RESUMO
Cell fate specification by lateral inhibition typically involves contact signaling through the Delta-Notch signaling pathway. However, whether this is the only signaling mode mediating lateral inhibition remains unclear. Here we show that in zebrafish oogenesis, a group of cells within the granulosa cell layer at the oocyte animal pole acquire elevated levels of the transcriptional coactivator TAZ in their nuclei. One of these cells, the future micropyle precursor cell (MPC), accumulates increasingly high levels of nuclear TAZ and grows faster than its surrounding cells, mechanically compressing those cells, which ultimately lose TAZ from their nuclei. Strikingly, relieving neighbor-cell compression by MPC ablation or aspiration restores nuclear TAZ accumulation in neighboring cells, eventually leading to MPC re-specification from these cells. Conversely, MPC specification is defective in taz-/- follicles. These findings uncover a novel mode of lateral inhibition in cell fate specification based on mechanical signals controlling TAZ activity.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Oogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Linhagem da Célula , Núcleo Celular/metabolismo , Feminino , Células da Granulosa/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oócitos/metabolismo , Oócitos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidoresRESUMO
Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.
Assuntos
Células Dendríticas , Homeostase , Megacariócitos , Trombopoese , Animais , Feminino , Humanos , Masculino , Camundongos , Apoptose , Plaquetas/citologia , Medula Óssea , Linhagem da Célula , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/citologia , Retroalimentação Fisiológica , Imunidade Inata , Microscopia Intravital , Megacariócitos/citologia , Megacariócitos/imunologia , Camundongos Endogâmicos C57BL , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/fisiopatologia , COVID-19/virologiaRESUMO
Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson's disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.
Assuntos
Proteínas de Drosophila/genética , Mitocôndrias/genética , Neurônios/metabolismo , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Modelos Animais de Doenças , Drosophila melanogaster/genética , Humanos , Luz , Mutação com Perda de Função/genética , Mitocôndrias/efeitos da radiação , Neurônios/patologia , Neurônios/efeitos da radiação , Optogenética/métodos , Doença de Parkinson/patologia , Fosfatidilinositol 3-Quinases/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Transdução de Sinais/genética , TransfecçãoRESUMO
Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/fisiologia , Endocitose/fisiologia , Células Vegetais/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clatrina , Corantes Fluorescentes , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência/métodos , PlântulaRESUMO
The goose barnacle Dosima fascicularis produces an excessive amount of adhesive (cement), which has a double function, being used for attachment to various substrata and also as a float (buoy). This paper focuses on the chemical composition of the cement, which has a water content of 92%. Scanning electron microscopy with EDX was used to measure the organic elements C, O and N in the foam-like cement. Vibrational spectroscopy (FTIR, Raman) provided further information about the overall secondary structure, which tended towards a ß-sheet. Disulphide bonds could not be detected by Raman spectroscopy. The cystine, methionine, histidine and tryptophan contents were each below 1% in the cement. Analyses of the cement revealed a protein content of 84% and a total carbohydrate content of 1.5% in the dry cement. The amino acid composition, 1D/2D-PAGE and MS/MS sequence analysis revealed a de novo set of peptides/proteins with low homologies with other proteins such as the barnacle cement proteins, largely with an acidic pI between 3.5 and 6.0. The biochemical composition of the cement of D. fascicularis is similar to that of other barnacles, but it shows interesting variations.
Assuntos
Proteínas de Artrópodes/química , Thoracica/química , Adesivos , Animais , Proteínas de Artrópodes/análise , Dinamarca , Microscopia Eletrônica de Varredura , Análise EspectralRESUMO
The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly.
Assuntos
Tomografia com Microscopia Eletrônica , Matriz Extracelular , Transporte Biológico , Movimento Celular , Citosol , Tomografia com Microscopia Eletrônica/métodos , Matriz Extracelular/ultraestruturaRESUMO
Contraction and flow of the actin cell cortex have emerged as a common principle by which cells reorganize their cytoplasm and take shape. However, how these cortical flows interact with adjacent cytoplasmic components, changing their form and localization, and how this affects cytoplasmic organization and cell shape remains unclear. Here we show that in ascidian oocytes, the cooperative activities of cortical actomyosin flows and deformation of the adjacent mitochondria-rich myoplasm drive oocyte cytoplasmic reorganization and shape changes following fertilization. We show that vegetal-directed cortical actomyosin flows, established upon oocyte fertilization, lead to both the accumulation of cortical actin at the vegetal pole of the zygote and compression and local buckling of the adjacent elastic solid-like myoplasm layer due to friction forces generated at their interface. Once cortical flows have ceased, the multiple myoplasm buckles resolve into one larger buckle, which again drives the formation of the contraction pole-a protuberance of the zygote's vegetal pole where maternal mRNAs accumulate. Thus, our findings reveal a mechanism where cortical actomyosin network flows determine cytoplasmic reorganization and cell shape by deforming adjacent cytoplasmic components through friction forces.
RESUMO
When crawling through the body, leukocytes often traverse tissues that are densely packed with extracellular matrix and other cells, and this raises the question: How do leukocytes overcome compressive mechanical loads? Here, we show that the actin cortex of leukocytes is mechanoresponsive and that this responsiveness requires neither force sensing via the nucleus nor adhesive interactions with a substrate. Upon global compression of the cell body as well as local indentation of the plasma membrane, Wiskott-Aldrich syndrome protein (WASp) assembles into dot-like structures, providing activation platforms for Arp2/3 nucleated actin patches. These patches locally push against the external load, which can be obstructing collagen fibers or other cells, and thereby create space to facilitate forward locomotion. We show in vitro and in vivo that this WASp function is rate limiting for ameboid leukocyte migration in dense but not in loose environments and is required for trafficking through diverse tissues such as skin and lymph nodes.
Assuntos
Actinas/fisiologia , Leucócitos/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.
Assuntos
Plaquetas/patologia , Vasos Sanguíneos/patologia , Quimiotaxia , Inflamação/patologia , Pneumonia/sangue , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Adulto , Animais , Movimento Celular , Microambiente Celular , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Humanos , Lipopolissacarídeos , Lesão Pulmonar/microbiologia , Lesão Pulmonar/patologia , Staphylococcus aureus Resistente à Meticilina/fisiologia , Camundongos Endogâmicos C57BL , Microvasos/patologia , Pneumonia/microbiologia , Pseudópodes/metabolismoRESUMO
BACKGROUND: Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. NEW METHOD: Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. RESULTS: We show that â¼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. COMPARISON WITH EXISTING METHODS: Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. CONCLUSIONS: These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.
Assuntos
Fracionamento Celular/métodos , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Engenharia Genética , Ácido Glutâmico/metabolismo , Lentivirus/fisiologia , Ratos WistarRESUMO
The stalked barnacle Dosima fascicularis secretes foam-like cement, the amount of which usually exceeds that produced by other barnacles. When Dosima settles on small objects, this adhesive is additionally used as a float which gives buoyancy to the animal. The dual use of the cement by D. fascicularis requires mechanical properties different from those of other barnacle species. In the float, two regions with different morphological structure and mechanical properties can be distinguished. The outer compact zone with small gas-filled bubbles (cells) is harder than the interior one and forms a protective rind presumably against mechanical damage. The inner region with large, gas-filled cells is soft. This study demonstrates that D. fascicularis cement is soft and visco-elastic. We show that the values of the elastic modulus, hardness and tensile stress are considerably lower than in the rigid cement of other barnacles.
RESUMO
Dosima fascicularis is the only barnacle which can drift autonomously at the water surface with a foam-like cement float. The cement secreted by the animal contains numerous gas-filled cells of different size. When several individuals share one float, their size and not their number is crucial for the production of both volume and mass of the float. The gas content within the cells of the foam gives positive static buoyancy to the whole float. The volume of the float, the gas volume and the positive static buoyancy are positively correlated. The density of the cement float without gas is greater than that of seawater. This study shows that the secreted cement consists of more than 90% water and the gas volume is on average 18.5%. Our experiments demonstrate that the intact foam-like cement float is sealed to the surrounding water.
RESUMO
Barnacles produce a proteinaceous adhesive called cement to attach permanently to rocks or to other hard substrata. The stalked barnacle Dosima fascicularis is of special interest as it produces a large amount of foam-like cement that can be used as a float. The morphology of the cement apparatus and of the polymerized cement of this species is almost unknown. The current study aims at filling these gaps in our knowledge using light and electron microscopy as well as x-ray microtomography. The shape of the cement gland cells changes from round to ovoid during barnacle development. The cytoplasm of the gland cells, unlike that of some other barnacles, does not have distinct secretory and storage regions. The cement canals, which transport the cement from the gland cells to the base of the stalk, end at different positions in juvenile and mature animals. With increasing size of the cement float, the exit of the cement canals shift from the centrally positioned attachment disk of the vestigial antennules to more lateral positions on the stalk. The bubbles enclosed in the foam-like float are most likely filled with CO(2) that diffuses from the hemolymph into the cement canal system and from there into the cement.