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1.
Nanoscale Horiz ; 7(4): 385-395, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-35289830

RESUMO

Clathrin-coated pits and caveolae are nanoscale invaginations of the plasma membrane of cells, forming through the assembly of membrane coat and accessory proteins in a tightly regulated process. We have analyzed the development of these membrane coat structures with high spatial and temporal resolution and sensitivity using super-resolution single-molecule localization microscopy (SMLM) on live cells. To this end, we developed a sophisticated clustering and data analysis workflow that automatically extracts the relevant information from SMLM image sequences taken on live cells. We quantified lifetime distributions of clathrin-coated and caveolar structures, and analyzed their growth dynamics. Moreover, we observed hotspots in the plasma membrane where coat structures appear repeatedly. The stunningly similar temporal development of clathrin-coated and caveolar structures suggests that key accessory proteins, some of which are shared between the two types of membrane coat structures, orchestrate the temporal evolution of these complex architectures.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Cavéolas/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Imagem Individual de Molécula
2.
Dev Cell ; 56(8): 1164-1181.e12, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33761321

RESUMO

Cells probe their surrounding matrix for attachment sites via integrins that are internalized by endocytosis. We find that SH3BP4 regulates integrin surface expression in a signaling-dependent manner via clathrin-coated pits (CCPs). Dephosphorylated SH3BP4 at S246 is efficiently recruited to CCPs, while upon Akt phosphorylation, SH3BP4 is sequestered by 14-3-3 adaptors and excluded from CCPs. In the absence of Akt activity, SH3BP4 binds GIPC1 and targets neuropilin-1 and α5/ß1-integrin for endocytosis, leading to inhibition of cell spreading. Similarly, chemorepellent semaphorin-3a binds neuropilin-1 to activate PTEN, which antagonizes Akt and thus recruits SH3BP4 to CCPs to internalize both receptors and induce cell contraction. In PTEN mutant non-small cell lung cancer cells with high Akt activity, expression of non-phosphorylatable active SH3BP4-S246A restores semaphorin-3a induced cell contraction. Thus, SH3BP4 links Akt signaling to endocytosis of NRP1 and α5/ß1-integrins to modulate cell-matrix interactions in response to intrinsic and extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose , Integrina alfa5/metabolismo , Neuropilina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas 14-3-3/metabolismo , Linhagem Celular Tumoral , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Mutantes/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Ligação Proteica , Semaforina-3A/metabolismo , Transdução de Sinais
3.
Proc Natl Acad Sci U S A ; 117(50): 31591-31602, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257546

RESUMO

Clathrin-mediated endocytosis (CME) begins with the nucleation of clathrin assembly on the plasma membrane, followed by stabilization and growth/maturation of clathrin-coated pits (CCPs) that eventually pinch off and internalize as clathrin-coated vesicles. This highly regulated process involves a myriad of endocytic accessory proteins (EAPs), many of which are multidomain proteins that encode a wide range of biochemical activities. Although domain-specific activities of EAPs have been extensively studied, their precise stage-specific functions have been identified in only a few cases. Using single-guide RNA (sgRNA)/dCas9 and small interfering RNA (siRNA)-mediated protein knockdown, combined with an image-based analysis pipeline, we have determined the phenotypic signature of 67 EAPs throughout the maturation process of CCPs. Based on these data, we show that EAPs can be partitioned into phenotypic clusters, which differentially affect CCP maturation and dynamics. Importantly, these clusters do not correlate with functional modules based on biochemical activities. Furthermore, we discover a critical role for SNARE proteins and their adaptors during early stages of CCP nucleation and stabilization and highlight the importance of GAK throughout CCP maturation that is consistent with GAK's multifunctional domain architecture. Together, these findings provide systematic, mechanistic insights into the plasticity and robustness of CME.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Análise por Conglomerados , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Microscopia Intravital/métodos , Substâncias Luminescentes/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , RNA Interferente Pequeno/metabolismo
4.
Nat Commun ; 11(1): 5108, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037189

RESUMO

The spectrin-based membrane skeleton is a major component of the cell cortex. While expressed by all metazoans, its dynamic interactions with the other cortex components, including the plasma membrane or the acto-myosin cytoskeleton, are poorly understood. Here, we investigate how spectrin re-organizes spatially and dynamically under the membrane during changes in cell mechanics. We find spectrin and acto-myosin to be spatially distinct but cooperating during mechanical challenges, such as cell adhesion and contraction, or compression, stretch and osmolarity fluctuations, creating a cohesive cortex supporting the plasma membrane. Actin territories control protrusions and contractile structures while spectrin territories concentrate in retractile zones and low-actin density/inter-contractile regions, acting as a fence that organize membrane trafficking events. We unveil here the existence of a dynamic interplay between acto-myosin and spectrin necessary to support a mesoscale organization of the lipid bilayer into spatially-confined cortical territories during cell mechanoresponse.


Assuntos
Actomiosina/metabolismo , Membrana Celular/metabolismo , Espectrina/metabolismo , Actinas/metabolismo , Animais , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Microscopia Confocal , Células NIH 3T3 , Espectrina/genética , Estresse Mecânico
5.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770195

RESUMO

Clathrin-mediated endocytosis occurs via the assembly of clathrin-coated pits (CCPs) that invaginate and pinch off to form clathrin-coated vesicles (CCVs). It is well known that adaptor protein 2 (AP2) complexes trigger clathrin assembly on the plasma membrane, and biochemical and structural studies have revealed the nature of these interactions. Numerous endocytic accessory proteins collaborate with clathrin and AP2 to drive CCV formation. However, many questions remain as to the molecular events involved in CCP initiation, stabilization, and curvature generation. Indeed, a plethora of recent evidence derived from cell perturbation, correlative light and EM tomography, live-cell imaging, modeling, and high-resolution structural analyses has revealed more complexity and promiscuity in the protein interactions driving CCP maturation than anticipated. After briefly reviewing the evidence supporting prevailing models, we integrate these new lines of evidence to develop a more dynamic and flexible model for how redundant, dynamic, and competing protein interactions can drive endocytic CCV formation and suggest new approaches to test emerging models.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/fisiologia , Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Endocitose/fisiologia , Humanos
6.
Traffic ; 21(9): 603-616, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32657003

RESUMO

Clathrin mediated endocytosis (CME) has been extensively studied in living cells by quantitative total internal reflection fluorescence microscopy (TIRFM). Fluorescent protein fusions to subunits of the major coat proteins, clathrin light chains or the heterotetrameric adaptor protein (AP2) complexes, have been used as fiduciary markers of clathrin coated pits (CCPs). However, the functionality of these fusion proteins has not been rigorously compared. Here, we generated stable cells lines overexpressing mRuby-CLCa and/or µ2-eGFP, σ2-eGFP, two markers currently in use, or a novel marker generated by inserting eGFP into the unstructured hinge region of the α subunit (α-eGFP). Using biochemical and TIRFM-based assays, we compared the functionality of the AP2 markers. All of the eGFP-tagged subunits were efficiently incorporated into AP2 and displayed greater accuracy in image-based CCP analyses than mRuby-CLCa. However, overexpression of either µ2-eGFP or σ2-eGFP impaired transferrin receptor uptake. In addition, µ2-eGFP reduced the rates of CCP initiation and σ2-eGFP perturbed AP2 incorporation into CCPs and CCP maturation. In contrast, CME and CCP dynamics were unperturbed in cells overexpressing α-eGFP. Moreover, α-eGFP was a more sensitive and accurate marker of CCP dynamics than mRuby-CLCa. Thus, our work establishes α-eGFP as a robust, fully functional marker for CME.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Ligação Proteica
7.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32642759

RESUMO

Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.


Assuntos
Processamento Alternativo/fisiologia , Cadeias Pesadas de Clatrina/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Adulto , Animais , Membrana Celular/metabolismo , Criança , Endocitose/fisiologia , Éxons/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Adulto Jovem
8.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32520988

RESUMO

Clathrin-mediated endocytosis (CME) occurs via the formation of clathrin-coated vesicles from clathrin-coated pits (CCPs). Clathrin is recruited to CCPs through interactions between the AP2 complex and its N-terminal domain, which in turn recruits endocytic accessory proteins. Inhibitors of CME that interfere with clathrin function have been described, but their specificity and mechanisms of action are unclear. Here we show that overexpression of the N-terminal domain with (TDD) or without (TD) the distal leg inhibits CME and CCP dynamics by perturbing clathrin interactions with AP2 and SNX9. TDD overexpression does not affect clathrin-independent endocytosis or, surprisingly, AP1-dependent lysosomal trafficking from the Golgi. We designed small membrane-permeant peptides that encode key functional residues within the four known binding sites on the TD. One peptide, Wbox2, encoding residues along the W-box motif binding surface, binds to SNX9 and AP2 and potently and acutely inhibits CME.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Peptídeos/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Sítios de Ligação/fisiologia , Linhagem Celular , Invaginações Revestidas da Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Nexinas de Classificação/metabolismo
9.
Angew Chem Int Ed Engl ; 59(37): 16173-16180, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32521111

RESUMO

In a conjugated polymer-based single-particle heterojunction, stochastic fluctuations of the photogenerated hole population lead to spontaneous fluorescence switching. We found that 405 nm irradiation can induce charge recombination and activate the single-particle emission. Based on these phenomena, we developed a novel class of semiconducting polymer dots that can operate in two superresolution imaging modes. The spontaneous switching mode offers efficient imaging of large areas, with <10 nm localization precision, while the photoactivation/deactivation mode offers slower imaging, with further improved localization precision (ca. 1 nm), showing advantages in resolving small structures that require high spatial resolution. Superresolution imaging of microtubules and clathrin-coated pits was demonstrated, under both modes. The excellent localization precision and versatile imaging options provided by these nanoparticles offer clear advantages for imaging of various biological systems.


Assuntos
Polímeros/química , Semicondutores , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Fluorescência , Microscopia de Fluorescência , Microtúbulos/metabolismo
10.
J Cell Sci ; 133(11)2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499318

RESUMO

Clathrin-mediated endocytosis is the main entry route for most cell surface receptors and their ligands. It is regulated by clathrin-coated structures that are endowed with the ability to cluster receptors and to locally bend the plasma membrane, resulting in the formation of receptor-containing vesicles that bud into the cytoplasm. This canonical role of clathrin-coated structures has been shown to play a fundamental part in many different aspects of cell physiology. However, it has recently become clear that the ability of clathrin-coated structures to deform membranes can be perturbed. In addition to chemical or genetic alterations, numerous environmental conditions can physically prevent or slow down membrane bending and/or budding at clathrin-coated structures. The resulting 'frustrated endocytosis' is emerging as not merely a passive consequence, but one that actually fulfils some very specific and important cellular functions. In this Review, we provide an historical and defining perspective on frustrated endocytosis in the clathrin pathway of mammalian cells, before discussing its causes and highlighting the possible functional consequences in physiology and diseases.


Assuntos
Clatrina , Endocitose , Animais , Membrana Celular , Vesículas Revestidas por Clatrina , Invaginações Revestidas da Membrana Celular
11.
Elife ; 92020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32352376

RESUMO

Clathrin-mediated endocytosis (CME) in mammalian cells is driven by resilient machinery that includes >70 endocytic accessory proteins (EAP). Accordingly, perturbation of individual EAPs often results in minor effects on biochemical measurements of CME, thus providing inconclusive/misleading information regarding EAP function. Live-cell imaging can detect earlier roles of EAPs preceding cargo internalization; however, this approach has been limited because unambiguously distinguishing abortive coats (ACs) from bona fide clathrin-coated pits (CCPs) is required but unaccomplished. Here, we develop a thermodynamics-inspired method, "disassembly asymmetry score classification (DASC)", that resolves ACs from CCPs based on single channel fluorescent movies. After extensive verification, we use DASC-resolved ACs and CCPs to quantify CME progression in 11 EAP knockdown conditions. We show that DASC is a sensitive detector of phenotypic variation in CCP dynamics that is uncorrelated to the variation in biochemical measurements of CME. Thus, DASC is an essential tool for uncovering EAP function.


Assuntos
Clatrina/fisiologia , Endocitose/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Humanos , Termodinâmica
12.
Cell Biol Int ; 44(5): 1252-1259, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31970864

RESUMO

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin-3 is the main cause of neurodegeneration in Machado-Joseph disease. Ataxin-3 contains an N-terminal catalytic domain, called Josephin domain, and a highly aggregation-prone C-terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin-mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin-3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin-3 could impact CME. Here, we report that both the wild-type and the expanded ataxin-3 reduce transferrin internalization and expanded ataxin-3 impacts dynamics of clathrin-coated pits (CCPs) by reducing CCP nucleation and increasing short-lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.


Assuntos
Ataxina-3/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Peptídeos/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular , Humanos , Doença de Machado-Joseph , Agregação Patológica de Proteínas
13.
Elife ; 92020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31971511

RESUMO

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.


Assuntos
Arabidopsis , Clatrina , Invaginações Revestidas da Membrana Celular , Endocitose/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Evolução Biológica , Clatrina/química , Clatrina/metabolismo , Clatrina/ultraestrutura , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Microscopia Eletrônica , Modelos Biológicos
14.
Nat Commun ; 10(1): 4974, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31672988

RESUMO

Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Leves de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Cadeias Leves de Clatrina/ultraestrutura , Cistos , Endocitose , Humanos , Espectroscopia de Ressonância Magnética , Cadeias Pesadas de Miosina/ultraestrutura , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas
15.
Dev Cell ; 50(4): 494-508.e11, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31430451

RESUMO

Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the µ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. µ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with µ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Endocitose/genética , Nexinas de Classificação/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Clatrina/genética , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/genética , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/genética , Invaginações Revestidas da Membrana Celular/metabolismo , Humanos , Fosforilação/genética , Ligação Proteica/genética
16.
Nat Commun ; 9(1): 2604, 2018 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-29973588

RESUMO

A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.


Assuntos
Ferritinas/genética , Corantes Fluorescentes/química , Microscopia Eletrônica/métodos , Sirolimo/química , Coloração e Rotulagem/métodos , Proteínas Adaptadoras de Transdução de Sinal , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Ferritinas/química , Ferritinas/metabolismo , Expressão Gênica , Células HeLa , Humanos , Proteínas dos Microfilamentos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Razão Sinal-Ruído , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
17.
Endocrinology ; 159(8): 2953-2965, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29931263

RESUMO

The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. In this study, we use fast multicolor single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SSTR2 clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SSTR2 expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.


Assuntos
Filaminas/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Citoesqueleto de Actina/metabolismo , Adenoma/tratamento farmacológico , Adenoma/genética , Adenoma/metabolismo , Animais , Células CHO , Invaginações Revestidas da Membrana Celular/metabolismo , Cricetulus , Filaminas/ultraestrutura , Adenoma Hipofisário Secretor de Hormônio do Crescimento/tratamento farmacológico , Adenoma Hipofisário Secretor de Hormônio do Crescimento/genética , Adenoma Hipofisário Secretor de Hormônio do Crescimento/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/ultraestrutura , Imagem Individual de Molécula
18.
Biochem Biophys Res Commun ; 501(1): 280-285, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29729269

RESUMO

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca2+ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacocinética , Peptídeos Catiônicos Antimicrobianos/toxicidade , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clorpromazina/farmacologia , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Invaginações Revestidas da Membrana Celular/metabolismo , Regulação para Baixo , Humanos , Osteoblastos/patologia
19.
Sci Rep ; 8(1): 5658, 2018 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-29618727

RESUMO

Polymerisation of clathrin is a key process that underlies clathrin-mediated endocytosis. Clathrin-coated vesicles are responsible for cell internalization of external substances required for normal homeostasis and life -sustaining activity. There are several hypotheses describing formation of closed clathrin structures. According to one of the proposed mechanisms cage formation may start from a flat lattice buildup on the cellular membrane, which is later transformed into a curved structure. Creation of the curved surface requires rearrangement of the lattice, induced by additional molecular mechanisms. Different potential mechanisms require a modeling framework that can be easily modified to compare between them. We created an extendable rule-based model that describes polymerisation of clathrin molecules and various scenarios of cage formation. Using Global Sensitivity Analysis (GSA) we obtained parameter sets describing clathrin pentagon closure and the emergence/production and closure of large-size clathrin cages/vesicles. We were able to demonstrate that the model can reproduce budding of the clathrin cage from an initial flat array.


Assuntos
Membrana Celular/química , Clatrina/química , Invaginações Revestidas da Membrana Celular/química , Modelos Teóricos , Polimerização , Conformação Proteica , Humanos , Termodinâmica
20.
Sci Rep ; 8(1): 5529, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615680

RESUMO

An available pathway to prepare the ionized covalent organic nanosheets (iCONs) has been proposed by a metal-assisted aqueous-phase exfoliation route from covalent organic frameworks. The soluble and belt-shaped iCONs could immobilize a large quantity of proteins (2.73 mg/mg, BSA/iCONs) and hence serve as transporters to enhance the protein uptake by cancer cells. Meanwhile, their energy-dependent endocytosis pathway via clathrin-coated pits has been proved as well.


Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Estruturas Metalorgânicas/química , Metais/química , Soroalbumina Bovina/metabolismo , Animais , Disponibilidade Biológica , Bovinos , Sobrevivência Celular , Células Hep G2 , Humanos , Transporte Proteico , Soroalbumina Bovina/química , Solubilidade , Água
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