Microscopes rely on lenses bending, or refracting, light in predictable ways to allow us to look at objects that are otherwise too small to see. Yet, for as long as scientists have used lenses to magnify and observe life, problems have occurred when light does not refract as predicted. Any departure from the norm is called an aberration, and modern imaging systems have complex arrays of lenses with specialized coatings to limit most optical aberrations. However, these highly engineered systems are designed to optimally focus on objects at a fixed distance, typically the surface of the coverslip placed over the sample on a microscopy slide (Cargille, 1985). Focusing any deeper into samples within a watery solution – like a living cell or tissue – raises problems once again because of a phenomenon known as spherical aberration.
Light travels faster through water than it travels through glass or most biological samples. The speed of light in a given material is described by a property called its refractive index; and the higher the refractive index the slower light will travel. Spherical aberration occurs when light from an object – such as a fluorescently tagged protein – crosses the boundary between two materials with different refractive indices – for example, the biological sample and the surrounding solution – at an angle, and then bends because it changes speed. As the sample moves deeper into an aqueous solution, the point where the light intersects with the coverslip moves as well, and further bending by refraction can prevent the light from being captured by the lens (Figure 1). This effectively decreases both the resolution and brightness of the image, making it too blurry and too dim to distinguish meaningful features of objects further into the sample.
Biologists looking at living samples and wanting to see deeper than about 10 micrometers from the surface have previously needed microscopes with lower resolution, corrective adjustments, or 'adaptive optics systems' to minimize the effects of spherical aberration (Booth, 2007). These technologies, however, have limited practicality and are often expensive. Now, in eLife, Jochen Rink of the Max Planck Institute of Molecular Cell Biology and Genetics and colleagues report a simpler and more affordable approach (Boothe et al., 2017).
For non-living – or fixed – specimens, the problem of spherical aberrations has long been overcome by replacing the water with an optically clear substance with a high refractive index to better match that of glass. Yet many of the substances currently used, such as glycerol, are toxic to living samples. Rink and colleagues – including Tobias Boothe as first author – instead looked for a water-soluble compound with a high refractive index that was not toxic. A compound called iodixanol met all their requirements and they showed that when added to the surrounding solution at the proper concentration the biological sample effectively became 'invisible'. This occurred because light from the object did not experience a change in refractive index when it traveled between the sample and the solution, which meant that fluorescent objects within could be seen more clearly. No change in refractive index meant that the light was no longer refracted when it exited the sample. In other words, spherical aberration was greatly reduced.
Boothe et al. demonstrate the benefits of decreasing the spherical aberration in live samples by imaging deep into developing zebrafish embryos and planarian flatworms. Fluorescent markers in animals mounted in a solution containing iodixanol looked sharper and brighter than those in a more traditional aqueous solution. As would be expected, the improvements in optical resolution and brightness were more pronounced for objects at greater depths away from the coverslip.
Boothe et al. confirm that iodixanol is compatible with living samples by showing that various zebrafish embryos, human cell cultures and planarian flatworms can develop, proliferate, and even regenerate in the presence of high concentrations of the substance. This method represents a breakthrough for scientists looking to obtain high-quality images from living organisms. Microscopists will, however, still face challenges in matching the refractive index of the surrounding solution to the sample, because most organisms consist of multiple materials of different refractive indices. As such, the technique presented by Boothe et al. is a step forward for the field, but opportunities remain to further improve image quality in complex organisms.
New York Microscopical Society YearbookImmersion oil and the microscope, New York Microscopical Society Yearbook, Cargille-Sacher Laboratories, Inc.
Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson’s disease (PD) and Crohn’s disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.
Activating mutations in the leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that these phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2-dependent and PPM1H phosphatase-reversible, and did not require Rab12 phosphorylation; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. As expected for a protein that regulates LRRK2 activity, Rab12 also influenced primary cilia formation. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain, and we show that residues predicted to be essential for Rab12 interaction at this site influence phosphoRab10 and phosphoRab12 levels in a manner distinct from Rab29 activation of LRRK2. Our data show that Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.