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1.
Science ; 385(6711): eado2032, 2024 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-39172837

RESUMO

Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.


Assuntos
Actinas , Axônios , Clatrina , Endocitose , Espectrina , Animais , Humanos , Ratos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Células HEK293 , Plasticidade Neuronal , Neurônios/metabolismo , Espectrina/metabolismo
2.
Cell Mol Life Sci ; 81(1): 43, 2024 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-38217571

RESUMO

Adherent cells ensure membrane homeostasis during de-adhesion by various mechanisms, including endocytosis. Although mechano-chemical feedbacks involved in this process have been studied, the step-by-step build-up and resolution of the mechanical changes by endocytosis are poorly understood. To investigate this, we studied the de-adhesion of HeLa cells using a combination of interference reflection microscopy, optical trapping and fluorescence experiments. We found that de-adhesion enhanced membrane height fluctuations of the basal membrane in the presence of an intact cortex. A reduction in the tether force was also noted at the apical side. However, membrane fluctuations reveal phases of an initial drop in effective tension followed by saturation. The area fractions of early (Rab5-labelled) and recycling (Rab4-labelled) endosomes, as well as transferrin-labelled pits close to the basal plasma membrane, also transiently increased. On blocking dynamin-dependent scission of endocytic pits, the regulation of fluctuations was not blocked, but knocking down AP2-dependent pit formation stopped the tension recovery. Interestingly, the regulation could not be suppressed by ATP or cholesterol depletion individually but was arrested by depleting both. The data strongly supports Clathrin and AP2-dependent pit-formation to be central to the reduction in fluctuations confirmed by super-resolution microscopy. Furthermore, we propose that cholesterol-dependent pits spontaneously regulate tension under ATP-depleted conditions.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Humanos , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Células HeLa , Endocitose/fisiologia , Colesterol/metabolismo , Trifosfato de Adenosina/metabolismo , Membrana Celular/metabolismo
3.
Nat Commun ; 14(1): 4602, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-37528083

RESUMO

Clathrin-mediated endocytosis is pivotal to signal transduction pathways between the extracellular environment and the intracellular space. Evidence from live-cell imaging and super-resolution microscopy of mammalian cells suggests an asymmetric distribution of actin fibres near the clathrin-coated pit, which induces asymmetric pit-closing rather than radial constriction. However, detailed molecular mechanisms of this 'asymmetricity' remain elusive. Herein, we used high-speed atomic force microscopy to demonstrate that CIP4, a multi-domain protein with a classic F-BAR domain and intrinsically disordered regions, is necessary for asymmetric pit-closing. Strong self-assembly of CIP4 via intrinsically disordered regions, together with stereospecific interactions with the curved membrane and actin-regulating proteins, generates a small actin-rich environment near the pit, which deforms the membrane and closes the pit. Our results provide mechanistic insights into how disordered and structured domain collaboration promotes spatio-temporal actin polymerisation near the plasma membrane.


Assuntos
Actinas , Endocitose , Animais , Actinas/metabolismo , Membrana Celular/metabolismo , Microscopia , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Mamíferos/metabolismo
4.
J Biol Chem ; 299(3): 102963, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36731797

RESUMO

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific cargo destined for internalization, generate membrane curvature, and in many cases undergo scission from the plasma membrane to yield intracellular vesicles. The diversity of functions of cell surface proteins controlled via internalization by CME may suggest that regulation of CCP formation could be effective to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked ß-GlcNAc (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. Here, we examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs are controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.


Assuntos
Invaginações Revestidas da Membrana Celular , Endocitose , N-Acetilglucosaminiltransferases , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo
5.
Elife ; 112022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35852853

RESUMO

The formation of a clathrin-coated vesicle (CCV) is a major membrane remodeling process that is crucial for membrane traffic in cells. Besides clathrin, these vesicles contain at least 100 different proteins although it is unclear how many are essential for the formation of the vesicle. Here, we show that intracellular clathrin-coated formation can be induced in living cells using minimal machinery and that it can be achieved on various membranes, including the mitochondrial outer membrane. Chemical heterodimerization was used to inducibly attach a clathrin-binding fragment 'hook' to an 'anchor' protein targeted to a specific membrane. Endogenous clathrin assembled to form coated pits on the mitochondria, termed MitoPits, within seconds of induction. MitoPits are double-membraned invaginations that form preferentially on high curvature regions of the mitochondrion. Upon induction, all stages of CCV formation - initiation, invagination, and even fission - were faithfully reconstituted. We found no evidence for the functional involvement of accessory proteins in this process. In addition, fission of MitoPit-derived vesicles was independent of known scission factors including dynamins and dynamin-related protein 1 (Drp1), suggesting that the clathrin cage generates sufficient force to bud intracellular vesicles. Our results suggest that, following its recruitment, clathrin is sufficient for intracellular CCV formation.


Assuntos
Clatrina , Invaginações Revestidas da Membrana Celular , Membrana Celular/metabolismo , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Dinaminas/metabolismo , Endocitose , Membranas Intracelulares/metabolismo
6.
Nat Commun ; 13(1): 3697, 2022 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-35760780

RESUMO

Membrane budding entails forces to transform flat membrane into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. By visualizing proteins in mediating endocytic budding in live neuroendocrine cells, performing in vitro protein reconstitution and physical modeling, we discovered how non-coated-membrane budding is mediated: actin filaments and dynamin generate a pulling force transforming flat membrane into Λ-shape; subsequently, dynamin helices surround and constrict Λ-profile's base, transforming Λ- to Ω-profile, and then constrict Ω-profile's pore, converting Ω-profiles to vesicles. These mechanisms control budding speed, vesicle size and number, generating diverse endocytic modes differing in these parameters. Their impact is widespread beyond secretory cells, as the unexpectedly powerful functions of dynamin and actin, previously thought to mediate fission and overcome tension, respectively, may contribute to many dynamin/actin-dependent non-coated-membrane buddings, coated-membrane buddings, and other membrane remodeling processes.


Assuntos
Actinas , Endocitose , Actinas/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Dinaminas/metabolismo
7.
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
8.
J Cell Biol ; 221(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35320342

RESUMO

Signaling by the activated epidermal growth factor receptor (EGFR) results in diverse cell fates. In this issue, Cabral-Dias et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.201808181) demonstrate how plasma membrane clathrin coated pits can act as a signaling platform for one branch of EGFR downstream signaling.


Assuntos
Vesículas Revestidas por Clatrina , Proteínas Proto-Oncogênicas c-akt , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Sci China Life Sci ; 65(2): 341-361, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34047913

RESUMO

Viruses utilize cellular lipids and manipulate host lipid metabolism to ensure their replication and spread. Therefore, the identification of lipids and metabolic pathways that are suitable targets for antiviral development is crucial. Using a library of compounds targeting host lipid metabolic factors and testing them for their ability to block pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) infection, we found that U18666A, a specific inhibitor of Niemann-Pick C1 (NPC1), is highly potent in suppressing the entry of diverse viruses including pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NPC1 deficiency markedly attenuates viral growth by decreasing cholesterol abundance in the plasma membrane, thereby inhibiting the dynamics of clathrin-coated pits (CCPs), which are indispensable for clathrin-mediated endocytosis. Significantly, exogenous cholesterol can complement the dynamics of CCPs, leading to efficient viral entry and infectivity. Administration of U18666A improves the survival and pathology of PRV- and influenza A virus-infected mice. Thus, our studies demonstrate a unique mechanism by which NPC1 inhibition achieves broad antiviral activity, indicating a potential new therapeutic strategy against SARS-CoV-2, as well as other emerging viruses.


Assuntos
Androstenos/farmacologia , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Vírus de DNA/efeitos dos fármacos , Proteína C1 de Niemann-Pick/fisiologia , Vírus de RNA/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Vírus de DNA/fisiologia , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Vírus de RNA/fisiologia
10.
Dev Cell ; 56(22): 3146-3159.e5, 2021 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-34774130

RESUMO

Sculpting a flat patch of membrane into an endocytic vesicle requires curvature generation on the cell surface, which is the primary function of the endocytosis machinery. Using super-resolved live cell fluorescence imaging, we demonstrate that curvature generation by individual clathrin-coated pits can be detected in real time within cultured cells and tissues of developing organisms. Our analyses demonstrate that the footprint of clathrin coats increases monotonically during the formation of pits at different levels of plasma membrane tension. These findings are only compatible with models that predict curvature generation at the early stages of endocytic clathrin pit formation. We also found that CALM adaptors associated with clathrin plaques form clusters, whereas AP2 distribution is more homogenous. Considering the curvature sensing and driving roles of CALM, we propose that CALM clusters may increase the strain on clathrin lattices locally, eventually giving rise to rupture and subsequent pit completion at the edges of plaques.


Assuntos
Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose/fisiologia , Sinapses/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Clatrina/farmacologia , Invaginações Revestidas da Membrana Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células HeLa , Humanos
11.
EMBO J ; 40(19): e108795, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34487371

RESUMO

Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the ß2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of ß2 hinge-appendage (ß2HA). We find that the ß2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, ß2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of ß2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that ß2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clatrina/química , Clatrina/metabolismo , Vesículas Revestidas/química , Vesículas Revestidas/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Sítios de Ligação , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/metabolismo , Endocitose , Imunofluorescência , Células HeLa , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Relação Estrutura-Atividade
12.
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
13.
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
14.
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
15.
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
16.
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
17.
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
18.
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
19.
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
20.
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
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