RESUMEN
Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT) due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-ß-carboline N^N' ligands have been synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and N^N' ligands on the photophysical and biological properties. All the compounds exhibit remarkable photostability under blue-light irradiation and are emissive (605 < λem < 720 nm), with the Ru(II) derivatives displaying higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1-3 exhibit the highest cytotoxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation, resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis. Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits moderate mitochondrial activity. Overall, Ir(III) complexes 1-3 show potential for selective light-controlled cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal cancer cell dissemination.
Asunto(s)
Antineoplásicos , Carbolinas , Complejos de Coordinación , Ensayos de Selección de Medicamentos Antitumorales , Iridio , Fotoquimioterapia , Fármacos Fotosensibilizantes , Rutenio , Humanos , Iridio/química , Iridio/farmacología , Rutenio/química , Rutenio/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Ligandos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/síntesis química , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Carbolinas/química , Carbolinas/farmacología , Carbolinas/síntesis química , Estructura Molecular , Relación Estructura-Actividad , Proliferación Celular/efectos de los fármacos , Piridinas/química , Piridinas/farmacología , Piridinas/síntesis química , Especies Reactivas de Oxígeno/metabolismo , Relación Dosis-Respuesta a Droga , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.
Asunto(s)
Actinas , Actinas/metabolismo , Membrana Celular/metabolismo , HomeostasisRESUMEN
Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.
Asunto(s)
Caveolinas , Proteostasis , Caveolinas/metabolismo , Caveolina 1/metabolismo , Proteínas de la Membrana/metabolismo , Caveolas/metabolismo , Membrana Celular/metabolismo , Aparato de Golgi/metabolismoRESUMEN
Maintaining lipid composition diversity in membranes from different organelles is critical for numerous cellular processes. However, many lipids are synthesized in the endoplasmic reticulum (ER) and require delivery to other organelles. In this scenario, formation of membrane contact sites (MCS) between neighbouring organelles has emerged as a novel non-vesicular lipid transport mechanism. Dissecting the molecular composition of MCS identified phosphoinositides (PIs), cholesterol, scaffolding/tethering proteins as well as Ca2+ and Ca2+-binding proteins contributing to MCS functioning. Compelling evidence now exists for the shuttling of PIs and cholesterol across MCS, affecting their concentrations in distinct membrane domains and diverse roles in membrane trafficking. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) not only controls endo-/exocytic membrane dynamics but is also critical in autophagy. Cholesterol is highly concentrated at the PM and enriched in recycling endosomes and Golgi membranes. MCS-mediated cholesterol transfer is intensely researched, identifying MCS dysfunction or altered MCS partnerships to correlate with de-regulated cellular cholesterol homeostasis and pathologies. Annexins, a conserved family of Ca2+-dependent phospholipid binding proteins, contribute to tethering and untethering events at MCS. In this chapter, we will discuss how Ca2+ homeostasis and annexins in the endocytic compartment affect the sensing and transfer of cholesterol and PIs across MCS.
Asunto(s)
Anexinas , Fosfatidilinositoles , Fosfatidilinositoles/metabolismo , Anexinas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Portadoras/metabolismo , Colesterol/metabolismoRESUMEN
Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.
Asunto(s)
Anexina A6/metabolismo , LDL-Colesterol/metabolismo , Adhesiones Focales/metabolismo , Proteína Niemann-Pick C1/metabolismo , Proteínas de Unión a GTP rab7/metabolismo , Animales , Células CHO , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular , Cricetulus , Humanos , Proteínas de la Membrana/metabolismoRESUMEN
Membrane contact sites (MCS) are specialized small areas of close apposition between two different organelles that have led researchers to reconsider the dogma of intercellular communication via vesicular trafficking. The latter is now being challenged by the discovery of lipid and ion transfer across MCS connecting adjacent organelles. These findings gave rise to a new concept that implicates cell compartments not to function as individual and isolated entities, but as a dynamic and regulated ensemble facilitating the trafficking of lipids, including cholesterol, and ions. Hence, MCS are now envisaged as metabolic platforms, crucial for cellular homeostasis. In this context, well-known as well as novel proteins were ascribed functions such as tethers, transporters, and scaffolds in MCS, or transient MCS companions with yet unknown functions. Intriguingly, we and others uncovered metabolic alterations in cell-based disease models that perturbed MCS size and numbers between coupled organelles such as endolysosomes, the endoplasmic reticulum, mitochondria, or lipid droplets. On the other hand, overexpression or deficiency of certain proteins in this narrow 10-30 nm membrane contact zone can enable MCS formation to either rescue compromised MCS function, or in certain disease settings trigger undesired metabolite transport. In this "Mini Review" we summarize recent findings regarding a subset of annexins and discuss their multiple roles to regulate MCS dynamics and functioning. Their contribution to novel pathways related to MCS biology will provide new insights relevant for a number of human diseases and offer opportunities to design innovative treatments in the future.
RESUMEN
The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.
Asunto(s)
Calmodulina/metabolismo , Membrana Celular/metabolismo , Animales , Células CHO , Señalización del Calcio/fisiología , Línea Celular , Cricetulus , Activación Enzimática/fisiología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.
Asunto(s)
Anexina A6/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Hepatopatías/patología , Longevidad , Animales , Conducta Animal , Hepatopatías/etiología , Hepatopatías/metabolismo , Ratones , Ratones Noqueados , Proteína Niemann-Pick C1RESUMEN
Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.
Asunto(s)
Bacterias/inmunología , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Gotas Lipídicas/inmunología , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Ácidos Grasos/metabolismo , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Humanos , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/inmunología , CatelicidinasRESUMEN
We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Two KFERQ-motifs are believed to target AnxA6 to lysosomes for chaperone-mediated autophagy (CMA), and we hypothesized that the cholesterol accumulation in endolysosomes (LE/Lys) triggered by the NPC1 inhibition could interfere with the CMA pathway. Therefore, AnxA6 protein amounts and cholesterol levels in the LE/Lys (LE-Chol) compartment were analyzed in NPC1 mutant cells ectopically expressing lysosome-associated membrane protein 2A (Lamp2A), which is well known to induce the CMA pathway. Strikingly, AnxA6 protein amounts were strongly decreased and coincided with significantly reduced LE-Chol levels in NPC1 mutant cells upon Lamp2A overexpression. Therefore, these findings suggest Lamp2A-mediated restoration of CMA in NPC1 mutant cells to lower LE-Chol levels with concomitant lysosomal AnxA6 degradation. Collectively, we propose CMA to permit a feedback loop between AnxA6 and cholesterol levels in LE/Lys, encompassing a novel mechanism for regulating cholesterol homeostasis in NPC1 disease.
Asunto(s)
Anexina A6/metabolismo , Colesterol/metabolismo , Endosomas/metabolismo , Retroalimentación Fisiológica , Péptidos y Proteínas de Señalización Intracelular/genética , Lisosomas/metabolismo , Mutación/genética , Proteolisis , Animales , Células CHO , Cricetulus , Endosomas/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/ultraestructura , Modelos Biológicos , Proteína Niemann-Pick C1RESUMEN
Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.
Asunto(s)
Calmodulina/metabolismo , Carcinogénesis/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Calmodulina/genética , Carcinogénesis/genética , Pleiotropía Genética , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Proteína de Unión al GTP rac1/genéticaRESUMEN
BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.
Asunto(s)
Anexina A6/metabolismo , Gluconeogénesis/fisiología , Regeneración Hepática/fisiología , Animales , Anexina A6/genética , Membrana Celular/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Glucólisis/fisiología , Hepatectomía , Hepatocitos/metabolismo , Humanos , Hígado/citología , Hígado/metabolismo , Hígado/cirugía , Masculino , Ratones , Ratones NoqueadosRESUMEN
Annexin A6 (AnxA6), a member of the calcium (Ca2+ ) and membrane binding annexins, is known to stabilize and establish the formation of multifactorial signaling complexes. At the plasma membrane, AnxA6 is a scaffold for protein kinase Cα (PKCα) and GTPase-activating protein p120GAP to promote downregulation of epidermal growth factor receptor (EGFR) and Ras/mitogen-activated protein kinase (MAPK) signaling. In human squamous A431 epithelial carcinoma cells, which overexpress EGFR, but lack endogenous AnxA6, restoration of AnxA6 expression (A431-A6) promotes PKCα-mediated threonine 654 (T654)-EGFR phosphorylation, which inhibits EGFR tyrosine kinase activity. This is associated with reduced A431-A6 cell growth, but also decreased migration and invasion in wound healing, matrigel, and organotypic matrices. Here, we show that A431-A6 cells display reduced EGFR activity in vivo, with xenograft analysis identifying increased pT654-EGFR levels, but reduced tyrosine EGFR phosphorylation compared to controls. In contrast, PKCα depletion in A431-A6 tumors is associated with strongly reduced pT654 EGFR levels, yet increased EGFR tyrosine phosphorylation and MAPK activity. Moreover, tyrosine kinase inhibitors (TKIs; gefitinib, erlotinib) more effectively inhibit cell viability, clonogenic growth, and wound healing of A431-A6 cells compared to controls. Likewise, the ability of AnxA6 to inhibit A431 motility and invasiveness strongly improves TKI efficacy in matrigel invasion assays. This correlates with a greatly reduced invasion of the surrounding matrix of TKI-treated A431-A6 when cultured in 3D spheroids. Altogether, these findings implicate that elevated AnxA6 scaffold levels contribute to improve TKI-mediated inhibition of growth and migration, but also invasive properties in EGFR overexpressing human squamous epithelial carcinoma.
Asunto(s)
Anexina A6/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Anexina A6/genética , Apoptosis , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Proliferación Celular , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Humanos , Ratones , Invasividad Neoplásica , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Fosforilación , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
Asunto(s)
Anexina A6/genética , Colesterol/genética , Proteínas Activadoras de GTPasa/genética , Enfermedad de Niemann-Pick Tipo C/genética , Proteínas de Unión al GTP rab/genética , Animales , Células CHO , Proteínas Portadoras/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Endosomas/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/genética , Enfermedad de Niemann-Pick Tipo C/metabolismo , Enfermedad de Niemann-Pick Tipo C/patología , Dominios Proteicos/genética , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , Proteínas de Unión a GTP rab7RESUMEN
The endocytic compartment is not only the functional continuity of the plasma membrane but consists of a diverse collection of intracellular heterogeneous complex structures that transport, amplify, sustain, and/or sort signaling molecules. Over the years, it has become evident that early, late, and recycling endosomes represent an interconnected vesicular-tubular network able to form signaling platforms that dynamically and efficiently translate extracellular signals into biological outcome. Cell activation, differentiation, migration, death, and survival are some of the endpoints of endosomal signaling. Hence, to understand the role of the endosomal system in signal transduction in space and time, it is therefore necessary to dissect and identify the plethora of decoders that are operational in the different steps along the endocytic pathway. In this chapter, we focus on the regulation of spatiotemporal signaling in cells, considering endosomes as central platforms, in which several small GTPases proteins of the Ras superfamily, in particular Ras and Rac1, actively participate to control cellular processes like proliferation and cell mobility.
Asunto(s)
Proliferación Celular/genética , Endosomas/genética , Proteína de Unión al GTP rac1/genética , Proteínas ras/genética , Movimiento Celular/genética , Endocitosis/genética , Humanos , Transporte de Proteínas , Transducción de Señal/genéticaRESUMEN
Clathrin-dependent and -independent pathways contribute for ß1-integrin endocytosis. This study defines a tubular membrane clathrin-independent endocytic network, induced with the calmodulin inhibitor W13, for ß1-integrin internalization. This pathway is dependent on increased phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) levels and dynamin activity at the plasma membrane. Exogenous addition of PI(4,5)P2 or phosphatidylinositol-4-phosphate 5-kinase (PIP5K) expression mimicked W13-generated-tubules which are inhibited by active Rac1. Therefore, the molecular mechanisms downstream of Rac1, that controls this plasma membrane tubulation, were analyzed biochemically and by the expression of different Rac1 mutants. The results indicate that phospholipase C and ROCK1 are the main Rac1 effectors that impair plasma membrane invagination and tubule formation, essentially by decreasing PI(4,5)P2 levels and promoting cortical actomyosin assembly respectively. Interestingly, among the plethora of proteins that participate in membrane remodeling, this study revealed that ROCK1, the well-known downstream RhoA effector, has an important role in Rac1 regulation of actomyosin at the cell cortex. This study provides new insights into Rac1 functioning on plasma membrane dynamics combining phosphatidylinositides and cytoskeleton regulation.
Asunto(s)
Endocitosis , Microtúbulos/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Quinasas Asociadas a rho/metabolismo , Actomiosina/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfolipasas de Tipo C/metabolismo , Células VeroRESUMEN
Despite the discovery of annexins 40 years ago, we are just beginning to understand some of the functions of these still enigmatic proteins. Defined and characterized by their ability to bind anionic membrane lipids in a Ca2+-dependent manner, each annexin has to be considered a multifunctional protein, with a multitude of cellular locations and diverse activities. Underlying causes for this considerable functional diversity include their capability to associate with multiple cytosolic and membrane proteins. In recent years, the increasingly recognized establishment of membrane contact sites between subcellular compartments opens a new scenario for annexins as instrumental players to link Ca2+ signalling with the integration of membrane trafficking in many facets of cell physiology. In this chapter, we review and discuss current knowledge on the contribution of annexins in the biogenesis and functioning of the late endocytic compartment, affecting endo- and exocytic pathways in a variety of physiological consequences ranging from membrane repair, lysosomal exocytosis, to cell migration.
Asunto(s)
Anexinas/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Membrana Celular/metabolismo , Animales , Movimiento Celular/fisiología , Endocitosis/fisiología , Exocitosis/fisiología , Humanos , Lisosomas/metabolismo , Lípidos de la Membrana/metabolismoRESUMEN
Niemann-Pick C disease is a neurovisceral disorder caused by mutations in the NPC gene that result in systemic accumulation of intracellular cholesterol. Although neurodegeneration defines the disease's severity, in most patients it is preceded by hepatic complications such as cholestatic jaundice or hepatomegaly. To analyze the contribution of the hepatic disease in Niemann-Pick C disease progression and to evaluate the degree of primary and secondary hepatic damage, we generated a transgenic mouse with liver-selective expression of NPC1 from embryonic stages. Hepatic NPC1 re-expression did not ameliorate the onset and progression of neurodegeneration of the NPC1-null animal. However, the mice showed reduced hepatomegalia and dramatic, although not complete, reduction of hepatic cholesterol and serum bile salts, bilirubin, and transaminase levels. Therefore, hepatic primary and secondary cholesterol deposition and damage occur simultaneously during Niemann-Pick C disease progression.
Asunto(s)
Colesterol/metabolismo , Modelos Animales de Enfermedad , Hepatopatías/complicaciones , Hígado/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Animales , Ácidos y Sales Biliares/sangre , Bilirrubina/sangre , Colesterol/análisis , Progresión de la Enfermedad , Células Madre Embrionarias , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Hígado/patología , Hepatopatías/genética , Hepatopatías/metabolismo , Hepatopatías/patología , Masculino , Ratones , Ratones Noqueados , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/complicaciones , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , Proteínas/metabolismo , Transaminasas/sangreRESUMEN
Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.
Asunto(s)
Anexina A6/metabolismo , Colesterol/metabolismo , Endosomas/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Qa-SNARE/metabolismo , Animales , Anexina A6/antagonistas & inhibidores , Anexina A6/genética , Células CHO , Línea Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Movimiento Celular , Células Cultivadas , Cricetulus , Endosomas/ultraestructura , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfaVbeta3/antagonistas & inhibidores , Ratones , Microscopía Confocal , Microscopía por Video , Proteínas Qa-SNARE/antagonistas & inhibidores , Proteínas Qa-SNARE/genética , Interferencia de ARN , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagen de Lapso de TiempoRESUMEN
Lipid droplets (LDs) are intracellular organelles that provide fatty acids (FAs) to cellular processes including synthesis of membranes and production of metabolic energy. While known to move bidirectionally along microtubules (MTs), the role of LD motion and whether it facilitates interaction with other organelles are unclear. Here we show that during nutrient starvation, LDs and mitochondria relocate on detyrosinated MT from the cell centre to adopt a dispersed distribution. In the cell periphery, LD-mitochondria interactions increase and LDs efficiently supply FAs for mitochondrial beta-oxidation. This cellular adaptation requires the activation of the energy sensor AMPK, which in response to starvation simultaneously increases LD motion, reorganizes the network of detyrosinated MTs and activates mitochondria. In conclusion, we describe the existence of a specialized cellular network connecting the cellular energetic status and MT dynamics to coordinate the functioning of LDs and mitochondria during nutrient scarcity.