The vesicle trafficking inhibitor Brefeldin A (BFA) changes the localization of plasma membrane localized PINs, proteins that function as polar auxin efflux carriers, by inducing their accumulation within cells. Pretreatment with the synthetic auxin 1-NAA reduces this BFA-induced PIN internalization, suggesting that auxinic compounds inhibit the endocytosis of PIN proteins. However, the most important natural auxin, IAA, did not substantially inhibit PIN internalization unless a supplementary antioxidant, butylated hydroxytoluene (BHT), was also included in the incubation medium. We asked whether the relatively small inhibition caused by IAA alone could be explained by its instability in the incubation solution or whether IAA might interact with BHT to inhibit endocytosis. Analysis of the IAA concentration in the incubation solution and of DR5 reporter activity in the roots showed that IAA is both stable and active in the medium. Therefore, IAA degradation was not able to explain the inability of IAA to inhibit endocytosis. Furthermore, when applied in the absence of auxin, BHT caused a strong increase in the rate of PIN1 internalization and a weaker increase in the rate of PIN2 internalization. These increases were unaffected by the simultaneous application of IAA, further indicating that endocytosis is not inhibited by the natural auxin IAA under physiologically relevant conditions. Endocytosis was inhibited at the same rate with 2-NAA, an inactive auxin analog, as was observed with 1-NAA and more strongly than with natural auxins, supporting the idea that this inhibition is not auxin specific.

Auxin is a molecule, which controls many aspects of plant development through both transcriptional and non-transcriptional signaling responses. AUXIN BINDING PROTEIN1 (ABP1) is a putative receptor for rapid non-transcriptional auxin-induced changes in plasma membrane depolarization and endocytosis rates. However, the mechanism of ABP1-mediated signaling is poorly understood. Here we show that membrane depolarization and endocytosis inhibition are ABP1-independent responses and that auxin-induced plasma membrane depolarization is instead dependent on the auxin influx carrier AUX1. AUX1 was itself not involved in the regulation of endocytosis. Auxin-dependent depolarization of the plasma membrane was also modulated by the auxin efflux carrier PIN2. These data establish a new connection between auxin transport and non-transcriptional auxin signaling.

Nephronophthisis (NPH) is an autosomal recessive form of cystic kidney disease and the leading cause of hereditary kidney failure in children and young adults. Like other NPH proteins, the NPHP16/Anks6-interacting protein Anks3 has been identified to cause laterality defects in humans. However, the cellular functions of Anks3 remain enigmatic. We investigated the metabolic impact of Anks3 depletion in cultured murine inner medullary collecting duct cells via GC-MS profiling and LC-MS/MS analysis. Combined metabolomics successfully identified 155 metabolites; 48 metabolites were identified to be significantly altered by decreasing Anks3 levels. Especially, amino acid and purine/pyrimidine metabolism were affected by loss of Anks3. Branched-chain amino acids were identified to be significantly downregulated suggesting disrupted nutrient signalling. Tryptophan and 1-ribosyl-imidazolenicotinamide accumulated whereas NAD+ and NADP+ concentrations were diminished indicating disturbances within the tryptophan-niacin pathway. Most strikingly, nucleosides were reduced upon Anks3 depletion, while 5-methyluridine and 6-methyladenosine accumulated over time. Hence, elevated PARP1 and cleaved PARP1 levels could be detected. Furthermore, living cell number and viability was significantly declined. In combination, these results suggest that Anks3 may be involved in DNA damage responses by balancing the intracellular nucleoside pool.

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