The asymmetric distribution of stable, posttranslationally modified microtubules (MTs) contributes to the polarization of many cell types, yet the factors controlling the formation of these MTs are not known. We have found that lysophosphatidic acid (LPA) is a major serum factor responsible for rapidly generating stable, detyrosinated (Glu) MTs in serum-starved 3T3 cells. Using C3 toxin and val14 rho we showed that rho was both necessary and sufficient for the induction of Glu MTs by LPA and serum. Unlike previously described factors that induce MT stability, rho induced the stabilization of only a subset of the MTs and, in wound-edge cells, these stable MTs were appropriately oriented toward the leading edge of the cell. LPA had little effect on individual parameters of MT dynamics, but did induce long states of pause in a subset of MTs near the edge of the cell. Rho stimulation of MT stability was independent of actin stress fiber formation. These results identify rho as a novel regulator of the MT cytoskeleton that selectively stabilizes MTs during cell polarization by acting as a switch between dynamic and stable states of MTs rather than as a modulator of MT assembly and disassembly.

Identifying mechanisms of diseases with complex inheritance patterns, such as macular telangiectasia type 2, is challenging. A link between macular telangiectasia type 2 and altered serine metabolism has been established previously. METHODS Through exome sequence analysis of a patient with macular telangiectasia type 2 and his family members, we identified a variant in SPTLC1 encoding a subunit of serine palmitoyltransferase (SPT). Because mutations affecting SPT are known to cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), we examined 10 additional persons with HSAN1 for ophthalmologic disease. We assayed serum amino acid and sphingoid base levels, including levels of deoxysphingolipids, in patients who had macular telangiectasia type 2 but did not have HSAN1 or pathogenic variants affecting SPT. We characterized mice with low serine levels and tested the effects of deoxysphingolipids on human retinal organoids. RESULTS Two variants known to cause HSAN1 were identified as causal for macular telangiectasia type 2: of 11 patients with HSAN1, 9 also had macular telangiectasia type 2. Circulating deoxysphingolipid levels were 84.2% higher among 125 patients with macular telangiectasia type 2 who did not have pathogenic variants affecting SPT than among 94 unaffected controls. Deoxysphingolipid levels were negatively correlated with serine levels, which were 20.6% lower than among controls. Reduction of serine levels in mice led to increases in levels of retinal deoxysphingolipids and compromised visual function. Deoxysphingolipids caused photoreceptor-cell death in retinal organoids, but not in the presence of regulators of lipid metabolism. CONCLUSIONS Elevated levels of atypical deoxysphingolipids, caused by variant SPTLC1 or SPTLC2 or by low serine levels, were risk factors for macular telangiectasia type 2, as well as for peripheral neuropathy.

Corneal epithelium of adult GFP mice exhibits a pattern of GFP expression that is suitable for studying cell movement in the normal cornea. Epithelial cells at the basal or suprabasal layers move centripetally in these mice at an average rate of 26 micro m/d.

Imaging of the human fundus of the eye with excitation wavelengths in the visible spectrum reveals a natural autofluorescence, that in a healthy retina originates primarily from the bisretinoids that constitute the lipofuscin of retinal pigment epithelial (RPE) cells. Since the intensity and distribution of fundus autofluorescence is altered in the presence of retinal disease, we have examined the fluorescence properties of the retinal bisretinoids with a view to aiding clinical interpretations. As is also observed for fundus autofluorescence, fluorescence emission from RPE lipofuscin was generated with a wide range of exciting wavelengths; with increasing excitation wavelength, the emission maximum shifted towards longer wavelengths and spectral width was decreased. These features are consistent with fluorescence generation from a mixture of compounds. While the bisretinoids that constitute RPE lipofuscin all fluoresced with maxima that were centered around 600 nm, fluorescence intensities varied when excited at 488 nm, the excitation wavelength utilized for fundus autofuorescence imaging. For instance the fluorescence efficiency of the bisretinoid A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE) was greater than A2E and relative to both of the latter, all-trans-retinal dimer-phosphatidylethanolamine was weakly fluorescent. On the other hand, certain photooxidized forms of the bisretinoids present in both RPE and photoreceptor cells were more strongly fluorescent than the parent compound. We also sought to evaluate whether diffuse puncta of autofluorescence observed in some retinal disorders of monogenic origin are attributable to retinoid accumulation. However, two retinoids of the visual cycle, all-trans-retinyl ester and all-trans-retinal, did not exhibit fluorescence at 488 nm excitation.

Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of the ABCA4 locus in STGD1 patients identifies two expected disease-causing alleles in ∼75% of patients and only one mutation in ∼15% of patients. Recently, many possibly pathogenic variants in deep intronic sequences of ABCA4 have been identified in the latter group. We extended our analyses of deep intronic ABCA4 variants and determined that one of these, c.4253+43G>A (rs61754045), is present in 29/1155 (2.6%) of STGD1 patients. The variant is found at statistically significantly higher frequency in patients with only one pathogenic ABCA4 allele, 23/160 (14.38%), MAF = 0.072, compared to MAF = 0.013 in all STGD1 cases and MAF = 0.006 in the matching general population (P < 1 × 10−7). The variant, which is not predicted to have any effect on splicing, is the first reported intronic “extremely hypomorphic allele” in the ABCA4 locus; that is, it is pathogenic only when in trans with a loss-of-function ABCA4 allele. It results in a distinct clinical phenotype characterized by late onset of symptoms and foveal sparing. In ∼70% of cases the variant was allelic with the c.6006-609T>A (rs575968112) variant, which was deemed nonpathogenic. Another rare deep intronic variant, c.5196+1056A>G (rs886044749), found in 5/834 (0.6%) of STGD1 cases is, conversely, a severe allele. This study determines pathogenicity for three noncoding variants in STGD1 patients of European descent accounting for ∼3% of the disease. Defining disease-associated alleles in the noncoding sequences of the ABCA4 locus can be accomplished by integrated clinical and genetic analyses.