De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.
Phosphate (Pi) deficiency induces a multitude of responses aimed at improving the acquisition of Pi, including an increased density of root hairs. To understand the mechanisms involved in Pi deficiency-induced alterations of the root hair phenotype in Arabidopsis (Arabidopsis thaliana), we analyzed the patterning and length of root epidermal cells under control and Pi-deficient conditions in wild-type plants and in four mutants defective in the expression of master regulators of cell fate, CAPRICE (CPC), ENHANCER OF TRY AND CPC 1 (ETC1), WEREWOLF (WER) and SCRAMBLED (SCM). From this analysis we deduced that the longitudinal cell length of root epidermal cells is dependent on the correct perception of a positional signal (‘cortical bias’) in both control and Pi-deficient plants; mutants defective in the receptor of the signal, SCM, produced short cells characteristic of root hair-forming cells (trichoblasts). Simulating the effect of cortical bias on the time-evolving probability of cell fate supports a scenario in which a compromised positional signal delays the time point at which non-hair cells opt out the default trichoblast pathway, resulting in short, trichoblast-like non-hair cells. Collectively, our data show that Pi-deficient plants increase root hair density by the formation of shorter cells, resulting in a higher frequency of hairs per unit root length, and additional trichoblast cell fate assignment via increased expression of ETC1.
Lateral segregation and the formation of lateral domains are well-known phenomena in ternary lipid bilayers composed of an unsaturated (low gel-to-liquid phase transition temperature (T m)) phospholipid, a saturated (high-T m) phospholipid, and cholesterol. The formation of lateral domains has been shown to be influenced by differences in phospholipid acyl chain unsaturation and length. Recently, we also showed that differential interactions of cholesterol with low-and high-T m phospholipids in the bilayer can facilitate phospholipid segregation. Now, we have investigated phospholipid-cholesterol interactions and their role in lateral segregation in ternary bilayers composed of different unsaturated phosphatidylcholines (PCs) with varying acyl chain lengths, N-palmitoyl-D-erythro-sphingomyelin (PSM), and cholesterol. Using deuterium NMR spectroscopy, we determined how PSM was influenced by the acyl chain composition in surrounding PC environments and correlated this with the affinity of cholestatrienol (a fluorescent cholesterol analog) for PSM in the different PC environments. Results from a combination of time-resolved fluorescence measurements of trans-parinaric acid and Fö rster resonance energy transfer experiments showed that the relative affinity of cholesterol for phospholipids determined the degree to which the sterol promoted domain formation. From Fö rster resonance energy transfer, deuterium NMR, and differential scanning calorimetry results, it was clear that cholesterol also influenced both the thermostability of the domains and the degree of order in and outside the PSM-rich domains. The results of this study have shown that the affinity of cholesterol for both low-T m and high-T m phospholipids and the effects of low-and high-T m phospholipids on each other influence both lateral structure and domain properties in complex bilayers. We envision that similar effects also contribute to lateral heterogeneity in even more complex biological membranes.
The segregation of lipids into lateral membrane domains has been extensively studied. It is well established that the structural differences between phospholipids play an important role in lateral membrane organization. When a high enough cholesterol concentration is present in the bilayer, liquid-ordered (L o ) domains, which are enriched in cholesterol and saturated phospholipids such as sphingomyelin (SM), may form. We have recently shown that such a formation of domains can be facilitated by the affinity differences of cholesterol for the saturated and unsaturated phospholipids present in the bilayer. In mammalian membranes, the saturated phospholipids are usually SMs with different acyl chains, the abundance of which vary with cell type. In this study, we investigated how the acyl chain structure of SMs affects the formation of SM-and cholesterol-enriched domains. From the analysis of trans-parinaric acid fluorescence emission lifetimes, we could determine that cholesterol facilitated lateral segregation most with the SMs that had 16 carbon-long acyl chains. Using differential scanning calorimetry and Fö rster resonance energy transfer techniques, we observed that the SM-and cholesterol-enriched domains with 16 carbonlong SMs were most thermally stabilized by cholesterol. The Fö rster resonance energy transfer technique also suggested that the same SMs also form the largest L o domains. In agreement with our previously published data, the extent of influence that cholesterol had on the propensity of lateral segregation and the properties of L o domains correlated with the relative affinity of cholesterol for the phospholipids present in the bilayers. Therefore, the specific SM species present in the membranes, together with unsaturated phospholipids and cholesterol, can be used by the cell to fine-tune the lateral structure of the membranes.
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