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1.
Cell ; 187(2): 219-224, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38242078

RESUMO

50 years ago, cell biology was a nascent field. Today, it is a vast discipline whose principles and tools are also applied to other disciplines; vice versa, cell biologists are inspired by other fields. So, the question begs: what is cell biology? The answers are as diverse as the people who define it.

2.
Cell ; 177(7): 1738-1756.e23, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31104842

RESUMO

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the ß1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.


Assuntos
Integrina beta1/metabolismo , Mecanotransdução Celular , Microdomínios da Membrana/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Cricetulus , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina beta1/genética , Microdomínios da Membrana/genética , Vinculina/genética , Vinculina/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo
3.
Cell ; 172(1-2): 305-317.e10, 2018 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-29328918

RESUMO

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.


Assuntos
Citoesqueleto de Actina/metabolismo , Membrana Celular/metabolismo , Receptores de Hialuronatos/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Animais , Sítios de Ligação , Células COS , Células Cultivadas , Chlorocebus aethiops , Feminino , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica
4.
Annu Rev Cell Dev Biol ; 35: 55-84, 2019 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-31283376

RESUMO

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.


Assuntos
Membrana Celular/metabolismo , Endocitose , Transdução de Sinais , Animais , Evolução Biológica , Membrana Celular/química , Homeostase , Humanos
5.
Cell ; 169(1): 108-119.e20, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28340336

RESUMO

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling.


Assuntos
Ligantes , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , DNA/metabolismo , Humanos , Células Jurkat , Fosforilação , Imagem Individual de Molécula , Proteína-Tirosina Quinase ZAP-70/análise
6.
Cell ; 161(3): 581-594, 2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25910209

RESUMO

Understanding how functional lipid domains in live cell membranes are generated has posed a challenge. Here, we show that transbilayer interactions are necessary for the generation of cholesterol-dependent nanoclusters of GPI-anchored proteins mediated by membrane-adjacent dynamic actin filaments. We find that long saturated acyl-chains are required for forming GPI-anchor nanoclusters. Simultaneously, at the inner leaflet, long acyl-chain-containing phosphatidylserine (PS) is necessary for transbilayer coupling. All-atom molecular dynamics simulations of asymmetric multicomponent-membrane bilayers in a mixed phase provide evidence that immobilization of long saturated acyl-chain lipids at either leaflet stabilizes cholesterol-dependent transbilayer interactions forming local domains with characteristics similar to a liquid-ordered (lo) phase. This is verified by experiments wherein immobilization of long acyl-chain lipids at one leaflet effects transbilayer interactions of corresponding lipids at the opposite leaflet. This suggests a general mechanism for the generation and stabilization of nanoscale cholesterol-dependent and actin-mediated lipid clusters in live cell membranes.


Assuntos
Proteínas Ligadas a Lipídeos/metabolismo , Actinas/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Glicosilfosfatidilinositóis/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilserinas/metabolismo
7.
Nat Rev Mol Cell Biol ; 18(6): 361-374, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28356571

RESUMO

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Animais , Humanos , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo
8.
Nat Rev Mol Cell Biol ; 16(5): 311-21, 2015 05.
Artigo em Inglês | MEDLINE | ID: mdl-25857812

RESUMO

How endocytic pits are built in clathrin- and caveolin-independent endocytosis still remains poorly understood. Recent insight suggests that different forms of clathrin-independent endocytosis might involve the actin-driven focusing of membrane constituents, the lectin-glycosphingolipid-dependent construction of endocytic nanoenvironments, and Bin-Amphiphysin-Rvs (BAR) domain proteins serving as scaffolding modules. We discuss the need for different types of internalization processes in the context of diverse cellular functions, the existence of clathrin-independent mechanisms of cargo recruitment and membrane bending from a biological and physical perspective, and finally propose a generic scheme for the formation of clathrin-independent endocytic pits.


Assuntos
Membrana Celular/química , Endocitose , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Clatrina/metabolismo , Humanos , Lectinas/metabolismo , Redes e Vias Metabólicas
9.
Cell ; 149(6): 1353-67, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22682254

RESUMO

Many lipid-tethered proteins and glycolipids exist as monomers and nanoclusters on the surface of living cells. The spatial distribution and dynamics of formation and breakup of nanoclusters does not reflect thermal and chemical equilibrium and is controlled by active remodeling of the underlying cortical actin. We propose a model for nanoclustering based on active hydrodynamics, wherein cell surface molecules bound to dynamic actin are actively driven to form transient clusters. This consistently explains all of our experimental observations. Using FCS and TIRF microscopy, we provide evidence for the existence of short, dynamic, polymerizing actin filaments at the cortex, a key assumption of the theoretical framework. Our theory predicts that lipid-anchored proteins that interact with dynamic actin must exhibit anomalous concentration fluctuations, and a cell membrane protein capable of binding directly to actin can form nanoclusters. These we confirm experimentally, providing an active mechanism for molecular organization and its spatiotemporal regulation on the plasma membrane.


Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Actinas/química , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Citoesqueleto/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Espectrometria de Fluorescência
10.
Cell ; 144(3): 323-4, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21295694

RESUMO

Caveolae are protein-driven membrane invaginations that regulate both the physical and chemical composition of the plasma membrane. Sinha et al. (2011) now show that caveolae are membrane reservoirs that are used to rapidly buffer against changes in membrane tension.

11.
Cell ; 142(4): 507-10, 2010 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-20723749

RESUMO

Lipid segregation occurs in biological membranes, but how this plays into cellular processes like endocytosis has been unclear. Here, we discuss how the active or passive induction of lipid-protein domain formation in membranes can alter membrane mechanics and thus affect processes such as the generation of curvature, the scission of buds and tubules, and lipid sorting.


Assuntos
Endocitose , Metabolismo dos Lipídeos , Animais , Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares , Humanos
12.
Proc Natl Acad Sci U S A ; 119(30): e2123056119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35867835

RESUMO

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.


Assuntos
Actinas , Actomiosina , Membrana Celular , Proteínas Ligadas por GPI , Estresse Mecânico , Actinas/química , Actomiosina/química , Animais , Células CHO , Membrana Celular/química , Cricetulus , Proteínas Ligadas por GPI/química , Lipídeos/química
13.
PLoS Pathog ; 17(7): e1009706, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252168

RESUMO

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.


Assuntos
Tratamento Farmacológico da COVID-19 , COVID-19/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Internalização do Vírus/efeitos dos fármacos , Cloreto de Amônio/farmacologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/fisiologia , Animais , Antivirais/administração & dosagem , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Cloroquina/farmacologia , Clatrina/metabolismo , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Hidroxicloroquina/administração & dosagem , Macrolídeos/farmacologia , Niclosamida/administração & dosagem , Niclosamida/farmacologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Células Vero
14.
Cell ; 133(7): 1214-27, 2008 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-18585355

RESUMO

Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform--incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.


Assuntos
Drosophila melanogaster/metabolismo , Proteínas Hedgehog/química , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Padronização Corporal , Membrana Celular/química , Membrana Celular/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/embriologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas Hedgehog/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Mutação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
15.
Cell ; 135(6): 1085-97, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-19070578

RESUMO

Several cell-surface lipid-tethered proteins exhibit a concentration-independent, cholesterol-sensitive organization of nanoscale clusters and monomers. To understand the mechanism of formation of these clusters, we investigate the spatial distribution and steady-state dynamics of fluorescently tagged GPI-anchored protein nanoclusters using high-spatial and temporal resolution FRET microscopy. These studies reveal a nonrandom spatial distribution of nanoclusters, concentrated in optically resolvable domains. Monitoring the dynamics of recovery of fluorescence intensity and anisotropy, we find that nanoclusters are immobile, and the dynamics of interconversion between nanoclusters and monomers, over a range of temperatures, is spatially heterogeneous and non-Arrhenius, with a sharp crossover coinciding with a reduction in the activity of cortical actin. Cholesterol depletion perturbs cortical actin and the spatial scale and interconversion dynamics of nanoclusters. Direct perturbations of cortical actin activity also affect the construction, dynamics, and spatial organization of nanoclusters. These results suggest a unique mechanism of complexation of cell-surface molecules regulated by cortical actin activity.


Assuntos
Actinas/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas/metabolismo , Animais , Células CHO , Colesterol/metabolismo , Cricetinae , Cricetulus , Microdomínios da Membrana/metabolismo , Microscopia Confocal , Miosinas/metabolismo
16.
Biophys J ; 118(8): 1946-1957, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32191863

RESUMO

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.


Assuntos
Actinas , Microscopia , Citoesqueleto de Actina , Miosina Tipo II , Miosinas
17.
Proc Natl Acad Sci U S A ; 114(40): 10648-10653, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-29073038

RESUMO

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVß3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Embrião de Mamíferos/metabolismo , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Integrina alfaVbeta3/metabolismo , Actinas/genética , Animais , Linhagem Celular , Movimento Celular/fisiologia , Citoesqueleto/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Adesões Focais/genética , Integrina alfaVbeta3/genética , Camundongos
18.
Proc Natl Acad Sci U S A ; 113(45): E6993-E7002, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27791132

RESUMO

Endocytosis of ligand-receptor complexes regulates signal transduction during development. In particular, clathrin and dynamin-dependent endocytosis has been well studied in the context of patterning of the Drosophila wing disc, wherein apically secreted Wingless (Wg) encounters its receptor, DFrizzled2 (DFz2), resulting in a distinctive dorso-ventral pattern of signaling outputs. Here, we directly track the endocytosis of Wg and DFz2 in the wing disc and demonstrate that Wg is endocytosed from the apical surface devoid of DFz2 via a dynamin-independent CLIC/GEEC pathway, regulated by Arf1, Garz, and class I PI3K. Subsequently, Wg containing CLIC/GEEC endosomes fuse with DFz2-containing vesicles derived from the clathrin and dynamin-dependent endocytic pathway, which results in a low pH-dependent transfer of Wg to DFz2 within the merged and acidified endosome to initiate Wg signaling. The employment of two distinct endocytic pathways exemplifies a mechanism wherein cells in tissues leverage multiple endocytic pathways to spatially regulate signaling.

19.
Proc Natl Acad Sci U S A ; 113(12): E1645-54, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26929326

RESUMO

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.


Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Citoesqueleto de Actina/ultraestrutura , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Bactérias/genética , Polaridade Celular , Quelantes , Galinhas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Técnicas In Vitro , Bicamadas Lipídicas , Proteínas Luminescentes/genética , Microscopia Eletrônica , Modelos Biológicos , Níquel , Ácido Nitrilotriacético/análogos & derivados , Fosfatidilcolinas , Fosfatidiletanolaminas , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Propriedades de Superfície
20.
Plant Cell ; 27(4): 1297-315, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25901088

RESUMO

Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity.


Assuntos
Arabidopsis/metabolismo , Clatrina/metabolismo , Raízes de Plantas/metabolismo , Cloreto de Sódio/farmacologia , Arabidopsis/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
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