Stromules, or stroma-filled tubules, are thin extensions of the plastid envelope membrane that are most frequently observed in undifferentiated or non-mesophyll cells. The formation of stromules is developmentally regulated and responsive to biotic and abiotic stress; however, the physiological roles and molecular mechanisms of the stromule formation remain enigmatic. Accordingly, we attempted to obtain Arabidopsis thaliana mutants with aberrant stromule biogenesis in the leaf epidermis. Here, we characterize one of the obtained mutants. Plastids in the leaf epidermis of this mutant were giant and pleomorphic, typically having one or more constrictions that indicated arrested plastid division, and usually possessed one or more extremely long stromules, which indicated the deregulation of stromule formation. Genetic mapping, whole-genome resequencing-aided exome analysis, and gene complementation identified PARC6/CDP1/ARC6H, which encodes a vascular plant-specific, chloroplast division site-positioning factor, as the causal gene for the stromule phenotype. Yeast two-hybrid assay and double mutant analysis also identified a possible interaction between PARC6 and MinD1, another known chloroplast division site-positioning factor, during the morphogenesis of leaf epidermal plastids. To the best of our knowledge, PARC6 is the only known A. thaliana chloroplast division factor whose mutations more extensively affect the morphology of plastids in non-mesophyll tissue than in mesophyll tissue. Therefore, the present study demonstrates that PARC6 plays a pivotal role in the morphology maintenance and stromule regulation of non-mesophyll plastids.

Mammalian sperm–egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) that mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second function for IZUMO1 as a unilateral mouse gamete fusogen.

Successful gamete fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a live imaging-based adhesion molecule (LIAM) assay to study gamete adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell–cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gamete adhesion molecule pair, IZUMO1 and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO1 or JUNO was partly translocated to the opposite cells as discrete spots; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the LIAM assay, we investigated the species specificity of IZUMO1 and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO1 and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO1 and JUNO from different species.

Successful gametic fusion requires species-specific membrane adhesion. However, the interaction of adhesion molecules in gametes is difficult to study in real time through low-throughput microscopic observation. Therefore, we developed a novel live imaging-based adhesion molecule (LIAM) assay to study gametic adhesion molecule interactions in cultured cells. First, we modified a fusion assay previously established for fusogens introduced into cultured cells, and confirmed that our live imaging technique could visualise cell-to-cell fusion in the modified fusion assay. Next, instead of fusogen, we introduced adhesion molecules including a mammalian gametic adhesion molecule pair, IZUMO and JUNO, and detected their temporal accumulation at the contact interfaces of adjacent cells. Accumulated IZUMO or JUNO was translocated to the opposite cells; the mutation in amino acids required for their interaction impaired accumulation and translocation. By using the novel LIAM assay, we investigated the species specificity of IZUMO and JUNO of mouse, human, hamster, and pig in all combinations. IZUMO and JUNO accumulation and translocation were observed in conspecific, and some interspecific, combinations, suggesting potentially interchangeable combinations of IZUMO and JUNO from different species.

Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Here we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. Using content mixing and live-imaging assays in heterologous cells we found that IZUMO1 is a unilateral fusogen that acts independently of JUNO. IZUMO1-mediated fusion is abrogated by deletion of a β-hairpin that was previously reported to be required for JUNO-IZUMO1 trans-interactions. Our findings suggest a dual function for IZUMO1 in mouse gamete fusion.