Vertebrate extracellular matrix protein hemicentin-1 interacts physically and genetically with basement membrane protein nidogen-2.

• The large extracellular matrix protein hemicentin-1 from mouse and zebrafish physically binds to the basement membrane protein nidogen-2. • Hemincentin-1 and laminins compete for binding to the G3 domain of nidogen-2, with binding between hemicentin-1 and nidogen-2 occurring kinetically faster, but binding between laminin and nidogen-2 being thermodynamically more stable. Hemicentin-1 and nidogen-2 display partially overlapping localization in. • different mouse and zebrafish tissues. • Loss-of-function studies in zebrafish embryos indicate that hemicentin-1, nidogen-2 and laminin-5 are all required for proper epidermal-dermal junction formation. • Synergistic enhancement studies via combined partial loss-of-function conditions in zebrafish embryos point to a tight genetic interaction between hemicentin-1 and nidogen-2 as well as between laminin-5 and nidogen-2, but not between hemicentin-1 and laminin-5, to promote epidermal-dermal junction formation, in line with the identified differential physical binding properties. • A model is proposed according to which hemicentin-1 plays a temporary, chaperone-like role during early steps of basement membrane formation, allowing molecular nidogen-2 networking before its G3-mediated binding to laminins. Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mouse and zebrafish hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 in basement membrane integrity. We show that HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately 3.5-fold lower affinity and in a competitive manner. Furthermore, immunofluorescence and immunogold labeling revealed that HMCN1/Hmcn1 is localized close to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as the loss of Laminin-α5 (Lama5) or Hmcn1. Finally, combined partial loss-of-function studies indicated that nid2a genetically interacts with both hmcn1 and lama5. Together, these findings suggest that despite their mutually exclusive physical binding, hemicentins, nidogens, and laminins tightly cooperate and support each other during formation, maintenance, and function of basement membranes to confer tissue linkage. [ABSTRACT FROM AUTHOR]