The Secret Lives of Fluorescent Membrane Probes as Revealed by Molecular Dynamics Simulations

Fluorescent probes have been employed for more than half a century to study the structure and dynamics of model and biological membranes, using spectroscopic and/or microscopic experimental approaches. While their utilization has led to tremendous progress in our knowledge of membrane biophysics and physiology, in some respects the behavior of bilayer-inserted membrane probes has long remained inscrutable. The location, orientation and interaction of fluorophores with lipid and/or water molecules are often not well known, and they are crucial for understanding what the probe is actually reporting. Moreover, because the probe is an extraneous inclusion, it may perturb the properties of the host membrane system, altering the very properties it is supposed to measure. For these reasons, the need for independent methodologies to assess the behavior of bilayer-inserted fluorescence probes has been recognized for a long time. Because of recent improvements in computational tools, molecular dynamics (MD) simulations have become a popular means of obtaining this important information. The present review addresses MD studies of all major classes of fluorescent membrane probes, focusing in the period between 2011 and 2020, during which such work has undergone a dramatic surge in both the number of studies and the variety of probes and properties accessed.

Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging

ABSTRACTThe plasma membrane (PM) plays a major role in many biological processes; therefore its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535 were found to display enhanced photophysical properties (reduced crosstalk, higher brightness, improved photostability) and unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell lines including neurons (primary culture and tissue imaging). Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we demonstrated that the modifications made to FM1-43 did not impair the ability of the new probes to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Soil Macronutrient Responses in Diverse Landscapes of Southern Tallgrass to Two Stocking Methods

Macronutrient (N, P, S, K, Ca, and Mg) availability and distribution in soils of grassland ecosystems are affected by diverse factors, including landscape position, climate, and forms of management. This study examined flux in plant-available macronutrients in production-scale (60 to 80 ha) paddocks of southern tallgrass prairie of central Oklahoma, United States, managed (2009–15) under two contrasting stocking methods (continuous yearlong; rotational stocking among 10 sub-paddocks). Macronutrient availability within the 0–7.5 cm and 7.5–15 cm soil depths were determined with sets of anion-cation exchange membrane probes at 16 locations within paddocks, oriented along transects from water sources to far corners. No clear overall effect related to stocking method was recorded for all macronutrient distributions. The only significant stocking method × location interaction occurred for K (p = 0.01). All other macronutrients displayed significant (p < 0.08) location effects that were common across stocking methods. Effects relatable to stocking method occurred in interactions with soil depth or time of year (p < 0.10), but responses of macronutrient flux to stocking method in these interactions varied. Higher flux occurred in available S, Ca, and Mg in proximity (<24 m) to water sources, which may be related to grazing, but local features of the landscape may also have been involved. More attention to landscape features included within paddocks, and standardized organization of water and other features within paddocks, would improve the potential to define grazing effects on macronutrient distribution.