RESUMEN
Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here, we present a method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with transient adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution.
Asunto(s)
Microscopía Fluorescente , Animales , ADN , Aparato de Golgi , Mamíferos , Microscopía Fluorescente/métodos , Oligonucleótidos , ProteínasRESUMEN
Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows. Here we present an integrated acquisition and analysis platform leveraging microscopy-specific data compression, distributed storage and distributed analysis to enable an acquisition and analysis throughput of 10,000 cells per day. The platform facilitates graphically reconfigurable analyses to be automatically initiated from the microscope during acquisition and remotely executed, and can even feed back and queue new acquisition tasks on the microscope. We demonstrate the utility of this framework by imaging hundreds of cells per well in multi-well sample formats. Our platform, implemented within the PYthon-Microscopy Environment (PYME), is easily configurable to control custom microscopes, and includes a plugin framework for user-defined extensions.
Asunto(s)
Imagenología Tridimensional , Programas Informáticos , Microscopía Fluorescente/métodos , Imagen Individual de Molécula/métodosRESUMEN
Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional ß1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image ß1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous ß1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal ß1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.
Asunto(s)
Integrina beta1 , Integrinas , Integrina beta1/metabolismo , Adhesión Celular , Endocitosis , ExocitosisRESUMEN
Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes ß1 integrin-dependent fibrillogenesis. We find that the site of FN secretion is regulated by cell polarization, which occurs in bursts under stabilized lamellipodia at the leading edge. Moreover, analysis of FN secretion and focal adhesion dynamics suggest that focal adhesion formation precedes FN deposition and that deposition continues during focal adhesion disassembly. Lastly, we show that the polarized FN deposition in spreading and migrating cells requires both intact microtubules and myosin II-mediated contractility. Thus, while FN secretion does not require integrin binding, the site of exocytosis is regulated by membrane and cytoskeletal dynamics with secretion occurring after new adhesion formation.
Asunto(s)
Fibronectinas , Microtúbulos , Miosina Tipo II , Seudópodos , Proteínas del Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Integrinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Seudópodos/genética , Seudópodos/metabolismo , Matriz Extracelular/metabolismo , ExocitosisRESUMEN
Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how ß-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress. We performed both in vitro studies with primary endothelial cells subjected to ß-arrestin knockdown, and in vivo studies using mice with endothelial specific deletion of ß-arrestin 1 and ß-arrestin 2. We found that ß-arrestins are localized to primary cilia in endothelial cells, which are present in subpopulations of endothelial cells in relatively low shear states. Recruitment of ß-arrestins to cilia involved its interaction with IFT81, a component of the flagellar transport protein complex in the cilia. ß-arrestin knockdown led to marked reduction in shear stress response, including induction of NOS3 expression. Within the cilia, ß-arrestins were found to associate with the type II bone morphogenetic protein receptor (BMPR-II), whose disruption similarly led to an impaired endothelial shear response. ß-arrestins also regulated Smad transcription factor phosphorylation by BMPR-II. Mice with endothelial specific deletion of ß-arrestin 1 and ß-arrestin 2 were found to have impaired retinal angiogenesis. In conclusion, we have identified a novel role for endothelial ß-arrestins as key transducers of ciliary mechanotransduction that play a central role in shear signaling by BMPR-II and contribute to vascular development.
RESUMEN
Insulin is synthesized by pancreatic ß-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid-liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a "cargo sponge" leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.
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Gránulos Citoplasmáticos , Aparato de Golgi , Células Secretoras de Insulina , Proinsulina , Vesículas Secretoras , Cromograninas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Vesículas Secretoras/metabolismoRESUMEN
A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function. Here, we present a probe based on the membrane-binding C2 domain of cytosolic phospholipase A2 (cPLA2) that fulfills this need. By conjugating the C2 domain with different detectable tags, we demonstrate that a single, modular probe can allow synaptic vesicles to be imaged at multiple levels of spatial and temporal resolution. Moreover, as a general endocytic marker, the C2 domain may also be used to study membrane recycling in many cell types.
Asunto(s)
Imagen Multimodal , Vesículas Sinápticas , Vesículas Sinápticas/químicaRESUMEN
Primary cilia are slender, cellular antennae that sense extracellular stimuli, and their absence or dysfunction plays a role in numerous human diseases. Prior work has indicated a role of the exocyst tethering complex in cilia biogenesis and maintenance,1-6 with the underlying paradigm that the exocyst targets vesicles to the ciliary base to deliver ciliary cargoes.7-9 However, the role of the exocyst vis-à-vis to primary cilia in living cells and during stimulation is unknown. Herein, using advanced imaging and quantitative analysis reveals that serum stimulation increases the exocyst's localization to cilia by three-fold. This serum-stimulated localization is highly dynamic, and FRAP experiments show that exocysts at the cilia are highly mobile (60%-80%). Super resolution imaging reveals that the xocyst extends past the cilia base to the entire ciliary pocket. To visualize cilia exocytosis, we conducted live cell imaging with pH-sensitive cilia reporters in combination with extracellular pH switching. Strikingly, we observed that an exocyst-positive internal cilia fuses with the cell surface. These live cell results support a novel and dynamic role of the exocyst complex in the delivery of internalized cilia to the cell surface. Moreover, they suggest a novel pathway may be used to recycle primary cilia to the cell surface that engages the exocyst in response to stimuli. This new remarkable plasticity in cilia presence on the surface in response to extracellular stimuli suggest new means to potentially modulate cilia signaling.
Asunto(s)
Cilios , Proteínas de Transporte Vesicular , Membrana Celular/metabolismo , Cilios/metabolismo , Citoplasma/metabolismo , Exocitosis , Humanos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion. We find that vesicles mainly undergo kiss-and-run instead of full fusion in the absence of functional exocyst. Full fusion is rescued by optogenetically restoring exocyst function, in a manner likely dependent on the stoichiometry of tether engagement with the plasma membrane. In contrast, a passive artificial tether produces mostly kissing events, suggesting that kiss-and-run is the default mode of vesicle fusion. Optogenetic control of tethering further shows that fusion mode has physiological relevance since only full fusion could trigger lamellipodial expansion. These findings demonstrate that active coupling between tethering and fusion is critical for robust membrane merger.
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Criptocromos/genética , Exosomas/metabolismo , Receptores de Transferrina/genética , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Criptocromos/metabolismo , Exosomas/ultraestructura , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Fusión de Membrana/genética , Microscopía Fluorescente , Optogenética/métodos , Receptores de Transferrina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Secretoras/ultraestructura , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteína Fluorescente RojaRESUMEN
Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect cell lines by super-resolution for over an order of magnitude longer than with tagged proteins. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.
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Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Orgánulos/metabolismo , Línea Celular , Colorantes Fluorescentes/química , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Imagenología Tridimensional , Microscopía Confocal , Fotoblanqueo , Imagen de Lapso de TiempoRESUMEN
Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs). Differentiation of monocytes into phDCs was concomitant with the formation of an "adhesion synapse," a biophysical junction enriched with platelet P-selectin and monocyte P-selectin glycoprotein ligand 1, followed by intracellular calcium fluxing and nuclear localization of nuclear factor κB. phDCs were more efficient than cytokine-derived DCs in generating tumor-specific T cell immunity. Our findings demonstrate that platelets mediate a cytokine-independent, physiologic maturation of DC and suggest a novel strategy for DC-based immunotherapies.
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Presentación de Antígeno , Plaquetas/inmunología , Señalización del Calcio/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/inmunología , Monocitos/inmunología , Selectina-P/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Señalización del Calcio/genética , Diferenciación Celular/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Transgénicos , FN-kappa B/genética , FN-kappa B/inmunología , Selectina-P/genética , Linfocitos T/inmunologíaRESUMEN
We report new lipid-based, high-density, environmentally sensitive (HIDE) probes that accurately and selectively image endo-lysosomes and their dynamics at super-resolution for extended times. Treatment of live cells with the small molecules DiIC16TCO or DiIC16'TCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC16-SiR and DiIC16'-SiR in the endo-lysosomal membrane. These new probes support the acquisition of super-resolution videos of organelle dynamics in primary cells for more than 7 min with no detectable change in endosome structure or function. Using DiIC16-SiR and DiIC16'-SiR, we describe direct evidence of endosome motility defects in cells from patients with Niemann-Pick Type-C disease. In wild-type fibroblasts, the probes reveal distinct but rare inter-endosome kiss-and-run events that cannot be observed using confocal methods. Our results shed new light on the role of NPC1 in organelle motility and cholesterol trafficking.
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Endosomas/metabolismo , Lisosomas/metabolismo , Microscopía Fluorescente/métodos , Transporte Biológico , Carbocianinas/química , Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Fibroblastos/metabolismo , Colorantes Fluorescentes , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Transporte de ProteínasRESUMEN
Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells. However, only a fraction of WBCs is exposed to ECP, and 8-MOP is inactive in the absence of UVA light, implying that other mechanisms underlie the anticancer effects of ECP. Recently, ECP has been shown to enable the physiological differentiation of monocytes into dendritic cells (DCs) that efficiently cross-present tumor-associated antigens (TAAs) to CD8+ T lymphocytes to initiate cognate immunity. However, the source of TAAs and immunostimulatory signals for such DCs remains to be elucidated. Here, we demonstrate that 8-MOP plus UVA light reduces melanoma cell viability along with the emission of ICD-associated danger signals including calreticulin (CALR) exposure on the cell surface and secretion of ATP, high mobility group box 1 (HMGB1) and type I interferon (IFN). Consistently, melanoma cells succumbing to 8-MOP plus UVA irradiation are efficiently engulfed by monocytes, ultimately leading to cross-priming of CD8+ T cells against cancer. Moreover, malignant cells killed by 8-MOP plus UVA irradiation in vitro vaccinate syngeneic immunocompetent mice against living cancer cells of the same type, and such a protection is lost when cancer cells are depleted of calreticulin or HMGB1, as well as in the presence of an ATP-degrading enzyme or antibodies blocking type I IFN receptors. ECP induces bona fide ICD, hence simultaneously providing monocytes with abundant amounts of TAAs and immunostimulatory signals that are sufficient to initiate cognate anticancer immunity.
Asunto(s)
Antígenos de Neoplasias/genética , Linfoma Cutáneo de Células T/inmunología , Linfoma Cutáneo de Células T/terapia , Metoxaleno/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de la radiación , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/efectos de la radiación , Proteína HMGB1/genética , Humanos , Muerte Celular Inmunogénica/efectos de los fármacos , Muerte Celular Inmunogénica/efectos de la radiación , Leucocitos/efectos de los fármacos , Leucocitos/inmunología , Leucocitos/efectos de la radiación , Linfoma Cutáneo de Células T/patología , Ratones , Monocitos/efectos de los fármacos , Monocitos/efectos de la radiación , Fotoféresis , Fármacos Fotosensibilizantes/farmacología , Receptor de Interferón alfa y beta/genética , Rayos UltravioletaRESUMEN
BACKGROUND: Fluorescent proteins (FPs) have widespread uses in cell biology. However, the practical applications of FPs are significantly limited due to their rapid photobleaching and misfolding when fused to target proteins. NEW METHOD: Using a combination of novel and known mutations to eGFP, we developed a well folded and very photostable variant, SiriusGFP. RESULTS: The fluorescence spectrum indicated that the excitation and emission peaks of SiriusGFP were red-shifted by 16 and 8 nm, respectively. Co- operative effects of two key mutations, S147R and S205 V, contribute to its photostability. SiriusGFP tagged to the mitochondrial outer membrane protein Omp25 showed sustained fluorescence during continuous 3D-scanning confocal imaging (4D confocal) compared to eGFP-tagged Omp25. Furthermore, with super-resolution structured illumination microscopy (SIM) we demonstrate marked improvements in image quality and resolution (130 nm in XY axis, and 310 nm in Z axis), as well as, decreased artifacts due to photobleaching. COMPARISON WITH EXISTING METHOD(S): Compared to eGFP. SiriusGFP shows a 2-fold increase in photostability in vitro, and folds well when fused to the N- and C- termini of cytoplasmic and membrane proteins. While its quantum yield is Ë3 fold lower than eGFP, its decreased brightness was more than compensated by its increasedphotostability in different experimental paradigms allowing practical experimentation without dynamic adjustment of light intensity or fluorescence sampling times. CONCLUSIONS: We have developed a variant of eGFP, SiriusGFP, that shows over a two fold increase in photostability with utility in methods requiring sustained or high intensity excitation as in 4D confocal or SIM imaging.
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Proteínas Fluorescentes Verdes , Microscopía Fluorescente/métodos , Células HEK293 , Células HeLa , Humanos , Microscopía Confocal/métodosRESUMEN
While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Lipoilación/fisiología , Transporte de Proteínas/fisiología , Transporte Biológico/fisiología , Células Cultivadas , Humanos , Membranas Intracelulares/metabolismoRESUMEN
As a master regulator of endothelial cell function, vascular endothelial growth factor receptor-2 (VEGFR2) activates multiple downstream signaling pathways that are critical for vascular development and normal vessel function. VEGFR2 trafficking through various endosomal compartments modulates its signaling output. Accordingly, proteins that regulate the speed and direction by which VEGFR2 traffics through endosomes have been demonstrated to be particularly important for arteriogenesis. However, little is known about how these proteins control VEGFR2 trafficking and about the implications of this control for endothelial cell function. Here, we show that Rab GTPase-binding effector protein 2 (RABEP2), a Rab-effector protein implicated in arteriogenesis, modulates VEGFR2 trafficking. By employing high-resolution microscopy and biochemical assays, we demonstrate that RABEP2 interacts with the small GTPase Rab4 and regulates VEGFR2 endosomal trafficking to maintain cell-surface expression of VEGFR2 and VEGF signaling. Lack of RABEP2 also led to prolonged retention of VEGFR2 in Rab5-positive sorting endosomes, which increased VEGFR2's exposure to phosphotyrosine phosphatase 1b (PTP1b), causing diminished VEGFR2 signaling. Finally, the loss of RABEP2 increased VEGFR2 degradation by diverting VEGFR2 to Rab7-positive endosomes destined for the lysosome. These results implicate RABEP2 as a key modulator of VEGFR2 endosomal trafficking, and demonstrate the importance of RABEP2 and Rab4 for VEGFR2 signaling in endothelial cells.
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Endosomas/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Endosomas/genética , Células Endoteliales/citología , Ratones , Ratones Endogámicos BALB C , Transporte de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab4/genética , Proteínas de Unión al GTP rab4/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Integrin function is dynamically modulated by endo-exocytic trafficking, however, major mysteries remain about where, when, and how this occurs in living cells. To address this, here we report the generation of functional recombinant ß1 integrins with traceable tags inserted in an extracellular loop. We demonstrate that these 'ecto-tagged' integrins are cell-surface expressed, localize to adhesions, exhibit normal integrin activation, and restore adhesion in ß1 integrin knockout fibroblasts. Importantly, ß1 integrins containing an extracellular pH-sensitive pHluorin tag allow direct visualization of integrin exocytosis in live cells and revealed targeted delivery of integrin vesicles to focal adhesions. Further, using ß1 integrins containing a HaloTag in combination with membrane-permeant and -impermeant Halo dyes allows imaging of integrin endocytosis and recycling. Thus, ecto-tagged integrins provide novel powerful tools to characterize integrin function and trafficking.Integrins are cell-surface adhesion receptors that are modulated by endo-exocytic trafficking, but existing tools to study this process can interfere with function. Here the authors develop ß1 integrins carrying traceable tags in the extracellular domain; a pH-sensitive pHlourin tag or a HaloTag to facilitate dye attachment.
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Endocitosis , Adhesiones Focales/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Integrina beta1/metabolismo , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Células HeLa , Humanos , Integrina beta1/genética , Ratones , Microscopía Confocal , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds. Our data reveal 2D dynamics of the mitochondria, plasma membrane and filopodia, and the 2D and 3D dynamics of the endoplasmic reticulum, in living cells. HIDE probes also facilitate acquisition of live-cell, two-color, super-resolution images, expanding the utility of nanoscopy to visualize dynamic processes and structures in living cells.
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Estructuras Celulares/ultraestructura , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodos , Nanotecnología/métodos , Células HeLa , HumanosRESUMEN
Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25â min at 0.5â s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9â min at 2â s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.
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Membrana Celular/química , Colorantes Fluorescentes/química , Nanotecnología , Imagen Óptica , Células HeLa , Humanos , Microscopía Fluorescente , Estructura Molecular , Células Tumorales CultivadasRESUMEN
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (â¼3 µm), thin (â¼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin. Instead, they contain ARF1 along their entire length at a density estimated to be in the range of close packing. Experiments using a mutant form of ARF1 affecting GTP hydrolysis suggest that ARF1[GTP] is functionally required for the tubules to form. Dynamic confocal and stimulated emission depletion imaging shows that ARF1-rich tubular compartments fall into two distinct classes containing 1) anterograde cargoes and clathrin clusters or 2) retrograde cargoes and coatomer clusters.