Biology subjects

Zajac, A. L.

Publications and source records attributed to Zajac, A. L..

3 recordsLinked to original sources

bioRxiv2025

Evidence for the major role of PH4EFB in the prolyl 4-hydroxylation of Drosophila collagen IV

Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4Hs), Drosophila melanogaster harbour at least 26 candidates for collagen P4Hs despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4Hs, PH4EFB shares the highest homology with vertebrate collagen P4Hs. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of PH4EFB but not the other P4H-related genes with the collagen IV genes. Moreover, expression of PH4EFB during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4EFB. Finally, biochemical assays confirm that PH4EFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4EFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in Drosophila. Our findings highlight the remarkably simple nature of Drosophila collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.

biochemistry↗

bioRxiv2021

Kinesin-3 and kinesin-1 motors direct basement membrane protein secretion to a basal sub-region of the basolateral plasma membrane in epithelial cells

Basement membranes (BMs) are sheet-like extracellular matrices that line the basal surfaces of all epithelia. Since BM proteins form networks, they likely need to be secreted near the basal surface. However, the location of their secretion site and how it is selected are unknown. Working in the Drosophila follicular epithelium, we identified two kinesins essential for normal BM formation. Our data suggest the two kinesins work together to transport Rab10+ BM protein-filled secretory vesicles towards the basal surface along the polarized microtubule array common to epithelia. This kinesin transport biases BM protein secretion basally. When kinesins are depleted, BM proteins are mis-secreted to more apical regions of the lateral membrane, creating ectopic BM protein networks between cells that disrupt cell movements and tissue architecture. These results introduce a new transport step in the BM protein secretion pathway and highlight the importance of controlling the sub-cellular exocytic site of network-forming proteins. HighlightsO_LIA kinesin-3 and a kinesin-1 are required for normal basement membrane (BM) assembly C_LIO_LIKinesins move Rab10+ BM secretory vesicles basally on polarized microtubule arrays C_LIO_LITransport biases BM exocytosis to basal subregions of the basolateral membrane C_LIO_LILoss of kinesins creates ectopic BM networks that disrupt tissue architecture C_LI

cell biology↗

bioRxiv2020

ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block exit of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescent secretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory protein ESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid transport out of the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used with many model organisms.

cell biology↗