Wednesday, April 24, 2024
4/24/2024
Apical extracellular matrices (aECMs) are associated with all epithelia and are essential for animal life. They form an outer protective layer against biotic and abiotic (physical damage) challenges from the environment. Human diseases associated with aECM dysfunction manifest a range of pathologies including, but not limited to, fibrosis, osteoarthritis, and cancer. aECMs also play important structural roles and are critical in mechanotransduction, such as in the vertebrate inner ear. However, in general we lack an understanding of the 3D organization of aECM at a molecular level, including its dynamics in development and disease states. The premise for this work is to create a systematic set of fluorescently tagged endogenous apical extracellular matrix (aECM) components, and further, through characterization using super-resolution microscopy, to reveal how these molecules are organized into nanoscale patterns in vivo. aECMs are associated with all epithelia and are essential for animal life. A hallmark of aECMs is their ability to form complex 3D structures patterned at the sub-micron scale. Such nanoscale architectures have been described by electron microscopy in a variety of systems but have for the most part lacked molecular correlates. Despite their recognized importance, the composition and biogenesis of aECMs remain poorly understood, partly because many aspects of aECM biology can only be fully studied in vivo. Moreover, a critical knowledge gap in aECM biology is how secreted molecules assemble in the extracellular environment to form and maintain complex 3D structures with nanoscale patterning. Basic features of the spatial and temporal localization of aECM components remain poorly understood because of these challenges. The increasing tractability of gene editing technology has enabled tagging of endogenous proteins (knockins); coupled with advanced imaging, knockin tagged proteins allow rigorous analysis of aECM nanoscale structure and biogenesis. We will exploit these technical advances to develop a standardized set of strains expressing fluorescently tagged aECM proteins in C. elegans, the ‘aECM toolkit’. These reagents will not only reveal novel aspects of aECM biology but will also establish principles for tagging and live imaging of the aECM in other systems. Our toolkit will open up the field of nanoscale aECM patterning to molecular genetic dissection, as well as leading to enhanced understanding of aECM in disease. We will generate a set of 100 knockin strains using CRISPR/Cas9 genome editing and make our toolkit and protocols widely available, generating a critical resource for the community. We will determine localization, biogenesis, and nanoscale architecture of aECM components. We will use live imaging to determine how aECM components are secreted and assemble into complex 3D structures during development. Our aECM toolkit will illuminate aECM assembly and dynamics and will fill critical knowledge gaps in aECM structure and dynamics in development and diseases.
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