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1.
Science ; 368(6487): 181-186, 2020 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-32273467

RESUMO

Embryonic development is a complex process that is unamenable to direct observation. In this study, we implanted a window to the mouse uterus to visualize the developing embryo from embryonic day 9.5 to birth. This removable intravital window allowed manipulation and high-resolution imaging. In live mouse embryos, we observed transient neurotransmission and early vascularization of neural crest cell (NCC)-derived perivascular cells in the brain, autophagy in the retina, viral gene delivery, and chemical diffusion through the placenta. We combined the imaging window with in utero electroporation to label and track cell division and movement within embryos and observed that clusters of mouse NCC-derived cells expanded in interspecies chimeras, whereas adjacent human donor NCC-derived cells shrank. This technique can be combined with various tissue manipulation and microscopy methods to study the processes of development at unprecedented spatiotemporal resolution.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário , Microscopia Intravital/métodos , Crista Neural , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Divisão Celular , Movimento Celular , Quimera/embriologia , Quimera/fisiologia , Eletroporação , Feminino , Técnicas de Transferência de Genes , Camundongos , Camundongos Transgênicos , Neovascularização Fisiológica , Crista Neural/irrigação sanguínea , Crista Neural/citologia , Crista Neural/embriologia , Placenta/fisiologia , Gravidez , Retina/embriologia , Retina/fisiologia , Transmissão Sináptica , Útero
2.
PLoS Negl Trop Dis ; 14(3): e0008007, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196491

RESUMO

Investigations into intracellular replication and differentiation of Trypanosoma cruzi within the mammalian host have been restricted by limitations in our ability to detect parasitized cells throughout the course of infection. We have overcome this problem by generating genetically modified parasites that express a bioluminescent/fluorescent fusion protein. By combining in vivo imaging and confocal microscopy, this has enabled us to routinely visualise murine infections at the level of individual host cells. These studies reveal that intracellular parasite replication is an asynchronous process, irrespective of tissue location or disease stage. Furthermore, using TUNEL assays and EdU labelling, we demonstrate that within individual infected cells, replication of both mitochondrial (kDNA) and nuclear genomes is not co-ordinated within the parasite population, and that replicating amastigotes and non-replicating trypomastigotes can co-exist in the same cell. Finally, we report the presence of distinct non-canonical morphological forms of T. cruzi in the mammalian host. These appear to represent transitional forms in the amastigote to trypomastigote differentiation process. Therefore, the intracellular life-cycle of T. cruzi in vivo is more complex than previously realised, with potential implications for our understanding of disease pathogenesis, immune evasion and drug development. Dissecting the mechanisms involved will be an important experimental challenge.


Assuntos
Doença de Chagas/parasitologia , Replicação do DNA , Estágios do Ciclo de Vida , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Modelos Animais de Doenças , Feminino , Genes Reporter , Microscopia Intravital/métodos , Camundongos SCID , Microscopia Confocal/métodos , Coloração e Rotulagem/métodos , Trypanosoma cruzi/genética
3.
PLoS One ; 15(2): e0219886, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023253

RESUMO

Förster Resonance Energy Transfer (FRET) has become an immensely powerful tool to profile intra- and inter-molecular interactions. Through fusion of genetically encoded fluorescent proteins (FPs) researchers have been able to detect protein oligomerization, receptor activation, and protein translocation among other biophysical phenomena. Recently, two bright monomeric red fluorescent proteins, mRuby3 and mScarlet-I, have been developed. These proteins offer much improved physical properties compared to previous generations of monomeric red FPs that should help facilitate more general adoption of Green/Red FRET. Here we assess the ability of these two proteins, along with mCherry, to act as a FRET acceptor for the bright, monomeric, green-yellow FP mNeonGreen using intensiometric FRET and 2-photon Fluorescent Lifetime Imaging Microscopy (FLIM) FRET techniques. We first determined that mNeonGreen was a stable donor for 2-photon FLIM experiments under a variety of imaging conditions. We then tested the red FP's ability to act as FRET acceptors using mNeonGreen-Red FP tandem construct. With these constructs we found that mScarlet-I and mCherry are able to efficiently FRET with mNeonGreen in spectroscopic and FLIM FRET. In contrast, mNeonGreen and mRuby3 FRET with a much lower efficiency than predicted in these same assays. We explore possible explanations for this poor performance and determine mRuby3's protein maturation properties are a major contributor. Overall, we find that mNeonGreen is an excellent FRET donor, and both mCherry and mScarlet-I, but not mRuby3, act as practical FRET acceptors, with the brighter mScarlet-I out performing mCherry in intensiometric studies, but mCherry out performing mScarlet-I in instances where consistent efficiency in a population is critical.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/normas , Transferência Ressonante de Energia de Fluorescência/normas , Células HEK293 , Humanos , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos
4.
PLoS Genet ; 16(2): e1008626, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32059017

RESUMO

Neuronal pruning is a commonly observed phenomenon for the developing nervous systems to ensure precise wiring of neural circuits. The function of Ik2 kinase and its downstream mediator, Spindle-F (Spn-F), are essential for dendrite pruning of Drosophila sensory neurons during development. However, little is known about how Ik2/Spn-F signaling is transduced in neurons and ultimately results in dendrite pruning. Our genetic analyses and rescue experiments demonstrated that the small GTPase Rab11, especially the active GTP-bound form, is required for dendrite pruning. We also found that Rab11 shows genetic interactions with spn-F and ik2 on pruning. Live imaging of single neurons and antibody staining reveal normal Ik2 kinase activation in Rab11 mutant neurons, suggesting that Rab11 could have a functional connection downstream of and/or parallel to the Ik2 kinase signaling. Moreover, we provide biochemical evidence that both the Ik2 kinase activity and the formation of Ik2/Spn-F/Rab11 complexes are central to promote Rab11 activation in cells. Together, our studies reveal that a critical role of Ik2/Spn-F signaling in neuronal pruning is to promote Rab11 activation, which is crucial for dendrite pruning in neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Quinase I-kappa B/metabolismo , Plasticidade Neuronal/genética , Células Receptoras Sensoriais/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Dendritos/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/metabolismo , Células Receptoras Sensoriais/citologia , Transdução de Sinais/fisiologia , Imagem com Lapso de Tempo , Proteínas rab de Ligação ao GTP/genética
5.
PLoS Genet ; 16(2): e1008633, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069276

RESUMO

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Clatrina/metabolismo , Quinases Relacionadas a NIMA/genética , Proteínas Quinases/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Endocitose , Microscopia Intravital , Larva/crescimento & desenvolvimento , Muda/genética , Mutação , Quinases Relacionadas a NIMA/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagem com Lapso de Tempo
6.
Nat Commun ; 11(1): 878, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054845

RESUMO

Macrophage-initiated inflammation is tightly regulated to eliminate threats such as infections while suppressing harmful immune activation. However, individual cells' signaling responses to pro-inflammatory cues are heterogeneous, with subpopulations emerging with high or low activation states. Here, we use single-cell tracking and dynamical modeling to develop and validate a revised model for lipopolysaccharide (LPS)-induced macrophage activation that invokes a mechanism we term quorum licensing. The results show that bimodal phenotypic partitioning of macrophages is primed during the resting state, dependent on cumulative history of cell density, predicted by extrinsic noise in transcription factor expression, and independent of canonical LPS-induced intercellular feedback in the tumor necrosis factor (TNF) response. Our analysis shows how this density-dependent coupling produces a nonlinear effect on collective TNF production. We speculate that by linking macrophage density to activation, this mechanism could amplify local responses to threats and prevent false alarms.


Assuntos
Comunicação Celular/imunologia , Inflamação/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Modelos Imunológicos , Animais , Fibroblastos , Citometria de Fluxo , Microscopia Intravital , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Microscopia Confocal , Cultura Primária de Células , Células RAW 264.7 , Transdução de Sinais/imunologia , Análise de Célula Única , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
7.
PLoS Biol ; 18(1): e3000567, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31986129

RESUMO

Cell- and tissue-level processes often occur across days or weeks, but few imaging methods can capture such long timescales. Here, we describe Bellymount, a simple, noninvasive method for longitudinal imaging of the Drosophila abdomen at subcellular resolution. Bellymounted animals remain live and intact, so the same individual can be imaged serially to yield vivid time series of multiday processes. This feature opens the door to longitudinal studies of Drosophila internal organs in their native context. Exploiting Bellymount's capabilities, we track intestinal stem cell lineages and gut microbial colonization in single animals, revealing spatiotemporal dynamics undetectable by previously available methods.


Assuntos
Anatomia Transversal/métodos , Drosophila/anatomia & histologia , Microbioma Gastrointestinal , Microscopia Intravital/métodos , Vísceras/anatomia & histologia , Fatores Etários , Animais , Drosophila/microbiologia , Intestinos/anatomia & histologia , Intestinos/diagnóstico por imagem , Imagem Óptica/métodos , Vísceras/diagnóstico por imagem
8.
Nat Commun ; 11(1): 395, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959752

RESUMO

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.


Assuntos
Capilares/fisiologia , Córtex Cerebral/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Capilares/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Feminino , Neuroimagem Funcional/métodos , Imageamento Tridimensional , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Modelos Animais , Modelos Cardiovasculares , Músculo Liso Vascular/diagnóstico por imagem , Fluxo Sanguíneo Regional/fisiologia , Crânio/cirurgia , Trepanação
9.
PLoS One ; 15(1): e0227286, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910228

RESUMO

In order to unravel rapid mechano-chemical feedback mechanisms in sprouting angiogenesis, we combine selective plane illumination microscopy (SPIM) and tailored image registration algorithms - further referred to as SPIM-based displacement microscopy - with an in vitro model of angiogenesis. SPIM successfully tackles the problem of imaging large volumes while upholding the spatial resolution required for the analysis of matrix displacements at a subcellular level. Applied to in vitro angiogenic sprouts, this unique methodological combination relates subcellular activity - minute to second time scale growing and retracting of protrusions - of a multicellular systems to the surrounding matrix deformations with an exceptional temporal resolution of 1 minute for a stack with multiple sprouts simultaneously or every 4 seconds for a single sprout, which is 20 times faster than with a conventional confocal setup. Our study reveals collective but non-synchronised, non-continuous activity of adjacent sprouting cells along with correlations between matrix deformations and protrusion dynamics.


Assuntos
Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Neovascularização Fisiológica/fisiologia , Imagem com Lapso de Tempo , Algoritmos , Técnicas de Cultura de Células/métodos , Colágeno Tipo I , Marcadores Fiduciais , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis , Microscopia de Fluorescência/métodos , Microesferas
10.
Nat Commun ; 11(1): 239, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932632

RESUMO

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Imagem Molecular/instrumentação , Animais , Linhagem Celular , Feminino , Fluorescência , Humanos , Microscopia Intravital , Camundongos , Imagem Molecular/métodos , Engenharia de Proteínas , Estabilidade Proteica , Espectroscopia de Luz Próxima ao Infravermelho
11.
Proc Natl Acad Sci U S A ; 117(1): 432-438, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871203

RESUMO

Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized Madin-Darby canine kidney (MDCK) cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells, while reformation of cadherin clusters across the cell-cell boundary correlates with microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as 3 actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNA interference (RNAi) results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Junções Aderentes/metabolismo , Caderinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Junções Íntimas/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Junções Aderentes/ultraestrutura , Animais , Adesão Celular , Cães , Microscopia Intravital , Células Madin Darby de Rim Canino , Microscopia Eletrônica , Miosina Tipo II/metabolismo , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética , Interferência de RNA , Junções Íntimas/ultraestrutura
12.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31824652

RESUMO

Image analysis in clinical research has evolved at fast pace in the last decade. This review discusses basic concepts ranging from immunohistochemistry to advanced techniques such as multiplex imaging, digital pathology, flow cytometry and intravital microscopy. Tissue imaging ex vivo is still one of the gold-standards in the field due to feasibility. We describe here different protocols and applications of digital analysis providing basic and clinical researchers with an overview on how to analyse tissue images. In vivo imaging is not accessible to researchers; however, it provides invaluable dynamic information easily. Overall, we discuss a plethora of techniques that - when combined - constitute a powerful platform for basic and translational cancer research.


Assuntos
Neoplasias , Humanos , Imuno-Histoquímica , Microscopia Intravital , Pesquisa
13.
Virol J ; 16(1): 159, 2019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856881

RESUMO

BACKGROUND: Live-cell imaging is a powerful tool for visualization of the spatio-temporal dynamics of moving signals in living cells. Although this technique can be utilized to visualize nucleocapsid transport in Marburg virus (MARV)- or Ebola virus-infected cells, the experiments require biosafety level-4 (BSL-4) laboratories, which are restricted to trained and authorized individuals. METHODS: To overcome this limitation, we developed a live-cell imaging system to visualize MARV nucleocapsid-like structures using fluorescence-conjugated viral proteins, which can be conducted outside BSL-4 laboratories. RESULTS: Our experiments revealed that nucleocapsid-like structures have similar transport characteristics to those of nucleocapsids observed in MARV-infected cells, both of which are mediated by actin polymerization. CONCLUSIONS: We developed a non-infectious live cell imaging system to visualize intracellular transport of MARV nucleocapsid-like structures. This system provides a safe platform to evaluate antiviral drugs that inhibit MARV nucleocapsid transport.


Assuntos
Transporte Biológico , Microscopia Intravital/métodos , Marburgvirus/crescimento & desenvolvimento , Microscopia de Fluorescência/métodos , Nucleocapsídeo/metabolismo , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos/virologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Coloração e Rotulagem/métodos , Proteínas Virais/análise
14.
Nat Commun ; 10(1): 5753, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848345

RESUMO

The coordination of cell movements across spatio-temporal scales ensures precise positioning of organs during vertebrate gastrulation. Mechanisms governing such morphogenetic movements have been studied only within a local region, a single germlayer or in whole embryos without cell identity. Scale-bridging imaging and automated analysis of cell dynamics are needed for a deeper understanding of tissue formation during gastrulation. Here, we report pan-embryo analyses of formation and dynamics of all three germlayers simultaneously within a developing zebrafish embryo. We show that a distinct distribution of cells in each germlayer is established during early gastrulation via cell movement characteristics that are predominantly determined by their position in the embryo. The differences in initial germlayer distributions are subsequently amplified by a global movement, which organizes the organ precursors along the embryonic body axis, giving rise to the blueprint of organ formation. The tools and data are available as a resource for the community.


Assuntos
Movimento Celular/fisiologia , Embrião não Mamífero/embriologia , Gastrulação/fisiologia , Camadas Germinativas/embriologia , Imagem Multimodal/métodos , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/diagnóstico por imagem , Camadas Germinativas/diagnóstico por imagem , Imageamento Tridimensional/métodos , Microscopia Intravital/métodos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos
15.
Nat Commun ; 10(1): 5744, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848350

RESUMO

During bacterial cell division, the tubulin-homolog FtsZ forms a ring-like structure at the center of the cell. This Z-ring not only organizes the division machinery, but treadmilling of FtsZ filaments was also found to play a key role in distributing proteins at the division site. What regulates the architecture, dynamics and stability of the Z-ring is currently unknown, but FtsZ-associated proteins are known to play an important role. Here, using an in vitro reconstitution approach, we studied how the well-conserved protein ZapA affects FtsZ treadmilling and filament organization into large-scale patterns. Using high-resolution fluorescence microscopy and quantitative image analysis, we found that ZapA cooperatively increases the spatial order of the filament network, but binds only transiently to FtsZ filaments and has no effect on filament length and treadmilling velocity. Together, our data provides a model for how FtsZ-associated proteins can increase the precision and stability of the bacterial cell division machinery in a switch-like manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Escherichia coli/citologia , Processamento de Imagem Assistida por Computador , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula
16.
Nat Commun ; 10(1): 5816, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862977

RESUMO

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood-brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.


Assuntos
Barreira Hematoencefálica/metabolismo , Circulação Cerebrovascular/imunologia , Células Endoteliais/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Microglia/imunologia , Animais , Astrócitos/imunologia , Astrócitos/metabolismo , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/imunologia , Claudina-5/imunologia , Claudina-5/metabolismo , Modelos Animais de Doenças , Células Endoteliais/imunologia , Humanos , Microscopia Intravital , Masculino , Camundongos , Microglia/metabolismo , Permeabilidade , Fagocitose/imunologia , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Técnicas Estereotáxicas , Junções Íntimas/imunologia , Junções Íntimas/metabolismo
17.
Nat Commun ; 10(1): 5725, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31844058

RESUMO

Many traits vary among isogenic individuals in homogeneous environments. In microbes, plants and animals, variation in the protein chaperone system affects many such traits. In the animal model C. elegans, the expression level of hsp-16.2 chaperone biomarkers correlates with or predicts the penetrance of mutations and lifespan after heat shock. But the physiological mechanisms causing cells to express different amounts of the biomarker were unknown. Here, we used an in vivo microscopy approach to dissect different contributions to cell-to-cell variation in hsp-16.2 expression in the intestines of young adult animals, which generate the most lifespan predicting signal. While we detected both cell autonomous intrinsic noise and signaling noise, we found both contributions were relatively unimportant. The major contributor to cell-to-cell variation in biomarker expression was general differences in protein dosage. The hsp-16.2 biomarker reveals states of high or low effective dosage for many genes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Dosagem de Genes , Proteínas de Choque Térmico/genética , Longevidade/genética , Penetrância , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/metabolismo , Genes Reporter/genética , Proteínas de Choque Térmico/metabolismo , Microscopia Intravital/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Modelos Animais , Imagem Molecular , Transdução de Sinais/genética
18.
Nat Commun ; 10(1): 4566, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594939

RESUMO

Fungal dissemination into the bloodstream is a critical step leading to invasive fungal infections. Here, using intravital imaging, we show that Kupffer cells (KCs) in the liver have a prominent function in the capture of circulating Cryptococcus neoformans and Candida albicans, thereby reducing fungal dissemination to target organs. Complement C3 but not C5, and complement receptor CRIg but not CR3, are involved in capture of C. neoformans. Internalization of C. neoformans by KCs is subsequently mediated by multiple receptors, including CR3, CRIg, and scavenger receptors, which work synergistically along with C5aR signaling. Following phagocytosis, the growth of C. neoformans is inhibited by KCs in an IFN-γ independent manner. Thus, the liver filters disseminating fungi from circulation via KCs, providing a mechanistic explanation for the enhanced risk of cryptococcosis among individuals with liver diseases, and suggesting a therapeutic strategy to prevent fungal dissemination through enhancing KC functions.


Assuntos
Infecções Fúngicas Invasivas/imunologia , Macrófagos do Fígado/imunologia , Fígado/imunologia , Fagocitose , Animais , Candida albicans/imunologia , Candida albicans/isolamento & purificação , Candida albicans/patogenicidade , Complemento C3/genética , Complemento C3/imunologia , Complemento C3/metabolismo , Cryptococcus neoformans/imunologia , Cryptococcus neoformans/isolamento & purificação , Cryptococcus neoformans/patogenicidade , Modelos Animais de Doenças , Feminino , Humanos , Microscopia Intravital , Infecções Fúngicas Invasivas/sangue , Infecções Fúngicas Invasivas/microbiologia , Macrófagos do Fígado/metabolismo , Macrófagos do Fígado/microbiologia , Fígado/citologia , Fígado/diagnóstico por imagem , Masculino , Camundongos , Camundongos Knockout , Microscopia Confocal , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo
19.
Nat Commun ; 10(1): 4580, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31594948

RESUMO

Photoactivatable fluorophores are important for single-particle tracking and super-resolution microscopy. Here we present a photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we create probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophore's outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.


Assuntos
Corantes Fluorescentes/efeitos da radiação , Microscopia Intravital/métodos , Rodaminas/efeitos da radiação , Silício/efeitos da radiação , Imagem Individual de Molécula/métodos , Animais , Células COS , Corantes Fluorescentes/química , Células HeLa , Humanos , Luz , Microscopia de Fluorescência/métodos , Processos Fotoquímicos/efeitos da radiação , Prótons , Rodaminas/química , Silício/química
20.
Nat Commun ; 10(1): 4513, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586073

RESUMO

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1ß-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.


Assuntos
Citocinese/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Mapas de Interação de Proteínas/fisiologia , Proteína Fosfatase 1/metabolismo , Aurora Quinase B/metabolismo , Sítios de Ligação/genética , Células HeLa , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Mutagênese Sítio-Dirigida , Fosforilação/fisiologia , Proteína Fosfatase 1/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Imagem com Lapso de Tempo
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