The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling, governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana) NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• dusičnany metabolismus   MeSH
• fosforylace MeSH
• kořeny rostlin růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející anionty genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vápníková signalizace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

• MeSH
• antiportéry biosyntéza   genetika   MeSH
• buněčná diferenciace MeSH
• dědičné dystrofie rohovky genetika   metabolismus   patologie   MeSH
• dítě MeSH
• dospělí MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• percepční nedoslýchavost genetika   metabolismus   patologie   MeSH
• předškolní dítě MeSH
• prekurzory RNA MeSH
• proteiny přenášející anionty biosyntéza   genetika   MeSH
• regulace genové exprese * MeSH
• RNA genetika   MeSH
• rodokmen MeSH
• rohovkový endotel metabolismus   patologie   MeSH
• senioři MeSH
• sestřih RNA MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Drosophila salivary glands (SGs) were well known for the puffing patterns of their polytene chromosomes and so became a tissue of choice to study sequential gene activation by the steroid hormone ecdysone. One well-documented function of these glands is to produce a secretory glue, which is released during pupariation to fix the freshly formed puparia to the substrate. Over the past two decades SGs have been used to address specific aspects of developmentally-regulated programmed cell death (PCD) as it was thought that they are doomed for histolysis and after pupariation are just awaiting their fate. More recently, however, we have shown that for the first 3-4 h after pupariation SGs undergo tremendous endocytosis and vacuolation followed by vacuole neutralization and membrane consolidation. Furthermore, from 8 to 10 h after puparium formation (APF) SGs display massive apocrine secretion of a diverse set of cellular proteins. Here, we show that during the period from 11 to 12 h APF, the prepupal glands are very active in calcium oxalate (CaOx) extrusion that resembles renal or nephridial excretory activity. We provide genetic evidence that Prestin, a Drosophila homologue of the mammalian electrogenic anion exchange carrier SLC26A5, is responsible for the instantaneous production of CaOx by the late prepupal SGs. Its positive regulation by the protein kinases encoded by fray and wnk lead to increased production of CaOx. The formation of CaOx appears to be dependent on the cooperation between Prestin and the vATPase complex as treatment with bafilomycin A1 or concanamycin A abolishes the production of detectable CaOx. These data demonstrate that prepupal SGs remain fully viable, physiologically active and engaged in various cellular activities at least until early pupal period, that is, until moments prior to the execution of PCD.

• MeSH
• aktivní transport fyziologie   MeSH
• Drosophila melanogaster MeSH
• protein-serin-threoninkinasy genetika   metabolismus   MeSH
• proteiny Drosophily genetika   metabolismus   MeSH
• proteiny přenášející anionty biosyntéza   genetika   metabolismus   MeSH
• slinné žlázy sekrece   MeSH
• šťavelan vápenatý metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND/AIMS: To identify the underlying molecular genetic cause of disease in a patient with Harboyan syndrome and to perform a detailed assessment of her renal function. We also assessed the influence of the SLC4A11 mutation identified on the corneal endothelium in the heterozygous state. METHODS: A 55-year-old female was examined ophthalmologically, audiologically and nephrologically including 24-hour urine collection. The coding region of SLC4A11 was directly sequenced. Specular microscopy was performed in the proband's 21-year-old daughter. RESULTS: The proband had bilateral iridectomy at the age of 3 months because of an initial diagnosis of congenital glaucoma and since the age of 12 years she underwent several keratoplasties in each eye. Nephrological examination did not reveal any abnormalities. Moderate bilateral sensorineural hearing loss was confirmed by audiometry. A novel homozygous mutation predicted to lead to a premature stop codon at the protein level, c.2188C>T; p.(Arg730*), was identified in SLC4A11. No changes in corneal endothelial cell morphology or density were observed in the heterozygous daughter. CONCLUSION: In contrast to the Slc4a11(-/-) mouse, no abnormalities in daily renal ion excretion or polyuria were observed in the Harboyan syndrome patient. The mutation identified does not affect corneal endothelial cell morphology or density in the heterozygous state.

• MeSH
• antiportéry genetika   MeSH
• audiometrie MeSH
• dědičné dystrofie rohovky diagnóza   genetika   patofyziologie   MeSH
• jaterní testy MeSH
• ledviny fyziologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mutační analýza DNA MeSH
• nesmyslný kodon * MeSH
• pachymetrie rohovky MeSH
• percepční nedoslýchavost diagnóza   genetika   patofyziologie   MeSH
• polymerázová řetězová reakce MeSH
• proteiny přenášející anionty genetika   MeSH
• rohovkový endotel patologie   MeSH
• vyšetření funkce ledvin MeSH
• zraková ostrost fyziologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

• MeSH
• cytosol metabolismus   MeSH
• finanční podpora výzkumu jako téma MeSH
• konformace proteinů MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• proteiny přenášející anionty genetika   chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH