FLCN KO HeLa cells also exhibited strong resistance to infection with 18PsVs and 16PsVs; nevertheless, they remained highly susceptible to infections with vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus. Immunofluorescence microscopy revealed that the numbers of virions binding to the cell surface were slightly increased in FLCN KO cells. However, virion internalization analysis showed that the internalized virions were rapidly degraded in FLCN KO cells. This degradation was blocked by treatment with the lysosome inhibitor bafilomycin A1. Furthermore, the virion degradation phenotype was also observed in Ras-related GTP-binding protein C (RagC) KO cells. These results suggest that FLCN prevents the lysosomal degradation of incoming HPV virions by enhancing lysosomal RagC activity. IMPORTANCE Cell entry by human papillomavirus (HPV) involves a cellular retrograde transport pathway from the endosome to the trans-Golgi network/Golgi apparatus. However, the mechanism by which this viral trafficking is safeguarded is poorly understood. This is the first study showing that the GTPase-activating protein folliculin (FLCN) protects incoming HPV virions from lysosomal degradation and supports infectious cell entry by activating the Rag GTPases, presumably through the suppression of excessive lysosomal biosynthesis. These findings provide new insights into the effects of small GTPase activity regulation on HPV cell entry and enhance our understanding of the HPV degradation pathway.
Asunto(s)
Virus del Papiloma Humano , Infecciones por Papillomavirus , Proteínas Proto-Oncogénicas , Proteínas Supresoras de Tumor , Internalización del Virus , Humanos , Proteínas Activadoras de GTPasa , Células HeLa , Virus del Papiloma Humano/fisiología , Lisosomas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Sphingolipids are ubiquitously expressed in eukaryotes and play various functional roles. The key characteristic of sphingolipids is their diversity of molecular species. Sphingomyelin (SM) and glycosphingolipids (GSLs) are the major components of sphingolipids in the plasma membrane, which are composed of ceramide and a polar head-group. SM is the most abundant sphingolipid species in mammalian cells, while GSLs have a wide variety of glycans as head groups. Various fatty acids in ceramide also contribute to the diversity of sphingolipid species. To analyze the cellular function of each sphingolipid species, precise gene manipulation is essential. Recent developments in genome editing technologies have facilitated complete gene disruption in cultured cells. This chapter describes protocols for the construction of various sphingolipid-related gene knockout HeLa cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and for confirmation of changes in their lipid composition using radioisotopes and thin layer chromatography. This sphingolipid-remodeled cell panel is a useful tool for analyzing the cellular functions of sphingolipid species and as a reference for lipid analysis.
Asunto(s)
Edición Génica , Esfingolípidos , Animales , Humanos , Esfingolípidos/metabolismo , Edición Génica/métodos , Células HeLa , Ceramidas/metabolismo , Esfingomielinas/metabolismo , Glicoesfingolípidos , Sistemas CRISPR-Cas , Mamíferos/metabolismoRESUMEN
We have previously shown that Rab34 is an important regulator of ciliogenesis and that its unique long N-terminal region (amino acids 1-49) is essential for ciliogenesis in certain cultured mammalian cells. In the present study, we performed an in-depth deletion analysis of the N-terminal region of Rab34 together with Ala-based site-directed mutagenesis to identify the essential amino acids that are required for serum-starvation-induced ciliogenesis in hTERT-RPE1 cells. The results showed that a Rab34 mutant lacking an N-terminal 18 amino acids and a Rab34 mutant carrying an LPQ-to-AAA mutation (amino acids 16-18) failed to rescue a Rab34-KO phenotype (i.e., defect in ciliogenesis). Our findings suggest that the LPQ sequence of Rab34 is crucial for ciliogenesis in hTERT-RPE1 cells.Abbreviations: AA, amino acid(s); ac-Tub, acetylated tubulin; bsr, blasticidin S-resistant gene; HRP, horseradish peroxidase; hTERT-RPE1, human telomerase reverse transcriptase retinal pigment epithelium 1; KO, knockout; NS, not significant; PBS, phosphate-buffered saline; puro, puromycin-resistant gene.
Asunto(s)
Cilios , Dipéptidos , Aminoácidos/metabolismo , Animales , Línea Celular , Cilios/metabolismo , Dipéptidos/metabolismo , MamíferosRESUMEN
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 18â h and 48â 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.
Asunto(s)
Endocitosis , Ácidos Indolacéticos , Animales , Endocitosis/genética , Endosomas/genética , Endosomas/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Endocytosis and endosome dynamics are controlled by proteins of the small GTPase Rab family. Besides possible recycling routes to the plasma membrane and various organelles, previously described endocytic pathways (e.g., clathrin-mediated endocytosis, macropinocytosis, CLIC/GEEC pathway) all appear to funnel the endocytosed material to Rab5-positive early endosomes that then mature into Rab7-positive late endosomes/lysosomes. By studying the uptake of a series of cell-penetrating peptides (CPPs), we identify an endocytic pathway that moves material to nonacidic Lamp1-positive late endosomes. Trafficking via this endocytic route is fully independent of Rab5 and Rab7 but requires the Rab14 protein. The pathway taken by CPPs differs from the conventional Rab5-dependent endocytosis at the stage of vesicle formation already, as it is not affected by a series of compounds that inhibit macropinocytosis or clathrin-mediated endocytosis. The Rab14-dependent pathway is also used by physiological cationic molecules such as polyamines and homeodomains found in homeoproteins.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Proteínas de Homeodominio/metabolismo , Poliaminas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7/metabolismo , Cationes , Endosomas/genética , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/genética , Lisosomas/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión a GTP rab7/genéticaRESUMEN
The Rab family small GTPases are key regulators of intracellular membrane traffic that are conserved in all eukaryotic cells. Rabs are thought to regulate various steps of membrane traffic, including the budding, transport, tethering, docking, and fusion of vesicles or organelles. Approximately 60 different Rabs have been identified in mammals, and each Rab is thought to localize to a specific membrane compartment and regulate its trafficking in a timely manner. Although a few mammalian Rabs have been thoroughly studied, the precise function of the majority of them remains poorly understood. In a recent study, we established a comprehensive collection of Rab-knockout (KO) renal epithelial cells (i.e., Madin-Darby canine kidney [MDCK] II cells) by using Cas9-mediated genome editing technology to analyze the function of each Rab or closely related Rabs in cell viability (or growth), organelle morphology, and epithelial morphogenesis. In this chapter, we describe the procedures for generating Rab-KO MDCK II cells in detail.
Asunto(s)
Proteínas de Unión al GTP rab/metabolismo , Animales , Perros , Membranas Intracelulares/metabolismo , Células de Riñón Canino Madin Darby , Orgánulos/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab/genéticaRESUMEN
Rab small GTPases regulate intracellular membrane trafficking by interacting with specific binding proteins called Rab effectors. Although Rab6 is implicated in basement membrane formation and secretory cargo trafficking, its precise regulatory mechanisms have remained largely unknown. In the present study we established five knockout cell lines for candidate Rab6 effectors and discovered that knockout of VPS52, a subunit of the GARP complex, resulted in attenuated secretion and lysosomal accumulation of secretory cargos, the same as Rab6-knockout does. We also evaluated the functional importance of the previously uncharacterized C-terminal region of VPS52 for restoring these phenotypes, as well as for the sorting of lysosomal proteins. Our findings suggest that VPS52 is an effector protein that is responsible for the Rab6-dependent secretory cargo trafficking.
Asunto(s)
Técnicas de Silenciamiento del Gen/métodos , Lisosomas/metabolismo , Vías Secretoras/fisiología , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Perros , Aparato de Golgi , Humanos , Membranas Intracelulares , Transporte de Proteínas , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Proteínas de Transporte Vesicular/genéticaRESUMEN
The primary cilium is an essential organizing center for signal transduction, and ciliary defects cause congenital disorders known collectively as ciliopathies.1-3 Primary cilia form by two pathways that are employed in a cell-type- and tissue-specific manner: an extracellular pathway in which the cilium grows out from the cell surface and an intracellular pathway in which the nascent cilium first forms inside the cell.4-8 After exposure to the external environment, cilia formed via the intracellular pathway may have distinct functional properties, as they often remain recessed within a ciliary pocket.9,10 However, the precise mechanism of intracellular ciliogenesis and its relatedness to extracellular ciliogenesis remain poorly understood. Here we show that Rab34, a poorly characterized GTPase recently linked to cilia,11-13 is a selective mediator of intracellular ciliogenesis. We find that Rab34 is required for formation of the ciliary vesicle at the mother centriole and that Rab34 marks the ciliary sheath, a unique sub-domain of assembling intracellular cilia. Rab34 activity is modulated by divergent residues within its GTPase domain, and ciliogenesis requires GTP binding and turnover by Rab34. Because Rab34 is found on assembly intermediates that are unique to intracellular ciliogenesis, we tested its role in the extracellular pathway used by polarized MDCK cells. Consistent with Rab34 acting specifically in the intracellular pathway, MDCK cells ciliate independently of Rab34 and its paralog Rab36. Together, these findings establish that different modes of ciliogenesis have distinct molecular requirements and reveal Rab34 as a new GTPase mediator of ciliary membrane biogenesis.
Asunto(s)
Membrana Celular/metabolismo , Cilios/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Línea Celular , Centriolos/metabolismo , Perros , Humanos , Hidrólisis , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rab/genéticaRESUMEN
The small GTPase Rab11 (herein referring to the Rab11A and Rab11B isoforms) plays pivotal roles in diverse physiological phenomena, including the recycling of membrane proteins, cytokinesis, neurite outgrowth and epithelial morphogenesis. One effective method of analyzing the function of endogenous Rab11 is to overexpress a Rab11-binding domain from one of its effectors, for example, the C-terminal domain of Rab11-FIP2 (Rab11-FIP2-C), as a dominant-negative construct. However, the drawback of this method is the broader Rab-binding specificity of the effector domain, because Rab11-FIP2-C binds to Rabs other than Rab11, for example, to Rab14 and Rab25. In this study, we bioengineered an artificial Rab11-specific binding domain, named RBD11. Expression of RBD11 allowed visualization of endogenous Rab11 without affecting its localization or function, whereas expression of a tandem RBD11, named 2×RBD11, inhibited epithelial morphogenesis and induced a multi-lumen phenotype characteristic of Rab11-deficient cysts. We also developed two tools for temporally and reversibly analyzing Rab11-dependent membrane trafficking - tetracycline-inducible 2×RBD11 and an artificially oligomerized domain (FM)-tagged RBD11.
Asunto(s)
Proteínas de la Membrana , Proteínas de Unión al GTP rab , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The acquisition of neuronal polarity is a complex molecular process that depends on changes in cytoskeletal dynamics and directed membrane traffic, regulated by the Rho and Rab families of small GTPases, respectively. However, during axon specification, a molecular link that couples these protein families has yet to be identified. In this paper, we describe a new positive feedback loop between Rab8a and Cdc42, coupled by Tuba, a Cdc42-specific guanine nucleotide-exchange factor (GEF), that ensures a single axon generation in rodent hippocampal neurons from embryos of either sex. Accordingly, Rab8a or Tuba gain-of-function generates neurons with supernumerary axons whereas Rab8a or Tuba loss-of-function abrogated axon specification, phenocopying the well-established effect of Cdc42 on neuronal polarity. Although Rab8 and Tuba do not interact physically, the activity of Rab8 is essential to generate a proximal to distal axonal gradient of Tuba in cultured neurons. Tuba-associated and Rab8a-associated polarity defects are also evidenced in vivo, since dominant negative (DN) Rab8a or Tuba knock-down impairs cortical neuronal migration in mice. Our results suggest that Tuba coordinates directed vesicular traffic and cytoskeleton dynamics during neuronal polarization.SIGNIFICANCE STATEMENT The morphologic, biochemical, and functional differences observed between axon and dendrites, require dramatic structural changes. The extension of an axon that is 1 µm in diameter and grows at rates of up to 500 µm/d, demands the confluence of two cellular processes: directed membrane traffic and fine-tuned cytoskeletal dynamics. In this study, we show that both processes are integrated in a positive feedback loop, mediated by the guanine nucleotide-exchange factor (GEF) Tuba. Tuba connects the activities of the Rab GTPase Rab8a and the Rho GTPase Cdc42, ensuring the generation of a single axon in cultured hippocampal neurons and controlling the migration of cortical neurons in the developing brain. Finally, we provide compelling evidence that Tuba is the GEF that mediates Cdc42 activation during the development of neuronal polarity.
Asunto(s)
Polaridad Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Neurogénesis/fisiología , Neuronas/citología , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Células COS , Movimiento Celular/fisiología , Chlorocebus aethiops , Retroalimentación Fisiológica/fisiología , Femenino , Hipocampo/embriología , Masculino , Ratones , Transporte de Proteínas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.
Asunto(s)
Proteínas Activadoras de GTPasa/genética , Factores de Intercambio de Guanina Nucleótido/genética , Orgánulos/metabolismo , Procesamiento Proteico-Postraduccional , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/genética , Animales , Transporte Biológico , Células Eucariotas/citología , Células Eucariotas/metabolismo , Proteínas Activadoras de GTPasa/clasificación , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/clasificación , Factores de Intercambio de Guanina Nucleótido/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Orgánulos/química , Fosforilación , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Terminología como Asunto , Vesículas Transportadoras/química , Proteínas de Unión al GTP rab/clasificación , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Melanin incorporated into keratinocytes plays an important role in photoprotection; however, abnormal melanin accumulation causes hyperpigmentary disorders. To understand the mechanism behind the accumulation of excess melanin in the skin, it is essential to clarify the spatial distribution of melanosomes or melanin in the epidermis. Although several markers have been used to detect melanosomes or melanin, no suitable markers to determine the precise localization of melanin in the epidermis have been reported. In this study, we showed that melanocore-interacting Kif1c-tail (M-INK), a recently developed fluorescent probe for visualizing mature melanosomes, binds to purified melanin in vitro, and applied it for detecting melanin in human skin tissues. Frozen skin sections from different phototypes were co-stained for the hemagglutinin (HA)-tagged M-INK probe and markers of melanocytes or keratinocytes, and a wide distribution of melanin was observed in the epidermis. Analysis of the different skin phototypes indicated that the fluorescent signals of HA-M-INK correlated well with skin color. The reconstruction of three-dimensional images of epidermal sheets enabled us to observe the spatial distribution of melanin in the epidermis. Thus, the HA-M-INK probe is an ideal tool to individually visualize melanin (or melanosome) distribution in melanocytes and in keratinocytes in skin tissues.
Asunto(s)
Melaninas/metabolismo , Melanosomas/metabolismo , Piel/metabolismo , Adolescente , Adulto , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Células Epidérmicas/metabolismo , Epidermis/metabolismo , Femenino , Humanos , Hiperpigmentación/metabolismo , Queratinocitos/metabolismo , Melanocitos/metabolismo , Persona de Mediana Edad , Pigmentación de la Piel/fisiologíaRESUMEN
Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e. ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g. Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g. Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.
Asunto(s)
Cilios/metabolismo , Células Epiteliales/enzimología , Epitelio Pigmentado de la Retina/enzimología , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Cilios/genética , Humanos , Proteínas Nucleares , Proteínas de Unión al GTP rab/genéticaRESUMEN
Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin-Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain-containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
Asunto(s)
Técnicas de Cultivo de Célula , Células Epiteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Sialoglicoproteínas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Perros , Factores de Intercambio de Guanina Nucleótido/química , Células de Riñón Canino Madin Darby , Modelos Biológicos , Dominios Proteicos , Transporte de ProteínasRESUMEN
Complexation of nickel(II) bromide with tert-butyl 5-phenyl-2-pyridyl nitroxide (phpyNO) gave two morphs of doubly chelated [Ni(phpyNO)2Br2] as a 2p-3d-2p heterospin triad. The α phase crystallizes in the orthorhombic space group Pbcn. An asymmetric unit involves a half-molecule. The torsion angle around Ni-O-N-C2py is as small as 6.5(3)° at 100 K and 7.0(6)° at 400 K, guaranteeing an orthogonal arrangement between the magnetic radical π* and metal 3dx2-y2 and 3dz2 orbitals. Magnetic study revealed the high-spin ground state with the exchange coupling constant 2J/kB = +288(5) K, on the basis of a symmetrical spin Hamiltonian. The ß phase crystallizes in the monoclinic space group P21/n. The whole molecule is an independent unit. The Ni-O-N-C2py torsion angles are 24.2(6) and 37.2(5)° at 100 K and 10.4(7) and 25.9(6)° at 400 K. A magnetic study revealed a very gradual and nonhysteretic spin transition. An analysis based on the van't Hoff equation gave a successful fit with the spin-crossover temperature of 134(1) K, although the susceptibility did not reach the theoretical high-spin value at 400 K. Density functional theory calculation on the ß phase showed ground Stotal = 0 in the low-temperature structure while Stotal = 2 in the high-temperature structure, supporting the synchronized exchange coupling switch on both sides. Consequently, the ß phase can be recognized as an "incomplete spin crossover" material, as a result of conflicting thermal depopulation effects in a high-temperature region.
RESUMEN
The Rab family of small GTPases comprises the largest number of proteins (â¼60 in mammals) among the regulators of intracellular membrane trafficking, but the precise function of many Rabs and the functional redundancy and diversity of Rabs remain largely unknown. Here, we generated a comprehensive collection of knockout (KO) MDCK cells for the entire Rab family. We knocked out closely related paralogs simultaneously (Rab subfamily knockout) to circumvent functional compensation and found that Rab1A/B and Rab5A/B/C are critical for cell survival and/or growth. In addition, we demonstrated that Rab6-KO cells lack the basement membrane, likely because of the inability to secrete extracellular matrix components. Further analysis revealed the general requirement of Rab6 for secretion of soluble cargos. Transport of transmembrane cargos to the plasma membrane was also significantly delayed in Rab6-KO cells, but the phenotype was relatively mild. Our Rab-KO collection, which shares the same background, would be a valuable resource for analyzing a variety of membrane trafficking events.
Asunto(s)
Membrana Celular/metabolismo , Células Epiteliales/citología , Guanosina Trifosfato/metabolismo , Complejos Multiproteicos/metabolismo , Orgánulos/fisiología , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Animales , Perros , Células Epiteliales/metabolismo , Técnicas de Inactivación de Genes/métodos , Células HEK293 , Humanos , Membranas Intracelulares , Células de Riñón Canino Madin Darby , Fenotipo , Transporte de Proteínas , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Macroautophagy (simply called autophagy hereafter) is an intracellular degradation mechanism that is activated by nutrient starvation. Although it is well known that starvation induces autophagosome formation in an mTORC1-dependent manner, whether starvation also regulates autophagosome or autolysosome maturation was unclear. In the present study, we succeeded in demonstrating that starvation activates autolysosome maturation in mammalian cells. We found that knockout (KO) of Rab7 (herein referring to the Rab7a isoform) caused an accumulation of a massive number of LC3-positive autolysosomes under nutrient-rich conditions, indicating that Rab7 is dispensable for autophagosome-lysosome fusion. Intriguingly, the autolysosomes that had accumulated in Rab7-KO cells matured and disappeared after starvation for a brief period (â¼10â min), and we identified glutamine as an essential nutrient for autolysosome maturation. In contrast, forced inactivation of mTORC1 through treatment with its inhibitor Torin2 failed to induce autolysosome maturation, suggesting that the process is controlled by an mTORC1-independent mechanism. Since starvation-induced autolysosome maturation was also observed in wild-type cells, the nutrient-starvation-induced maturation of autolysosomes is likely to be a generalized mechanism in the same manner as starvation-induced autophagosome formation. Such multistep regulatory mechanisms would enable efficient autophagic flux during starvation.
Asunto(s)
Autofagosomas/metabolismo , Autofagia/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas de Unión al GTP rab/genética , Animales , Perros , Técnicas de Inactivación de Genes , Glutamina/metabolismo , Células HeLa , Humanos , Lisosomas/química , Lisosomas/genética , Células de Riñón Canino Madin Darby , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Fusión de Membrana/genética , Naftiridinas/farmacología , Inanición , Proteínas de Unión a GTP rab7RESUMEN
Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.
Asunto(s)
Catalasa/metabolismo , Cilios/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Peroxisomas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Células Cultivadas , Citoplasma/metabolismo , Células HEK293 , Humanos , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Epitelio Pigmentado de la Retina/citología , Transducción de SeñalRESUMEN
Many aspects of membrane-trafficking events are regulated by Rab-family small GTPases. Neurite outgrowth requires massive addition of proteins and lipids to the tips of growing neurites by membrane trafficking, and although several Rabs, including Rab8, Rab10, and Rab11, have been implicated in this process, their regulatory mechanisms during neurite outgrowth are poorly understood. Here, we show that Rabin8, a Rab8-guanine nucleotide exchange factor (GEF), regulates nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells. Knockdown of Rabin8 results in inhibition of neurite outgrowth, whereas overexpression promotes it. We also find that Rab10 is a novel substrate of Rabin8 and that both Rab8 and Rab10 function during neurite outgrowth downstream of Rabin8. Surprisingly, however, a GEF activity-deficient isoform of Rabin8 also promotes neurite outgrowth, indicating the existence of a GEF activity-independent role of Rabin8. The Arf6/Rab8-positive recycling endosomes (Arf6/Rab8-REs) and Rab10/Rab11-positive REs (Rab10/Rab11-REs) in NGF-stimulated PC12 cells are differently distributed. Rabin8 localizes on both RE populations and appears to activate Rab8 and Rab10 there. These localizations and functions of Rabin8 are Rab11 dependent. Thus Rabin8 regulates neurite outgrowth both by coordinating with Rab8, Rab10, and Rab11 and by a GEF activity-independent mechanism.