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The transient receptor potential vanilloid-1 (TRPV1) ion channel is essential for sensation of thermal and chemical pain. TRPV1 activation is accompanied by Ca2+-dependent desensitization; acute desensitization reflects rapid reduction in channel activity during stimulation, whereas tachyphylaxis denotes the diminution in TRPV1 responses to repetitive stimulation. Acute desensitization has been attributed to conformational changes of the TRPV1 channel; however, the mechanisms underlying the establishment of tachyphylaxis remain to be defined. Here, we report that the degree of whole-cell TRPV1 tachyphylaxis is regulated by the strength of inducing stimulation. Using light-sheet microscopy and pH-sensitive sensor pHluorin to follow TRPV1 endocytosis and exocytosis trafficking, we provide real-time information that tachyphylaxis of different degrees concurs with TRPV1 recycling to the plasma membrane in a proportional manner. This process controls TRPV1 surface expression level thereby the whole-cell nociceptive response. We further show that activity-gated TRPV1 trafficking associates with intracellular Ca2+ signals of distinct kinetics, and recruits recycling routes mediated by synaptotagmin 1 and 7, respectively. These results suggest that activity-dependent TRPV1 recycling contributes to the establishment of tachyphylaxis.
Astrocytes are a major type of glial cell in the mammalian brain, essentially regulating neuronal development and function. Quantitative imaging represents an important approach to study astrocytic signaling in neural circuits. Focusing on astrocytic Ca2+ activity, a key pathway implicated in astrocye-neuron interaction, we here report a strategy combining fast light sheet fluorescence microscopy (LSFM) and correlative screening-based time series analysis, to map activity domains in astrocytes in living mammalian nerve tissue. Light sheet of micron-scale thickness enables wide-field optical sectioning to image astrocytes in acute mouse brain slices. Using both chemical and genetically encoded Ca2+ indicators, we demonstrate the complementary advantages of LSFM in mapping Ca2+ domains in astrocyte populations as compared to epifluorescence and two-photon microscopy. Our approach then revealed distinct kinetics of Ca2+ signals between cortical and hypothalamic astrocytes in resting conditions and following the activation of adrenergic G protein coupled receptor (GPCR). This observation highlights the activity heterogeneity across regionally distinct astrocyte populations, and indicates the potential of our method for investigating dynamic signals in astrocytes.
Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation–contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation–contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.
Диссертации
Matthieu Cortes
In plants, the development of aerial organs is indeterminate: it takes place throughout their lifespan. In contrast, the development of floral organs is determinate in Arabidopsis thaliana, each flower has the same number of floral organs. This difference in development is due to the maintenance or not of the pool of stem cells present in the stem cell niches, the meristems. During my thesis I showed that the transcriptional regulator VIP3 contributes to the regulation of the switch from indeterminate to determinate in flowers. This also revealed that the control of flower termination is not as robust as classically thought. Because VIP3 is also involved in the regulation of epigenetic marks and response to external mechanical stimuli, this work opens new questions on the role of mechanical signals in indeterminacy. On a more technical standpoint, the analysis of shoot development suffers from a lack of imaging methods with high temporal resolution and in-depth optical sectioning. During the last decade, light sheet microscopy has emerged as a competitive imaging modality in developmental biology. However, in plants, the technique has mainly been used in roots because of limits in the microscope design.
Учебные материалы
Рабочий процесс визуализации стеков изображений получаемых при использовании микроскопа Alpha3
PhaseView
В этом видеоролике показан типичный процесс визуализации наборов данных, полученных с помощью микроскопа планарного освещения Alpha3 с помощью программы визуализации данных Imaris.
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PhaseView
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Учебные материалы о микроскопах Alpha3, PhaseView, 2021
Монтаж образцов больших размеров при использовании микроскопа Alpha3
PhaseView
В этом видеоролике демонстрируется, как установить и закрепить большой образец на стеклянной пластине держателя образцов для получения изображений с помощью микроскопа планарного освещения Alpha3.
Учебные материалы о микроскопах Alpha3, PhaseView, 2021
Olympus Australia
В этом видеоролике представлен пятиэтапный обзор процедуры запуска микроскопа планарного освещения Alpha3.
Презентации
Визуализация данных с помощью программного обеспечения Imaris
Daniel Reisen
В этом видеоролике показано, как визуализировать наборы данных изображений, полученных с помощью микроскопа Alpha3, с использованием Imaris — универсального программного решения для визуализации, анализа и интерпретации данных многомерной микроскопии.
Презентации о микроскопах Alpha3, Bitplane AG, 2020