RESUMO
Color vision deficiency (CVD) is a frequent condition that alters color perception to such an extent that many people encounter serious difficulties on their everyday lives. In this work, we present a strategy to analyze the effectiveness of light filters aimed to improve color discriminability of persons with CVD. The calculations are based on a simple model of color discrimination which has been successfully applied to several animal species. We first tested the calculations on three well-known commercial lenses designed for persons with CVD. In agreement with results of clinical studies, the calculations show that the highly colored lenses (VINO® and X-Chrom®) enhance chromaticity contrasts between problematic colors, whereas the more neutral Enchroma® do not provide any benefit. Also, we predict that two light filters proposed in recent works for novel lenses would not improve the performance of the commercial ones. Since the mathematical approach presented in this work enables predictive filter assessment, it opens the door to future research on the design of more effective lenses to improve color discriminability of persons with CVD. The calculations allow for large-scale screening of numerous light filters and different colored stimuli, CVD conditions, light sources, etc.
RESUMO
Microtubule-dependent motors usually work together to transport organelles through the crowded intracellular milieu. Thus, transport performance depends on how motors organize on the cargo. Unfortunately, the lack of methodologies capable of measuring this organization in cells determines that many aspects of the collective action of motors remain elusive. Here, we combined fluorescence fluctuations and single particle tracking techniques to address how kinesins organize on rod-like mitochondria moving along microtubules in cells. This methodology simultaneously provides mitochondria trajectories and EGFP-tagged kinesin-1 intensity at different mitochondrial positions with millisecond resolution. We show that kinesin exchange at the mitochondrion surface is within ~100â¯ms and depends on the organelle speed. During anterograde transport, the mitochondrial leading tip presents slower motor exchange in comparison to the rear tip. In contrast, retrograde mitochondria show similar exchange rates of kinesins at both tips. Numerical simulations provide theoretical support to these results and evidence that motors do not share the load equally during intracellular transport.
Assuntos
Cinesinas/metabolismo , Microtúbulos/fisiologia , Organelas/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Drosophila , Fluorescência , Cinética , Microtúbulos/metabolismo , Espectrometria de FluorescênciaRESUMO
The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.