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1.
Cell ; 184(3): 655-674.e27, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33497611

RESUMEN

Ras GTPase-activating protein-binding proteins 1 and 2 (G3BP1 and G3BP2, respectively) are widely recognized as core components of stress granules (SGs). We report that G3BPs reside at the cytoplasmic surface of lysosomes. They act in a non-redundant manner to anchor the tuberous sclerosis complex (TSC) protein complex to lysosomes and suppress activation of the metabolic master regulator mechanistic target of rapamycin complex 1 (mTORC1) by amino acids and insulin. Like the TSC complex, G3BP1 deficiency elicits phenotypes related to mTORC1 hyperactivity. In the context of tumors, low G3BP1 levels enhance mTORC1-driven breast cancer cell motility and correlate with adverse outcomes in patients. Furthermore, G3bp1 inhibition in zebrafish disturbs neuronal development and function, leading to white matter heterotopia and neuronal hyperactivity. Thus, G3BPs are not only core components of SGs but also a key element of lysosomal TSC-mTORC1 signaling.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , ADN Helicasas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Esclerosis Tuberosa/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , ADN Helicasas/química , Evolución Molecular , Femenino , Humanos , Insulina/farmacología , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Proteínas de Unión a Poli-ADP-Ribosa/química , ARN Helicasas/química , Proteínas con Motivos de Reconocimiento de ARN/química , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Pez Cebra/metabolismo
2.
J Cell Sci ; 137(13)2024 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-38881365

RESUMEN

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.


Asunto(s)
Matriz Extracelular , Adhesiones Focales , Familia-src Quinasas , Adhesiones Focales/metabolismo , Matriz Extracelular/metabolismo , Humanos , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Animales , Proteína Tirosina Quinasa CSK/metabolismo , Transducción de Señal , Células Endoteliales/metabolismo , Células Endoteliales/patología , Metaloproteinasas de la Matriz/metabolismo
3.
Development ; 149(23)2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36314606

RESUMEN

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.


Asunto(s)
Adhesiones Focales , Talina , Animales , Adhesiones Focales/metabolismo , Talina/genética , Talina/metabolismo , Actinas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Células Endoteliales/metabolismo , Integrinas/genética , Integrinas/metabolismo , Adhesión Celular
4.
Traffic ; 20(9): 674-696, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31314175

RESUMEN

Mechanisms that control lysosomal function are essential for cellular homeostasis. Lysosomes adapt in size and number to cellular needs but little is known about the underlying molecular mechanism. We demonstrate that the late endosomal/lysosomal multimeric BLOC-1-related complex (BORC) regulates the size of these organelles via PIKfyve-dependent phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2 ] production. Deletion of the core BORC component Diaskedin led to increased levels of PI(3,5)P2 , suggesting activation of PIKfyve, and resulted in enhanced lysosomal reformation and subsequent reduction in lysosomal size. This process required AMP-activated protein kinase (AMPK), a known PIKfyve activator, and was additionally dependent on the late endosomal/lysosomal adaptor, mitogen-activated protein kinases and mechanistic target of rapamycin activator (LAMTOR/Ragulator) complex. Consistently, in response to glucose limitation, AMPK activated PIKfyve, which induced lysosomal reformation with increased baseline autophagy and was coupled to a decrease in lysosomal size. These adaptations of the late endosomal/lysosomal system reversed under glucose replete growth conditions. In summary, our results demonstrate that BORC regulates lysosomal reformation and size in response to glucose availability.


Asunto(s)
Endosomas/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Autofagia , Células HEK293 , Células HeLa , Humanos , Proteína 1 de la Membrana Asociada a los Lisosomas/metabolismo , Proteínas de Membrana de los Lisosomas/genética , Sistema de Señalización de MAP Quinasas , Ratones , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/genética , Proteínas/metabolismo
5.
Traffic ; 19(8): 639-649, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29673018

RESUMEN

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.


Asunto(s)
Microscopía Electrónica de Transmisión/métodos , Microscopía Inmunoelectrónica/métodos , Tomografía/métodos , Animales , Antígenos/química , Células CACO-2 , Criopreservación/métodos , Compuestos Epoxi/química , Congelación , Oro/química , Células HeLa , Humanos , Inmunohistoquímica , Nanopartículas/química , Presión , Plata/química
6.
APL Bioeng ; 8(1): 016108, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38352162

RESUMEN

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell-matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

7.
Sci Signal ; 16(782): eabq1366, 2023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-37098119

RESUMEN

Macrophages are key cellular contributors to the pathogenesis of COVID-19, the disease caused by the virus SARS-CoV-2. The SARS-CoV-2 entry receptor ACE2 is present only on a subset of macrophages at sites of SARS-CoV-2 infection in humans. Here, we investigated whether SARS-CoV-2 can enter macrophages, replicate, and release new viral progeny; whether macrophages need to sense a replicating virus to drive cytokine release; and, if so, whether ACE2 is involved in these mechanisms. We found that SARS-CoV-2 could enter, but did not replicate within, ACE2-deficient human primary macrophages and did not induce proinflammatory cytokine expression. By contrast, ACE2 overexpression in human THP-1-derived macrophages permitted SARS-CoV-2 entry, processing and replication, and virion release. ACE2-overexpressing THP-1 macrophages sensed active viral replication and triggered proinflammatory, antiviral programs mediated by the kinase TBK-1 that limited prolonged viral replication and release. These findings help elucidate the role of ACE2 and its absence in macrophage responses to SARS-CoV-2 infection.


Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/fisiología , Enzima Convertidora de Angiotensina 2/genética , Citocinas , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Macrófagos/metabolismo , Virión/metabolismo
8.
Clin Transl Immunology ; 10(10): e1350, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34721846

RESUMEN

OBJECTIVES: Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. METHODS: Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier. RESULTS: We show that primary human endothelial cells express very low levels of the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS-CoV-2. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. CONCLUSIONS: Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

9.
Front Immunol ; 11: 2010, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32922405

RESUMEN

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.


Asunto(s)
Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Integrina alfaXbeta2/metabolismo , Antígeno de Macrófago-1/metabolismo , Antígeno CD11b/genética , Antígeno CD11c/genética , Antígenos CD18/genética , Sistemas CRISPR-Cas , Proteínas del Sistema Complemento/metabolismo , Humanos , Inmunidad , Integrina alfaXbeta2/genética , Antígeno de Macrófago-1/genética , Eliminación de Secuencia/genética , Transducción de Señal , Células THP-1
10.
Virulence ; 10(1): 957-969, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-30372658

RESUMEN

Complement system and dendritic cells (DCs) form - beside neutrophils and macrophages - the first line of defense to combat fungal infections. Therefore, we here studied interactions of these first immune elements with Aspergillus fumigatus lacking ß-1,3-glucans (fks1tetOnrep under repressed conditions) to mechanistically explain the mode of action of echinocandins in more detail. Echinocandins are cell wall active agents blocking ß-glucan synthase, making the A. fumigatus fks1tetOn mutant a good model to study immune-modulatory actions of these drugs. We now demonstrate herein, that complement was activated to significantly higher levels by the fks1-deficient strain compared to its respective wild type. This enhanced covalent linking of complement fragments to the A. fumigatus fks1tetOnrep mutant further resulted in enhanced DC binding and internalization of the fungus. Additionally, we found that fks1tetOnrep induced a Th1-/Th17-polarizing cytokine profile program in DCs. The effect was essentially dependent on massive galactomannan shedding, since blocking of DC-SIGN significantly reduced the fks1tetOnrep-mediated induction of an inflammatory cytokine profile.Our data demonstrate that lack of ß-1,3-glucan, also found under echinocandin therapy, results in improved recognition of Aspergillus fumigatus by complement and DCs and therefore not only directly affects the fungus by its fungistatic actions, but also is likely to exert indirect antifungal mechanisms by strengthening innate host immune mechanisms.Abbreviations: C: complement; CR:complement receptor; DC: dendritic cell; iDC: immature dendritic cell; DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; ERK: extracellular signal-regulated kinases; JNK : c-Jun N-terminal kinases; MAPK: mitogen-activated protein kinase; NHS: normal human serum; PRR: pattern recognition receptor; Th :T helper; TLR :Toll-like receptor; WT: wild type.


Asunto(s)
Aspergillus fumigatus/química , Aspergillus fumigatus/inmunología , Activación de Complemento , Células Dendríticas/inmunología , beta-Glucanos , Aspergilosis/microbiología , Aspergillus fumigatus/genética , Citocinas/inmunología , Equinocandinas/uso terapéutico , Humanos , Inmunidad Innata , Mutación , Esporas Fúngicas/inmunología , Células THP-1
11.
J Cell Biol ; 216(12): 4199-4215, 2017 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-28993467

RESUMEN

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Ribosilacion-ADP/genética , Proteínas Portadoras/genética , Endosomas/efectos de los fármacos , Endosomas/ultraestructura , Factor de Crecimiento Epidérmico/farmacología , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Movimiento , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Transducción de Señal
12.
J Cell Biol ; 205(4): 525-40, 2014 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-24841562

RESUMEN

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14-MP1 (LAMTOR2/3) complex in FA dynamics. p14-MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end-directed traffic of p14-MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14-MP1 scaffold complex to the vicinity of FAs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Movimiento Celular/fisiología , Endosomas/metabolismo , Adhesiones Focales/metabolismo , Proteínas/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Línea Celular , Fibroblastos/citología , Células HeLa , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Células 3T3 NIH , Proteínas/genética , Transducción de Señal/fisiología , Proteínas Activadoras de ras GTPasa/genética , Proteínas Activadoras de ras GTPasa/metabolismo
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