Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.DOI: http://dx.doi.org/10.7554/eLife.10415.001

The study of cellular glycosylation presents many challenges due, in large part, to the non-template driven nature of glycan biosynthesis and their structural complexity. Chemoenzymatic glycan labeling (CEGL) has emerged as a new technique to address the limitations of existing methods for glycan detection. CEGL combines glycosyltransferases and unnatural nucleotide sugar donors equipped with a bioorthogonal chemical tag to directly label specific glycan acceptor substrates in situ within biological samples. This article reviews the current CEGL strategies that are available to characterize cell-surface and intracellular glycans. Applications include imaging glycan expression status in live cells and tissue samples, proteomic analysis of glycoproteins, and target validation. Combined with genetic and biochemical tools, CEGL provides new opportunities to elucidate the functional roles of glycans in human health and disease.

We study the spreading of viscous nonvolatile liquids on smooth horizontal substrates using a phase-modulated interference microscope with sufficient dynamic range to enable the simultaneous measurement of both the inner ("microscopic") length scale and the outer ("macroscopic") flow scale in addition to the intermediate matching region. The resulting measurements of both the apparent contact angle and the lateral scale of the precursor "wetting" film agree quantitatively with theoretical predictions for a van der Waal's liquid over a wide range of capillary numbers.

Using a combination of metabolically labeled glycans, bioorthogonal Cu(I)-catalyzed azide-alkyne cycloaddition and controlled bleaching of fluorescent probes conjugated to azide or alkyne tagged glycans, we achieve a sufficiently low spatial density of dye labeled glycans enabling dynamic single-molecule tracking and super-resolution imaging of N-linked sialic acids and O-linked GalNAc on the membrane of live cells. Analysis of the trajectories of these dye labeled glycans in mammary cancer cells reveal constrained diffusion of both N- and O-linked glycans which we interpret as reflecting the mobility of the glycan rather than caused by transient immobilization due to spatial inhomogeneities on the plasma membrane. Stochastic optical reconstruction microscopy (STORM) imaging reveals the structure of dynamic membrane nanotubes.

Laser confocal microscopy is used to non-invasively investigate the steady and unsteady evolution of viscous microdroplets on solid substrates. Three characteristic dynamical regimes of spreading drops (viscous-capillary, viscous-inertia-capillary, and inertia-capillary) are studied using this non-invasive optical technique. It is shown that the dynamics of each regime depend on the Ohnesorge number, Oh = v/(zR|) 1 2 , and on the relative magnitudes of the droplet height, radius, compared with the capillary length, l cap = |/zg. The power-law relationships between the extent of spreading and elapsed time that are extracted from the experiments are in excellent agreement with available analytical results. We also study the onset and evolution of surface instabilities of the slightly volatile liquid films as they spread across the thermally-conductive surfaces. When the fluid droplet is a volatile silicone oil and the surface is a smooth silicon wafer, an evaporatively-driven thermocapillary instability leads to onset of a time-dependent free surface motion. Below a certain critical thickness ( 20 mm), waves can be observed on the free surface of the film, and the confocal technique is used to measure the amplitude, the frequency, and non-linear evolution of these waves. We interpret these waves in terms of evaporatively-driven Marangoni instabilities induced by surface tension gradients close to the moving contact line. Experiments show that the amplitude and the critical onset thickness of the disturbances vary with the viscosity and the volatility of the liquid, and also with the surface roughness and thermal diffusivity of the substrate. The critical onset conditions for this evaporatively driven instability can be characterized by a dimensionless interfacial thermal resistance, R, which has to be larger than a critical value at the onset of instability. We also demonstrate that this evaporatively-driven Marangoni instability can be eliminated by reducing the volatility of the liquid or the thermal diffusivity of the substrate.