scholarly journals Live imaging of apoptosis in a novel transgenic mouse highlights its role in neural tube closure

2011 ◽  
Vol 195 (6) ◽  
pp. 1047-1060 ◽  
Author(s):  
Yoshifumi Yamaguchi ◽  
Naomi Shinotsuka ◽  
Keiko Nonomura ◽  
Kiwamu Takemoto ◽  
Keisuke Kuida ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Neural Tube ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Neural Tube Closure ◽  
Caspase Activation ◽  
Fluorescent Reporter ◽  
Time Lapse Imaging ◽  
Tube Closure

Many cells die during development, tissue homeostasis, and disease. Dysregulation of apoptosis leads to cranial neural tube closure (NTC) defects like exencephaly, although the mechanism is unclear. Observing cells undergoing apoptosis in a living context could help elucidate their origin, behavior, and influence on surrounding tissues, but few tools are available for this purpose, especially in mammals. In this paper, we used insulator sequences to generate a transgenic mouse that stably expressed a genetically encoded fluorescence resonance energy transfer (FRET)–based fluorescent reporter for caspase activation and performed simultaneous time-lapse imaging of apoptosis and morphogenesis in living embryos. Live FRET imaging with a fast-scanning confocal microscope revealed that cells containing activated caspases showed typical and nontypical apoptotic behavior in a region-specific manner during NTC. Inhibiting caspase activation perturbed and delayed the smooth progression of cranial NTC, which might increase the risk of exencephaly. Our results suggest that caspase-mediated cell removal facilitates NTC completion within a limited developmental window.

scholarly journals Live-imaging analysis of apoptosis and caspase activation reveals that apoptosis is a facilitator of the neural tube closure

2010 ◽  
Vol 344 (1) ◽  
pp. 442
Author(s):  
Yoshifumi Yamaguchi ◽  
Naomi Shinotsuka ◽  
Keiko Nonomura ◽  
Masayuki Miura
Keyword(s):  
Neural Tube ◽  
Live Imaging ◽  
Neural Tube Closure ◽  
Imaging Analysis ◽  
Caspase Activation ◽  
Tube Closure

scholarly journals Spatial distribution and functional significance of activated vinculin in living cells

2005 ◽  
Vol 169 (3) ◽  
pp. 459-470 ◽  
Author(s):  
Hui Chen ◽  
Daniel M. Cohen ◽  
Dilshad M. Choudhury ◽  
Noriyuki Kioka ◽  
Susan W. Craig
Keyword(s):  
Conformational Change ◽  
Direct Evidence ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Time Lapse ◽  
Living Cells ◽  
Actin Binding ◽  
Binding Conformation ◽  
Time Lapse Imaging

Conformational change is believed to be important to vinculin's function at sites of cell adhesion. However, nothing is known about vinculin's conformation in living cells. Using a Forster resonance energy transfer probe that reports on changes in vinculin's conformation, we find that vinculin is in the actin-binding conformation in a peripheral band of adhesive puncta in spreading cells. However, in fully spread cells with established polarity, vinculin's conformation is variable at focal adhesions. Time-lapse imaging reveals a gradient of conformational change that precedes loss of vinculin from focal adhesions in retracting regions. At stable or protruding regions, recruitment of vinculin is not necessarily coupled to the actin-binding conformation. However, a different measure of vinculin conformation, the recruitment of vinexin β by activated vinculin, shows that autoinhibition of endogenous vinculin is relaxed at focal adhesions. Beyond providing direct evidence that vinculin is activated at focal adhesions, this study shows that the specific functional conformation correlates with regional cellular dynamics.

scholarly journals Global analysis of cell behavior and protein localization dynamics reveals region-specific functions for Shroom3 and N-cadherin during neural tube closure

2021 ◽  
Author(s):  
Austin T. Baldwin ◽  
Juliana Kim ◽  
John B. Wallingford
Keyword(s):  
Neural Tube ◽  
Cell Biology ◽  
Protein Localization ◽  
Global Analysis ◽  
Time Lapse ◽  
Tissue Level ◽  
Neural Tube Closure ◽  
Cell Behaviors ◽  
Regional Specificity ◽  
Tube Closure

AbstractFailures of neural tube closure are common and serious birth defects, yet we have a poor understanding of the interaction of genetics and cell biology during neural tube closure. Additionally, mutations that cause neural tube defects (NTDs) tend to affect anterior or posterior regions of the neural tube but rarely both, indicating a regional specificity to NTD genetics. To better understand the regional specificity of cell behaviors during neural tube closure, we analyzed the dynamic localization of actin and N-cadherin via high-resolution tissue-level time-lapse microscopy during Xenopus neural tube closure. To investigate the regionality of gene function, we generated mosaic mutations in shroom3, a key regulator or neural tube closure This approach elucidates new differences between cell behaviors during cranial/anterior and spinal/posterior neural tube closure, provides mechanistic insight into the function of shroom3 and demonstrates the ability of tissue-level imaging and analysis to generate cell-biological mechanistic insights into neural tube closure.

scholarly journals In vivo time-lapse analysis of cell divisions during neural tube closure

2006 ◽  
Vol 295 (1) ◽  
pp. 444
Author(s):  
Esther K. Kieserman ◽  
Julian M. Tyszka ◽  
John B. Wallingford
Keyword(s):  
Neural Tube ◽  
Time Lapse ◽  
Neural Tube Closure ◽  
Cell Divisions ◽  
Tube Closure

scholarly journals Multiplexed bioluminescence microscopy via phasor analysis

2021 ◽  
Author(s):  
Zi Yao ◽  
Caroline K. Brennan ◽  
Lorenzo Scipioni ◽  
Hongtao Chen ◽  
Kevin Ng ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Bioluminescence Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Lapse ◽  
Detection Methods ◽  
Transfer Processes ◽  
Time Lapse Imaging ◽  
Luciferase Reporters ◽  
Phasor Analysis

Microscopic bioluminescence imaging has been historically challenging due to a lack of detection methods and easily resolved probes. Here we combine bioluminescence with phasor analysis, an optical method commonly used to distinguish spectrally similar fluorophores. Bioluminescent phasor enabled rapid differentiation of multiple luciferase reporters and resonance energy transfer processes. The merger of bioluminescence and phasor analysis provides a platform for routine, time-lapse imaging of collections of cellular features.

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