C-terminal EMB-9 and LET-2 fluorophore knock-ins are secreted and incorporated into BMs more efficiently than internal knock-ins. (A) Top: the schematic indicates location of tissues and surrounding BMs (dashed lines) analyzed in an L1 larva. Bottom: representative max projections from confocal fluorescence z-stacks of L1 larvae with fluorescently tagged type IV collagen α-chains. Internally tagged α-chains aggregate in body wall muscles, while C-terminally tagged α-chains do not (n = 10 animals per strain). The scale bar is 10 μm. (B) Single z-slice confocal images show that aggregates of EMB-9::mScarlet-I (IS1) (magenta in merge) are localized within the ER membrane (visualized with ELO-1::mNG, cyan in merge) in L1 larvae. Yellow arrows indicate aggregates in the ER (100/100 aggregates localized to the ER, 10 aggregates counted per animal). The scale bar is 5 μm. (C) Left: Single z-slice confocal images show reduced EMB-9::mNG (IS1) localized to the pharyngeal BM compared with EMB-9::mNG (C) in L1 larvae (∼28% reduction; yellow arrows indicate pharyngeal BM). Right: Boxplots show mean fluorescence intensity measurements (a.u.) of the pharyngeal BM surrounding the proximal bulb (median of data denoted by horizontal line within each box for this and all subsequent boxplots) for EMB-9::mNG (C) versus EMB-9::mNG (IS1) animals (n = 10 animals each, ***P < 0.001, unpaired two-tailed Student’s t test). The scale bar is 10 μm.