A congenital neutrophil defect syndrome associated with mutations in VPS45.

BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).

TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development.

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process.