RESUMO
The epithelial tissues of the distal lung are continuously exposed to inhaled air, and are of research interest in studying respiratory exposure to both hazardous and therapeutic materials. Pharmaco-toxicological research depends on the development of sophisticated models of the alveolar epithelium, which better represent the different cell types present in the native lung and interactions between them. We developed an air-liquid interface (ALI) model of the alveolar epithelium which incorporates cell lines which bear features of type I (hAELVi) and type II (NCI-H441) epithelial cells. We compared morphology of single cells and the structure of cell layers of the two lines using light and electron microscopy. Working both in monotypic cultures and cocultures, we measured barrier function by trans-epithelial electrical resistance (TEER), and demonstrated that barrier properties can be maintained for 30 days. We created a mathematical model of TEER development over time based on these data in order to make inferences about the interactions occurring in these culture systems. We assessed expression of a panel of relevant genes that play important roles in barrier function and differentiation. The coculture model was observed to form a stable barrier akin to that seen in hAELVi, while expressing surfactant protein C, and having a profile of expression of claudins and aquaporins appropriate for the distal lung. We described cavities which arise within stratified cell layers in NCI-H441 and cocultured cells, and present evidence that these cavities represent an aberrant apical surface. In summary, our results support the coculture of these two cell lines to produce a model which better represents the breadth of functions seen in native alveolar epithelium.
Assuntos
Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/fisiologia , Técnicas de Cocultura/métodos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Cavéolas/fisiologia , Linhagem Celular , Claudinas/genética , Claudinas/metabolismo , Impedância Elétrica , Expressão Gênica , Humanos , Surfactantes Pulmonares/metabolismoRESUMO
Inside living cells, the remodelling of membrane tubules by actomyosin networks is crucial for processes such as intracellular trafficking or organelle reshaping. In this review, we first present various in vivo situations in which actin affects membrane tubule remodelling, then we recall some results on force production by actin dynamics and on membrane tubules physics. Finally, we show that our knowledge of the underlying mechanisms by which actomyosin dynamics affect tubule morphology has recently been moved forward. This is thanks to in vitro experiments that mimic cellular membranes and actin dynamics and allow deciphering the physics of tubule remodelling in biochemically controlled conditions, and shed new light on tubule shape regulation.
Assuntos
Citoesqueleto de Actina , Membrana Celular , Células Eucarióticas , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Cavéolas/fisiologia , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Vesículas Revestidas por Clatrina/fisiologia , Endocitose/fisiologia , Células Eucarióticas/fisiologia , Células Eucarióticas/ultraestrutura , Transporte ProteicoRESUMO
H-1 protoparvovirus (H-1PV) is a self-propagating virus that is non-pathogenic in humans and has oncolytic and oncosuppressive activities. H-1PV is the first member of the Parvoviridae family to undergo clinical testing as an anticancer agent. Results from clinical trials in patients with glioblastoma or pancreatic carcinoma show that virus treatment is safe, well-tolerated and associated with first signs of efficacy. Characterisation of the H-1PV life cycle may help to improve its efficacy and clinical outcome. In this study, we investigated the entry route of H-1PV in cervical carcinoma HeLa and glioma NCH125 cell lines. Using electron and confocal microscopy, we detected H-1PV particles within clathrin-coated pits and vesicles, providing evidence that the virus uses clathrin-mediated endocytosis for cell entry. In agreement with these results, we found that blocking clathrin-mediated endocytosis using specific inhibitors or small interfering RNA-mediated knockdown of its key regulator, AP2M1, markedly reduced H-1PV entry. By contrast, we found no evidence of viral entry through caveolae-mediated endocytosis. We also show that H-1PV entry is dependent on dynamin, while viral trafficking occurs from early to late endosomes, with acidic pH necessary for a productive infection. This is the first study that characterises the cell entry pathways of oncolytic H-1PV.
Assuntos
Clatrina/fisiologia , Endocitose , Parvovirus H-1 , Neoplasias/terapia , Terapia Viral Oncolítica , Cavéolas/fisiologia , Linhagem Celular Tumoral , Dinaminas/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Internalização do VírusRESUMO
Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important for multiple signaling processes such as production of nitric oxide and caveolae-mediated intracellular trafficking. Using superresolution microscopy, fluorescence resonance energy transfer, and biochemical analysis, we observed that the EphB1 receptor tyrosine kinase constitutively interacts with caveolin-1 (Cav-1), the key structural protein of caveolae. Activation of EphB1 with its ligand Ephrin B1 induced EphB1 phosphorylation and the uncoupling EphB1 from Cav-1 and thereby promoted phosphorylation of Cav-1 by Src. Deletion of Cav-1 scaffold domain binding (CSD) motif in EphB1 prevented EphB1 binding to Cav-1 as well as Src-dependent Cav-1 phosphorylation, indicating the importance of CSD in the interaction. We also observed that Cav-1 protein expression and caveolae numbers were markedly reduced in ECs from EphB1-deficient (EphB1-/-) mice. The loss of EphB1 binding to Cav-1 promoted Cav-1 ubiquitination and degradation, and hence the loss of Cav-1 was responsible for reducing the caveolae numbers. These studies identify the crucial role of EphB1/Cav-1 interaction in the biogenesis of caveolae and in coordinating the signaling function of Cav-1 in ECs.
Assuntos
Cavéolas/metabolismo , Receptor EphB1/metabolismo , Animais , Cavéolas/fisiologia , Caveolina 1/metabolismo , Células Endoteliais/metabolismo , Efrina-B1/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Receptor EphB1/fisiologia , Transdução de Sinais/fisiologiaAssuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Transporte/fisiologia , Cavéolas/fisiologia , Mecanotransdução Celular/fisiologia , Animais , Caveolina 1/metabolismo , Caveolina 1/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Microambiente Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Mecanotransdução Celular/genética , Multimerização Proteica/fisiologia , Estresse MecânicoRESUMO
Caveolae are flask-shaped invaginations of the cell membrane rich in cholesterol and sphingomyelin, with caveolin proteins acting as their primary structural components that allow compartmentalization and orchestration of various signalling molecules. In this review, we discuss how pleiotropic functions of caveolin-1 (Cav1) and its intricate roles in numerous cellular functions including lipid trafficking, signalling, cell migration and proliferation, as well as cellular senescence, infection and inflammation, are integral for normal development and functioning of skin and its appendages. We then examine how disruption of the homeostatic levels of Cav1 can lead to development of various cutaneous pathophysiologies including skin cancers, cutaneous fibroses, psoriasis, alopecia, age-related changes in skin and aberrant wound healing and propose how levels of Cav1 may have theragnostic value in skin physiology/pathophysiology.
Assuntos
Cavéolas/fisiologia , Caveolina 1/metabolismo , Neoplasias Cutâneas/metabolismo , Fenômenos Fisiológicos da Pele , Pele/metabolismo , Infecções Bacterianas/metabolismo , Movimento Celular , Proliferação de Células , Senescência Celular , Fibrose/metabolismo , Cabelo/metabolismo , Humanos , Inflamação/metabolismo , Metabolismo dos Lipídeos , Psoríase/metabolismo , Transdução de Sinais , Pele/patologia , CicatrizaçãoRESUMO
In development, wound healing, and cancer metastasis, vertebrate cells move through 3D interstitial matrix, responding to chemical and physical guidance cues. Protrusion at the cell front has been extensively studied, but the retraction phase of the migration cycle is not well understood. Here, we show that fast-moving cells guided by matrix cues establish positive feedback control of rear retraction by sensing membrane tension. We reveal a mechanism of rear retraction in 3D matrix and durotaxis controlled by caveolae, which form in response to low membrane tension at the cell rear. Caveolae activate RhoA-ROCK1/PKN2 signaling via the RhoA guanidine nucleotide exchange factor (GEF) Ect2 to control local F-actin organization and contractility in this subcellular region and promote translocation of the cell rear. A positive feedback loop between cytoskeletal signaling and membrane tension leads to rapid retraction to complete the migration cycle in fast-moving cells, providing directional memory to drive persistent cell migration in complex matrices.
Assuntos
Movimento Celular/fisiologia , Pseudópodes/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Cavéolas/fisiologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Polaridade Celular/fisiologia , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/fisiologia , Citoesqueleto/metabolismo , Citosol/metabolismo , Matriz Extracelular/metabolismo , Humanos , Camundongos , Proteína Quinase C/metabolismo , Pseudópodes/metabolismo , Ratos , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.
Assuntos
Adenocarcinoma de Pulmão/patologia , Cavéolas/fisiologia , Endocitose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/patologia , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Células COS , Cavéolas/metabolismo , Sobrevivência Celular/genética , Células Cultivadas , Chlorocebus aethiops , Endocitose/genética , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ligação Proteica/fisiologia , Proto-Oncogene Mas , Células Sf9 , Transdução de Sinais/genética , SpodopteraRESUMO
To comprehensively understand the endocytosis of Sapelovirus A (PSV) entry into PK-15 cells, we studied PSV infection in the context of cell perturbations through drug inhibition, siRNA silencing and overexpression of dominant negative (DN) mutants. We showed here that PSV infection of PK-15 cells was unaffected by pretreated with chlorpromazine, EIPA, knockdown of the clathrin heavy chain or overexpression of Eps15 DN mutant. Conversely, PSV infection was sensitive to NH4Cl, chloroquine, dynasore, nystatin, MßCD and wortmannin with reduced PSV VP1 expression levels and virus titer. Additionally, PSV invasion leaded to rapid actin rearrangement and disruption of the cellular actin network enhanced PSV infection. After internalization the virus was transported to late endosomes and/or cycling endosomes that requires the participation of Rab7 and Rab11. Our findings demonstrate that PSV uses caveolae-dependent endocytosis as the predominant entry portal into PK-15 cells which requires low pH, dynamin, Rab7 and Rab11.
Assuntos
Cavéolas/fisiologia , Endocitose/fisiologia , Picornaviridae/fisiologia , Internalização do Vírus , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular , Concentração de Íons de Hidrogênio , Suínos , proteínas de unión al GTP Rab7RESUMO
Although many studies have reported that the breakdown of the blood-brain barrier (BBB) represents one of the major pathological changes in aging, the mechanism underlying this process remains relatively unexplored. In this study, we described that acid sphingomyelinase (ASM) derived from endothelial cells plays a critical role in BBB disruption in aging. ASM levels were elevated in the brain endothelium and plasma of aged humans and mice, resulting in BBB leakage through an increase in caveolae-mediated transcytosis. Moreover, ASM caused damage to the caveolae-cytoskeleton via protein phosphatase 1-mediated ezrin/radixin/moesin dephosphorylation in primary mouse brain endothelial cells. Mice overexpressing brain endothelial cell-specific ASM exhibited acceleration of BBB impairment and neuronal dysfunction. However, genetic inhibition and endothelial specific knock-down of ASM in mice improved BBB disruption and neurocognitive impairment during aging. Results of this study revealed a novel role of ASM in the regulation of BBB integrity and neuronal function in aging, thus highlighting the potential of ASM as a new therapeutic target for anti-aging. [BMB Reports 2019; 52(2): 111-112].
Assuntos
Envelhecimento/fisiologia , Barreira Hematoencefálica/fisiologia , Esfingomielina Fosfodiesterase/fisiologia , Animais , Encéfalo/metabolismo , Cavéolas/fisiologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Esfingomielina Fosfodiesterase/genética , Junções Íntimas/metabolismo , Transcitose/fisiologiaRESUMO
Plasma membrane (PM) of smooth muscle cells hosts channel molecules regulating the flow of various ions. An intact architecture of PM is essential to orchestrate proper channel functions in order to complete agonist-mediated contraction, which includes Ca2+ release from the sarcoplasmic reticulum (SR) to initiate contraction, and subsequent Ca2+ refilling into SR through PM to sustain muscle contraction. The Junctional Complex (JC), comprising of junctional SR, and its apposing PM and neighboring caveolae, provides a quasi-enclosed microdomain housing receptors as well as ion channels and also restricting ion diffusions into the cytosol so the cell achieves optimal performance. The spatial arrangement of the JC is believed to ensure an uninterrupted Ca2+ cycling route. Full understanding of the functional role of the JC is the key to elucidating the contractile mechanisms of vascular smooth muscle and the physiological function of vessel contraction. The JC can be further divided into two sub-divisions, namely the PM-SR and caveolar regions. Previously, we demonstrated the role of the PM-SR region in the initiation of muscle contraction using pharmacological tools on the inferior vena cava (IVC) of rabbit. In the current study, we further dissected the caveolar region using a cholesterol-disrupting agent to investigate the role of the caveolar region. We conclude that disruption of the caveolar region in rabbit IVC smooth muscle results in augmented muscle contraction in response to adrenergic stimulation and the altered Ca2+ signaling may underlie the augmented contractility. Anat Rec, 302:186-192, 2019. © 2018 Wiley Periodicals, Inc.
Assuntos
Cálcio/metabolismo , Cavéolas/fisiologia , Membrana Celular/química , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Cavéolas/efeitos dos fármacos , Colesterol/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Coelhos , beta-Ciclodextrinas/farmacologiaRESUMO
Transcytosis of nanoparticles (NPs) is emerging as an attractive alternative to the paracellular route in cancer drug delivery with studies suggesting targeting caveolae-mediated endocytosis to maximize NP transcytosis. However, there are limited studies on transcytosis of NPs, especially for corona-coated NPs. Most studies focused on cellular uptake as an indirect measure of the NP's transcellular permeability (Pd). Here, we probed the effect of protein corona on the uptake and transcytosis of 20, 40, 100, and 200 nm polystyrene nanoparticles (pNP-PC) across HUVECs in a microfluidic channel that modelled the microvasculature. We observed increased cell uptake with size of pNP-PC although it was the smallest 20 nm pNP-PC that exhibited the highest transcellular Pd. In the absence of corona however, cell uptake decreased with size, and the largest 200 nm pNP-PEG exhibited the lowest transcellular Pd. By inhibiting caveolae-mediated endocytosis in HUVECs, smaller pNPs had a larger drop in cell uptake than larger pNPs, regardless of surface coating. However, only the smallest (20 nm) and largest (200 nm) pNP-PC had a decrease in Pd following inhibition with MßCD. Our findings showed that the protein corona affected the transcytosis of NPs, and their uptake by caveolae-mediated endocytosis did not necessarily lead to transcytosis.
Assuntos
Cavéolas/fisiologia , Endocitose , Nanopartículas/metabolismo , Coroa de Proteína/química , Transcitose , Células Endoteliais da Veia Umbilical Humana , Humanos , Dispositivos Lab-On-A-Chip , PoliestirenosRESUMO
Caveolae are one of the most abundant and striking features of the plasma membrane of many mammalian cell types. These surface pits have fascinated biologists since their discovery by the pioneers of electron microscopy in the middle of the last century, but we are only just starting to understand their multiple functions. Molecular understanding of caveolar formation is advancing rapidly and we now know that sculpting the membrane to generate the characteristic bulb-shaped caveolar pit involves the coordinated action of integral membrane proteins and peripheral membrane coat proteins in a process dependent on their multiple interactions with membrane lipids. The resulting structure is further stabilised by protein complexes at the caveolar neck. Caveolae can bud to generate an endocytic carrier but can also be disassembled in response to specific stimuli to function as a mechanoprotective device. These structures have also been linked to numerous signalling pathways. Here, we will briefly summarise the current molecular and structural understanding of caveolar formation and dynamics, discuss how the crucial structural components of caveolae work together to generate a dynamic sensing domain, and discuss the implications of recent studies on the diverse roles proposed for caveolae in different cells and tissues.
Assuntos
Cavéolas/metabolismo , Cavéolas/fisiologia , Membrana Celular/fisiologia , Animais , Caveolinas/metabolismo , Estruturas da Membrana Celular/fisiologia , Humanos , Mamíferos , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologiaRESUMO
Cell-penetrating peptide (CPP) is a promising cargo for delivering bioactive molecules. In this study, the N terminus of VP1 from chicken anemia virus, designated as CVP1, was found to carry enriched arginine residues with α-helix. By confocal imaging, flow cytometry and MTT assay, we identified CVP1 as a novel, safe and efficient CPP. CVP1-FITC peptide could entry different types of cells tested with dose dependence, but without cytotoxic effects. Compared with TAT-FITC peptide, the CVP1-FITC peptide showed much higher cell-penetrating activity. Moreover, CVP1 could successfully deliver ß-glycosidase, poly (I:C) and plasmid into HCT116 cells. Inhibitors and temperature sensitivity analysis further indicated that the cell-penetrating activity of CVP1 was based on ATP-dependent and caveolae-mediated endocytosis. All these data demonstrate that CVP1 has efficient cell-penetrating activity and great potential for developing a novel delivery vector.
Assuntos
Cavéolas/fisiologia , Peptídeos Penetradores de Células/administração & dosagem , Vírus da Anemia da Galinha/fisiologia , Animais , Cavéolas/virologia , Linhagem Celular , Galinhas , Cães , Sistemas de Liberação de Medicamentos/veterinária , Endocitose/fisiologia , Células HCT116 , Células HEK293 , Humanos , Células Madin Darby de Rim CaninoRESUMO
Effective suppression of JAK-STAT signalling by the inducible inhibitor "suppressor of cytokine signalling 3" (SOCS3) is essential for limiting signalling from cytokine receptors. Here we show that cavin-1, a component of caveolae, is a functionally significant SOCS3-interacting protein. Biochemical and confocal imaging demonstrate that SOCS3 localisation to the plasma membrane requires cavin-1. SOCS3 is also critical for cavin-1 stabilisation, such that deletion of SOCS3 reduces the expression of cavin-1 and caveolin-1 proteins, thereby reducing caveola abundance in endothelial cells. Moreover, the interaction of cavin-1 and SOCS3 is essential for SOCS3 function, as loss of cavin-1 enhances cytokine-stimulated STAT3 phosphorylation and abolishes SOCS3-dependent inhibition of IL-6 signalling by cyclic AMP. Together, these findings reveal a new functionally important mechanism linking SOCS3-mediated inhibition of cytokine signalling to localisation at the plasma membrane via interaction with and stabilisation of cavin-1.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Animais , Cavéolas/fisiologia , Deleção de Genes , Regulação da Expressão Gênica , Células HEK293 , Humanos , Janus Quinases/genética , Janus Quinases/metabolismo , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Proteínas de Ligação a RNA/genética , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genéticaRESUMO
During early pregnancy, uterine epithelial cells (UECs) become less adherent to the underlying basal lamina and are subsequently removed so the blastocyst can invade the underlying stroma. This process involves the removal of focal adhesions from the basal plasma membrane of UECs. These focal adhesions are thought to be internalized by caveolae, which significantly increase in abundance at the time of blastocyst implantation. A recent in vitro study indicated that prominin-2 prevents the formation of caveolae by sequestering membrane cholesterol. The present study examines whether prominin-2 affects the formation of caveolae and loss of focal adhesions in UECs during normal and ovarian hyperstimulation (OH) pregnancy in the rat. At the time of fertilization during normal pregnancy, prominin-2 is distributed throughout the basolateral plasma membrane. However, at the time of implantation and coincident with an increase in caveolae, prominin-2 is lost from the basal plasma membrane. In contrast, prominin-2 remains in the basolateral plasma membrane throughout OH pregnancy. Transmission electron microscopy showed that this membrane contained few caveolae throughout OH pregnancy. Our results indicate that prominin-2 prevents the formation of caveolae. We suggest the retention of prominin-2 in the basal plasma membrane during OH pregnancy prevents the formation of caveolae and is responsible for the retention of focal adhesions in this membrane, thereby contributing to the reduced implantation rate observed after such treatments.
Assuntos
Cavéolas/fisiologia , Glicoproteínas de Membrana/fisiologia , Útero/fisiologia , Animais , Cavéolas/metabolismo , Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Estrogênios/administração & dosagem , Estrogênios/fisiologia , Feminino , Adesões Focais/fisiologia , Glicoproteínas de Membrana/metabolismo , Ovariectomia , Gravidez , Progesterona/administração & dosagem , Progesterona/fisiologia , Ratos Wistar , Útero/metabolismo , Útero/ultraestruturaRESUMO
INTRODUCTION: Studies in animal models have shown that unidirectional vesicular transport of amniotic fluid across the amnion plays a primary role in regulating amniotic fluid volume. Our objective was to explore vesicle type, vesicular uptake and intracellular distribution of vesicles in human amnion cells using high- and super-resolution fluorescence microscopy. METHODS: Placental amnion was obtained at cesarean section and amnion cells were prepared and cultured. At 20%-50% confluence, the cells were incubated with fluorophore conjugated macromolecules for 1-30 min at 22 °C or 37 °C. Fluorophore labeled macromolecules were selected as markers of receptor-mediated caveolar and clathrin-coated vesicular uptake as well as non-specific endocytosis. After fluorophore treatment, the cells were fixed, imaged and vesicles counted using Imaris® software. RESULTS: Vesicular uptake displayed first order saturation kinetics with half saturation times averaging 1.3 min at 37 °C compared to 4.9 min at 22 °C, with non-specific endocytotic uptake being more rapid at both temperatures. There was extensive cell-to-cell variability in uptake rate. Under super-resolution microscopy, the pattern of intracellular spatial distribution was distinct for each macromolecule. Co-localization of fluorescently labeled macromolecules was very low at vesicular dimensions. CONCLUSIONS: In human placental amnion cells, 1) vesicular uptake of macromolecules is rapid, consistent with the concept that vesicular transcytosis across the amnion plays a role in the regulation of amniotic fluid volume; 2) uptake is temperature dependent and variable among individual cells; 3) the unique intracellular distributions suggest distinct functions for each vesicle type; 4) non-receptor mediated vesicular uptake may be a primary vesicular uptake mechanism.
Assuntos
Âmnio/citologia , Cavéolas/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Endocitose , Células Epiteliais/fisiologia , Feminino , Humanos , Substâncias Macromoleculares , GravidezRESUMO
Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.
Assuntos
Proteínas de Transporte/genética , Cavéolas/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Proteínas de Transporte Vesicular/genética , Animais , Fenômenos Biomecânicos , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Camundongos , Células NIH 3T3 , Proteínas Nucleares/metabolismo , Estresse Mecânico , Proteínas de Transporte Vesicular/metabolismoRESUMO
Caveolin-1 (CAV1) is an essential component of caveolae and is implicated in numerous physiological processes. Recent studies have identified heterozygous mutations in the CAV1 gene in patients with pulmonary arterial hypertension (PAH), but the mechanisms by which these mutations impact caveolae assembly and contribute to disease remain unclear. To address this question, we examined the consequences of a familial PAH-associated frameshift mutation in CAV1, P158PfsX22, on caveolae assembly and function. We show that C-terminus of the CAV1 P158 protein contains a functional ER-retention signal that inhibits ER exit and caveolae formation and accelerates CAV1 turnover in Cav1-/- MEFs. Moreover, when coexpressed with wild-type (WT) CAV1 in Cav1-/- MEFs, CAV1-P158 functions as a dominant negative by partially disrupting WT CAV1 trafficking. In patient skin fibroblasts, CAV1 and caveolar accessory protein levels are reduced, fewer caveolae are observed, and CAV1 complexes exhibit biochemical abnormalities. Patient fibroblasts also exhibit decreased resistance to a hypo-osmotic challenge, suggesting the function of caveolae as membrane reservoir is compromised. We conclude that the P158PfsX22 frameshift introduces a gain of function that gives rise to a dominant negative form of CAV1, defining a new mechanism by which disease-associated mutations in CAV1 impair caveolae assembly.
Assuntos
Caveolina 1/genética , Caveolina 1/metabolismo , Cavéolas/fisiologia , Endocitose , Retículo Endoplasmático , Fibroblastos/metabolismo , Mutação da Fase de Leitura/genética , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Mutação , Transporte ProteicoRESUMO
Caveolae are signal transduction centers, yet their subcellular distribution and preservation in cardiac myocytes after cell isolation are not well documented. Here, we quantify caveolae located within 100 nm of the outer cell surface membrane in rabbit single-ventricular cardiomyocytes over 8 h post-isolation and relate this to the presence of caveolae in intact tissue. Hearts from New Zealand white rabbits were either chemically fixed by coronary perfusion or enzymatically digested to isolate ventricular myocytes, which were subsequently fixed at 0, 3, and 8 h post-isolation. In live cells, the patch-clamp technique was used to measure whole-cell plasma membrane capacitance, and in fixed cells, caveolae were quantified by transmission electron microscopy. Changes in cell-surface topology were assessed using scanning electron microscopy. In fixed ventricular myocardium, dual-axis electron tomography was used for three-dimensional reconstruction and analysis of caveolae in situ. The presence and distribution of surface-sarcolemmal caveolae in freshly isolated cells matches that of intact myocardium. With time, the number of surface-sarcolemmal caveolae decreases in isolated cardiomyocytes. This is associated with a gradual increase in whole-cell membrane capacitance. Concurrently, there is a significant increase in area, diameter, and circularity of sub-sarcolemmal mitochondria, indicative of swelling. In addition, electron tomography data from intact heart illustrate the regular presence of caveolae not only at the surface sarcolemma, but also on transverse-tubular membranes in ventricular myocardium. Thus, caveolae are dynamic structures, present both at surface-sarcolemmal and transverse-tubular membranes. After cell isolation, the number of surface-sarcolemmal caveolae decreases significantly within a time frame relevant for single-cell research. The concurrent increase in cell capacitance suggests that membrane incorporation of surface-sarcolemmal caveolae underlies this, but internalization and/or micro-vesicle loss to the extracellular space may also contribute. Given that much of the research into cardiac caveolae-dependent signaling utilizes isolated cells, and since caveolae-dependent pathways matter for a wide range of other study targets, analysis of isolated cell data should take the time post-isolation into account.