Multiscale patterning of a model apical extracellular matrix revealed by systematic endogenous protein tagging

Area of Science:

• Cell Biology
• Biochemistry
• Developmental Biology

Background:

• Apical extracellular matrices (aECMs) protect barrier epithelia but their complex molecular organization in vivo remains poorly understood.
• Understanding aECM structure is crucial for comprehending epithelial barrier function and tissue integrity.

Purpose of the Study:

• To define the molecular architecture of a model aECM using a comprehensive protein tagging approach.
• To generate a standardized toolkit of fluorescently tagged proteins for visualizing aECM components in vivo.

Main Methods:

• Generated a toolkit of 101 fluorescently tagged apical extracellular matrix components in C. elegans using CRISPR gene editing.
• Developed efficient pipelines for modular protein tagging and rapid fluorophore swapping.
• Utilized tagged proteins to define reference markers for key aECM substructures, including the cortical layer and helical crossed fiber arrays.

Main Results:

• Most tagged collagens were functional and displayed highly specific localization patterns across different developmental stages, cell types, and matrix substructures.
• Defined multiple reference markers for established aECM compartments and identified markers for newly characterized substructures.
• Successfully tagged over 30 additional key aECM protein classes, including proteases, protease inhibitors, and lipid transporters.

Conclusions:

• The developed toolkit provides the first large-scale protein tagging resource for the apical extracellular matrix.
• Standardized markers enable detailed dissection of the spatiotemporal patterning mechanisms governing aECM organization in vivo.
• This resource facilitates future studies on the role of aECM structure in maintaining organismal shape and barrier function.