Assemblies of actin and its regulators underlie the dynamic morphology of all eukaryotic cells. To understand how actin regulatory proteins work together to generate actin-rich structures such as filopodia, we analyzed the localization of diverse actin regulators within filopodia in Drosophila embryos and in a complementary in vitro system of filopodia-like structures (FLSs). We found that the composition of the regulatory protein complex where actin is incorporated (the filopodial tip complex) is remarkably heterogeneous both in vivo and in vitro. Our data reveal that different pairs of proteins correlate with each other and with actin bundle length, suggesting the presence of functional subcomplexes. This is consistent with a theoretical framework where three or more redundant subcomplexes join the tip complex stochastically, with any two being sufficient to drive filopodia formation. We provide an explanation for the observed heterogeneity and suggest that a mechanism based on multiple components allows stereotypical filopodial dynamics to arise from diverse upstream signaling pathways.
Purpose To evaluate the potential use of anterior segment spectral domain optical coherence tomography (AS-SD-OCT) combined with an automated grading of fluorescein staining for assessment of corneal erosions in a rabbit short-term dry eye model. Methods Twenty-one New Zealand white rabbits were anesthetized and eyes were kept open for 140 minutes to induce acute corneal desiccation. Rectangular scans of the cornea were performed using Spectralis AS-SD-OCT. Total corneal thickness, corneal epithelial thickness, and the percentage of epithelial erosion area (PEEA) were evaluated. Corneas were stained with fluorescein and graded automatically using EpiView and semi-automatically using ImageJ. Spearman's rank-order correlations were calculated to compare the AS-SD-OCT PEEA and the two corneal staining scores. Results Eye desiccation resulted in corneal epithelium erosions that covered 0.67% to 14.2% of the central cornea (mean ± SD: 3.95% ± 3.2%) by AS-SD-OCT. The percentage of corneal area positively stained with fluorescein ranged from 0.24% to 38.01% (mean ± SD: 12.24% ± 9.7%) by using ImageJ, correlating with the AS-SD-OCT PEEA (Spearman's ρ, 0.574; P = 0.007). The EpiView score ranged from 0.5 to 10.17 and was better correlated with the AS-SD-OCT PEEA score (Spearman's ρ, 0.795; P = 0.000017). Conclusions Our study suggests that multimodal analysis of AS-SD-OCT and grading of fluorescein staining using EpiView software may enable quantitative assessment of corneal epithelial erosions in a rabbit short-term dry eye model. Translational Relevance This multimodal imaging analysis may be applied for evaluation of superficial punctate keratitis associated with dry eye.
Purpose Development of a method for noninvasive longitudinal follow-up of retinal degeneration in the whole retina for Royal College of Surgeons (RCS) rats, a commonly used model of retinitis pigmentosa associated with mutations in the MER-proto-oncogene tyrosine kinase ( MERTK ) gene. Methods Pigmented RCS rats at postnatal (p) days p28 to p84 were subjected to a biweekly spectral-domain optical coherence tomography (SD-OCT), blue laser fundus autofluorescence (BL-FAF) imaging, and multicolor fundus imaging. Wild-type (WT; Long Evans) rats were tested as control. Results Hyperautofluorescence developed throughout the fundus at p42, concomitant with a significant increase in SD-OCT thickness and reflectivity of the debris zone (DZ) layer as well as thinning of the photoreceptor outer nuclear layer (ONL). From p56 to p84, discrete hypofluorescent lesions surrounded by hyperfluorescent flecks were demonstrated around the optic disc that gradually spread throughout the retina. The hypofluorescent lesions were associated with loss of ONL and gradual thinning of the DZ layer. No hypofluorescent BL-FAF lesions were observed in WT rats. Conclusions This study suggests that BL-FAF imaging may present a new method for noninvasive longitudinal follow-up of retinal degeneration in nearly the whole retina in RCS rats. Translational Relevance A clinical test was developed that may be implemented in translational studies in the RCS rat model of MERTK -associated retinitis pigmentosa.
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