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1.
Nat Commun ; 15(1): 2093, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38453931

RESUMEN

Adhesions are critical for anchoring cells in their environment, as signaling platforms and for cell migration. In line with these diverse functions different types of cell-matrix adhesions have been described. Best-studied are the canonical integrin-based focal adhesions. In addition, non-canonical integrin adhesions lacking focal adhesion proteins have been discovered. These include reticular adhesions also known as clathrin plaques or flat clathrin lattices, that are enriched in clathrin and other endocytic proteins, as well as extensive adhesion networks and retraction fibers. How these different adhesion types that share a common integrin backbone are related and whether they can interconvert is unknown. Here, we identify the protein stonin1 as a marker for non-canonical αVß5 integrin-based adhesions and demonstrate by live cell imaging that canonical and non-canonical adhesions can reciprocally interconvert by the selective exchange of components on a stable αVß5 integrin scaffold. Hence, non-canonical adhesions can serve as points of origin for the generation of canonical focal adhesions.


Asunto(s)
Adhesiones Focales , Integrinas , Integrinas/metabolismo , Adhesiones Focales/metabolismo , Uniones Célula-Matriz/metabolismo , Movimiento Celular , Clatrina/metabolismo , Adhesión Celular
2.
Elife ; 112022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-35014951

RESUMEN

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Conflicting models have been proposed regarding the mechanisms of SV endocytosis, most notably clathrin/adaptor protein complex 2 (AP-2)-mediated endocytosis and clathrin-independent ultrafast endocytosis. Partitioning between these pathways has been suggested to be controlled by temperature and stimulus paradigm. We report on the comprehensive survey of six major SV proteins to show that SV endocytosis in mouse hippocampal neurons at physiological temperature occurs independent of clathrin while the endocytic retrieval of a subset of SV proteins including the vesicular transporters for glutamate and GABA depend on sorting by the clathrin adaptor AP-2. Our findings highlight a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic cell surface and suggest a revised model for the endocytosis of SV membranes at mammalian central synapses.


Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Clatrina/metabolismo , Endocitosis , Sinapsis/fisiología , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Ratones
3.
Sci Rep ; 11(1): 18435, 2021 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-34531445

RESUMEN

The significance of crosstalks among constituents of plasma membrane protein clusters/complexes in cellular proteostasis and protein quality control (PQC) remains incompletely understood. Examining the glial (enriched) cell adhesion molecule (CAM), we demonstrate its chaperone-like role in the biosynthetic processing of the megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1)-heteromeric regulatory membrane protein complex, as well as the function of the GlialCAM/MLC1 signalling complex. We show that in the absence of GlialCAM, newly synthesized MLC1 molecules remain unfolded and are susceptible to polyubiquitination-dependent proteasomal degradation at the endoplasmic reticulum. At the plasma membrane, GlialCAM regulates the diffusional partitioning and endocytic dynamics of cluster members, including the ClC-2 chloride channel and MLC1. Impaired folding and/or expression of GlialCAM or MLC1 in the presence of diseases causing mutations, as well as plasma membrane tethering compromise the functional expression of the cluster, leading to compromised endo-lysosomal organellar identity. In addition, the enlarged endo-lysosomal compartments display accelerated acidification, ubiquitinated cargo-sorting and impaired endosomal recycling. Jointly, these observations indicate an essential and previously unrecognized role for CAM, where GliaCAM functions as a PQC factor for the MLC1 signalling complex biogenesis and possess a permissive role in the membrane dynamic and cargo sorting functions with implications in modulations of receptor signalling.


Asunto(s)
Astrocitos/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de la Membrana/metabolismo , Proteostasis , Animales , Células Cultivadas , Canales de Cloruro/metabolismo , Endosomas/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Ratas
4.
Cell Stress ; 4(10): 230-247, 2020 Aug 18.
Artículo en Inglés | MEDLINE | ID: mdl-33024932

RESUMEN

Cellular life is challenged by a multitude of stress conditions, triggered for example by alterations in osmolarity, oxygen or nutrient supply. Hence, cells have developed sophisticated stress responses to cope with these challenges. Some of these stress programs such as the heat shock response are understood in great detail, while other aspects remain largely elusive including potential stress-dependent adaptations of the plasma membrane proteome. The plasma membrane is not only the first point of encounter for many types of environmental stress, but given the diversity of receptor proteins and their associated molecules also represents the site at which many cellular signal cascades originate. Since these signaling pathways affect virtually all aspects of cellular life, changes in the plasma membrane proteome appear ideally suited to contribute to the cellular adaptation to stress. The most rapid means to alter the cell surface proteome in response to stress is by alterations in endocytosis. Changes in the overall endocytic flux or in the endocytic regulation of select proteins conceivably can help to counteract adverse environmental conditions. In this review we summarize recent data regarding stress-induced changes in endocytosis and discuss how these changes might contribute to the cellular adaptation to stress in different systems. Future studies will be needed to uncover the underlying mechanisms in detail and to arrive at a coherent picture.

5.
Autophagy ; 16(10): 1921-1922, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32684085

RESUMEN

Osmotic stress is a critical challenge for mammalian cells as loss of water triggered by a hyperosmotic environment promotes harmful protein aggregation and impairs cell survival. How the degradative capacity of cells, in particular the macroautophagy/autophagy-lysosome system, is adapted to meet the proteolytic demands induced by osmotic challenge remains poorly understood. We have identified a hitherto unknown pathway that is activated by hyperosmotic stress and serves to link alterations in cellular ion homeostasis to the induction of autophagy and lysosomal gene expression and, thereby, to lysosome biogenesis.


Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Animales , Homeostasis , Lisosomas , Presión Osmótica
6.
Nat Cell Biol ; 22(7): 815-827, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32601373

RESUMEN

Lysosomes serve as cellular degradation and signalling centres that coordinate metabolism in response to intracellular cues and extracellular signals. Lysosomal capacity is adapted to cellular needs by transcription factors, such as TFEB and TFE3, which activate the expression of lysosomal and autophagy genes. Nuclear translocation and activation of TFEB are induced by a variety of conditions such as starvation, lysosome stress and lysosomal storage disorders. How these various cues are integrated remains incompletely understood. Here, we describe a pathway initiated at the plasma membrane that controls lysosome biogenesis via the endocytic regulation of intracellular ion homeostasis. This pathway is based on the exo-endocytosis of NHE7, a Na+/H+ exchanger mutated in X-linked intellectual disability, and serves to control intracellular ion homeostasis and thereby Ca2+/calcineurin-mediated activation of TFEB and downstream lysosome biogenesis in response to osmotic stress to promote the turnover of toxic proteins and cell survival.


Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Membrana Celular/metabolismo , Endocitosis , Lisosomas/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Calcineurina/genética , Calcineurina/metabolismo , Calcio/metabolismo , Clatrina/metabolismo , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Intercambiadores de Sodio-Hidrógeno/genética
7.
Cells ; 8(11)2019 10 29.
Artículo en Inglés | MEDLINE | ID: mdl-31671891

RESUMEN

Cells need to exchange material and information with their environment. This is largely achieved via cell-surface receptors which mediate processes ranging from nutrient uptake to signaling responses. Consequently, their surface levels have to be dynamically controlled. Endocytosis constitutes a powerful mechanism to regulate the surface proteome and to recycle vesicular transmembrane proteins that strand at the plasma membrane after exocytosis. For efficient internalization, the cargo proteins need to be linked to the endocytic machinery via adaptor proteins such as the heterotetrameric endocytic adaptor complex AP-2 and a variety of mostly monomeric endocytic adaptors. In line with the importance of endocytosis for nutrient uptake, cell signaling and neurotransmission, animal models and human mutations have revealed that defects in these adaptors are associated with several diseases ranging from metabolic disorders to encephalopathies. This review will discuss the physiological functions of the so far known adaptor proteins and will provide a comprehensive overview of their links to human diseases.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Enfermedad , Endocitosis , Salud , Proteínas de la Membrana/fisiología , Mutación/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Vesículas Cubiertas por Clatrina/fisiología , Enfermedad/etiología , Enfermedad/genética , Endocitosis/genética , Endocitosis/fisiología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Animales
8.
Am J Hum Genet ; 104(6): 1060-1072, 2019 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-31104773

RESUMEN

The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the µ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the µ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2µ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.


Asunto(s)
Complejo 2 de Proteína Adaptadora/genética , Subunidades mu de Complejo de Proteína Adaptadora/genética , Encefalopatías/etiología , Clatrina/metabolismo , Endocitosis , Epilepsia/etiología , Mutación Missense , Trastornos del Neurodesarrollo/etiología , Adolescente , Animales , Encefalopatías/patología , Niño , Preescolar , Clatrina/genética , Epilepsia/patología , Femenino , Humanos , Lactante , Ratones , Ratones Noqueados , Trastornos del Neurodesarrollo/patología , Secuenciación del Exoma
9.
Eur J Med Genet ; 61(1): 50-60, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29079544

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.


Asunto(s)
Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Proteínas/genética , Animales , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Quistes/patología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Proteínas/química , Proteínas/metabolismo
10.
Hum Mol Genet ; 26(13): 2436-2450, 2017 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-28398517

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.


Asunto(s)
Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Encefalopatías/patología , Canales de Cloruro CLC-2 , Canales de Calcio Tipo L/genética , Canales de Cloruro , Quistes/genética , Células HEK293 , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Proteínas de la Membrana/genética , Ratones , Transporte de Proteínas/genética
11.
J Biol Chem ; 291(27): 14170-14184, 2016 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-27226546

RESUMEN

Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond.


Asunto(s)
Lisosomas/metabolismo , Nanopartículas , Dióxido de Silicio/metabolismo , Endocitosis , Células HeLa , Humanos
12.
Biophys J ; 107(5): 1105-1116, 2014 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-25185546

RESUMEN

GlialCAM, a glial cell adhesion molecule mutated in megalencephalic leukoencephalopathy with subcortical cysts, targets the CLC-2 Cl(-) channel to cell contacts in glia and activates CLC-2 currents in vitro and in vivo. We found that GlialCAM clusters all CLC channels at cell contacts in vitro and thus studied GlialCAM interaction with CLC channels to investigate the mechanism of functional activation. GlialCAM slowed deactivation kinetics of CLC-Ka/barttin channels and increased CLC-0 currents opening the common gate and slowing its deactivation. No functional effect was seen for common gate deficient CLC-0 mutants. Similarly, GlialCAM targets the common gate deficient CLC-2 mutant E211V/H816A to cell contacts, without altering its function. Thus, GlialCAM is able to interact with all CLC channels tested, targeting them to cell junctions and activating them by stabilizing the open configuration of the common gate. These results are important to better understand the physiological role of GlialCAM/CLC-2 interaction.


Asunto(s)
Canales de Cloruro/metabolismo , Proteínas/metabolismo , Animales , Proteínas de Ciclo Celular , Canales de Cloruro/genética , Células HEK293 , Humanos , Iones/química , Cinética , Potenciales de la Membrana/fisiología , Mutación , Oocitos , Ratas , Temperatura , Torpedo , Transfección , Xenopus laevis , Zinc/química
13.
Hum Mutat ; 35(10): 1175-8, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25044933

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by white matter edema. Autosomal-recessive mutations in MLC1 cause MLC type 1, and autosomal-recessive or dominant mutations in HEPACAM (also called GLIALCAM) cause MLC type 2A and type 2B, respectively. The role of MLC1 and HEPACAM is unknown, although they have been related with the processes of cell-volume regulation and potassium siphoning by astrocytes. Previous studies with some of the mutations identified in HEPACAM showed that most of them are associated with a trafficking defect. Here, we analyzed biochemically and functionally most mutations identified up-to-date in HEPACAM. Our results allow classifying the effect of mutations in different subtypes and we indicate different cellular mechanisms that lead to MLC pathogenesis.


Asunto(s)
Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Mutación , Proteínas/genética , Animales , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Canales de Cloruro/genética , Quistes/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Ratones , Proteínas/metabolismo
14.
Hum Mol Genet ; 23(19): 5069-86, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-24824219

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by myelin vacuolization and caused by mutations in MLC1 or GLIALCAM. Patients with recessive mutations in either MLC1 or GLIALCAM show the same clinical phenotype. It has been shown that GLIALCAM is necessary for the correct targeting of MLC1 to the membrane at cell junctions, but its own localization was independent of MLC1 in vitro. However, recent studies in Mlc1(-/-) mice have shown that GlialCAM is mislocalized in glial cells. In order to investigate whether the relationship between Mlc1 and GlialCAM is species-specific, we first identified MLC-related genes in zebrafish and generated an mlc1(-/-) zebrafish. We have characterized mlc1(-/-) zebrafish both functionally and histologically and compared the phenotype with that of the Mlc1(-/-) mice. In mlc1(-/-) zebrafish, as in Mlc1(-/-) mice, Glialcam is mislocalized. Re-examination of a brain biopsy from an MLC patient indicates that GLIALCAM is also mislocalized in Bergmann glia in the cerebellum. In vitro, impaired localization of GlialCAM was observed in astrocyte cultures from Mlc1(-/-) mouse only in the presence of elevated potassium levels, which mimics neuronal activity. In summary, here we demonstrate an evolutionary conserved role for MLC1 in regulating glial surface levels of GLIALCAM, and this interrelationship explains why patients with mutations in either gene (MLC1 or GLIALCAM) share the same clinical phenotype.


Asunto(s)
Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/metabolismo , Neuroglía/metabolismo , Proteínas/metabolismo , Animales , Animales Modificados Genéticamente , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular , Línea Celular , Membrana Celular/metabolismo , Quistes/genética , Modelos Animales de Enfermedad , Epéndimo/citología , Epéndimo/metabolismo , Epéndimo/ultraestructura , Expresión Génica , Genotipo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Uniones Intercelulares/metabolismo , Uniones Intercelulares/ultraestructura , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Mutación , Fenotipo , Transporte de Proteínas , Proteínas/genética , Retina/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo
15.
Hum Mol Genet ; 22(21): 4405-16, 2013 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23793458

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes and is associated with myelin and astrocyte vacuolation. It has been suggested that MLC is caused by impaired cell volume regulation as a result of defective activation of astrocytic volume-regulated anion currents (VRAC). GlialCAM brings MLC1 and the ClC-2 Cl(-) channel to cell-cell junctions, even though the role of ClC-2 in MLC disease remains incompletely understood. To gain insights into the biological role of GlialCAM in the pathogenesis of MLC disease, here we analyzed the gain- and loss-of-function phenotypes of GlialCAM in Hela cells and primary astrocytes, focusing on its interaction with the MLC1 protein. Unexpectedly, GlialCAM ablation provoked intracellular accumulation and reduced expression of MLC1 at the plasma membrane. Conversely, over-expression of GlialCAM increased the cellular stability of mutant MLC1 variants. Reduction in GlialCAM expression resulted in defective activation of VRAC and augmented vacuolation, phenocopying MLC1 mutations. Importantly, over-expression of GlialCAM together with MLC1 containing MLC-related mutations was able to reactivate VRAC currents and to reverse the vacuolation caused in the presence of mutant MLC1. These results indicate a previously unrecognized role of GlialCAM in facilitating the biosynthetic maturation and cell surface expression of MLC1, and suggest that pharmacological strategies aimed to increase surface expression of MLC1 and/or VRAC activity may be beneficial for MLC patients.


Asunto(s)
Astrocitos/fisiología , Canales de Cloruro/fisiología , Quistes/fisiopatología , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Proteínas/genética , Proteínas/metabolismo , Animales , Astrocitos/ultraestructura , Canales de Cloruro CLC-2 , Proteínas de Ciclo Celular , Quistes/genética , Variación Genética , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Fenotipo , Estabilidad Proteica , Interferencia de ARN , Ratas , Vacuolas/fisiología
16.
Neuron ; 73(5): 951-61, 2012 Mar 08.
Artículo en Inglés | MEDLINE | ID: mdl-22405205

RESUMEN

Ion fluxes mediated by glial cells are required for several physiological processes such as fluid homeostasis or the maintenance of low extracellular potassium during high neuronal activity. In mice, the disruption of the Cl(-) channel ClC-2 causes fluid accumulation leading to myelin vacuolation. A similar vacuolation phenotype is detected in humans affected with megalencephalic leukoencephalopathy with subcortical cysts (MLC), a leukodystrophy which is caused by mutations in MLC1 or GLIALCAM. We here identify GlialCAM as a ClC-2 binding partner. GlialCAM and ClC-2 colocalize in Bergmann glia, in astrocyte-astrocyte junctions at astrocytic endfeet around blood vessels, and in myelinated fiber tracts. GlialCAM targets ClC-2 to cell junctions, increases ClC-2 mediated currents, and changes its functional properties. Disease-causing GLIALCAM mutations abolish the targeting of the channel to cell junctions. This work describes the first auxiliary subunit of ClC-2 and suggests that ClC-2 may play a role in the pathology of MLC disease.


Asunto(s)
Canales de Cloruro/fisiología , Neuroglía/metabolismo , Animales , Biofisica , Canales de Cloruro CLC-2 , Células Cultivadas , Canales de Cloruro/genética , Canales de Cloruro/ultraestructura , Conexinas/metabolismo , Estimulación Eléctrica , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Espectrometría de Masas , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Transgénicos , Microinyecciones/métodos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Moleculares , Mutación/genética , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Cadenas Ligeras de Miosina/genética , Neuroglía/ultraestructura , Oocitos , Técnicas de Placa-Clamp , Transporte de Proteínas/genética , Ratas , Transfección , Xenopus
17.
Anal Biochem ; 423(1): 109-18, 2012 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-22342621

RESUMEN

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.


Asunto(s)
Bioensayo/métodos , Endopeptidasas/metabolismo , Mediciones Luminiscentes , Proteínas de la Membrana/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Potyvirus/enzimología , Mapeo de Interacción de Proteínas , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
18.
Hum Mol Genet ; 20(16): 3266-77, 2011 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-21624973

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or targeting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC.


Asunto(s)
Quistes/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Proteínas de la Membrana/genética , Mutación/genética , Proteínas/genética , Adulto , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Ciclo Celular , Quistes/patología , Resultado Fatal , Femenino , Células HEK293 , Células HeLa , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/patología , Humanos , Persona de Mediana Edad , Proteínas Mutantes/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Interferencia de ARN , Ratas , Transfección
19.
Am J Hum Genet ; 88(4): 422-32, 2011 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-21419380

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly.


Asunto(s)
Trastorno Autístico/genética , Moléculas de Adhesión Celular Neuronal/genética , Discapacidad Intelectual/genética , Megalencefalia/genética , Mutación , Proteínas/genética , Secuencia de Aminoácidos , Animales , Trastorno Autístico/metabolismo , Encéfalo/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Quistes/genética , Quistes/metabolismo , Genes Dominantes , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Humanos , Discapacidad Intelectual/metabolismo , Megalencefalia/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/metabolismo , Ratas , Homología de Secuencia de Aminoácido
20.
Neurobiol Dis ; 43(1): 228-38, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21440627

RESUMEN

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.


Asunto(s)
Astrocitos/metabolismo , Quistes/genética , Quistes/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Vacuolas/genética , Adolescente , Animales , Astrocitos/patología , Células Cultivadas , Quistes/fisiopatología , Regulación hacia Abajo/genética , Líquido Extracelular/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/fisiopatología , Humanos , Proteínas de la Membrana/fisiología , Ratones , Ratas , Ratas Sprague-Dawley , Vacuolas/patología
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