Molecular profiling of enteric nervous system cell lineages.

The enteric nervous system (ENS) is an extensive network of enteric neurons and glial cells that is intrinsic to the gut wall and regulates almost all aspects of intestinal physiology. While considerable advancement has been made in understanding the genetic programs regulating ENS development, there is limited understanding of the molecular pathways that control ENS function in adult stages. One of the limitations in advancing the molecular characterization of the adult ENS relates to technical difficulties in purifying healthy neurons and glia from adult intestinal tissues. To overcome this, we developed novel methods for performing transcriptomic analysis of enteric neurons and glia, which are based on the isolation of fluorescently labeled nuclei. Here we provide a step-by-step protocol for the labeling of adult mouse enteric neuronal nuclei using adeno-associated-virus-mediated gene transfer, isolation of the labeled nuclei by fluorimetric analysis, RNA purification and nuclear RNA sequencing. This protocol has also been adapted for the isolation of enteric neuron and glia nuclei from myenteric plexus preparations from adult zebrafish intestine. Finally, we describe a method for visualization and quantification of RNA in myenteric ganglia: Spatial Integration of Granular Nuclear Signals (SIGNS). By following this protocol, it takes ~3 d to generate RNA and create cDNA libraries for nuclear RNA sequencing and 4 d to carry out high-resolution RNA expression analysis on ENS tissues.

Force-velocity relationship for multiple kinesin motors pulling a magnetic bead.

Although the velocity of single kinesin motors against an opposing force F of 0-10 pN is well known, the behavior of multiple kinesin motors working to overcome a larger load is still poorly understood. We have carried out gliding assays in which 3-7 Drosophila kinesin-1 motors moved a microtubule at 200-700 µm/s against a 0-31 pN load at saturating [ATP]. The load F was generated by applying a spatially uniform magnetic field gradient to a superparamagnetic bead attached to the (+) end of the microtubule. When F was scaled by the average number of motors [Symbol: see text]n[Symbol: see text], the force-velocity relationship for multiple motors was similar to the force-velocity relationship for a single motor, supporting a minimal load-sharing model. The velocity distribution at low load has a single mode consistent with rapid fluctuations of n. However, against a load of 2.5-4.7 pN/motor, additional modes appeared at lower velocity. These observations support the Klumpp-Lipowsky model of multimotor transport [Proc Natl Acad Sci USA 102. 17284-17289 (2005)].

Measuring the number and spacing of molecular motors propelling a gliding microtubule.

The molecular motor gliding assay, in which a microtubule or other filament moves across a surface coated with motors, has provided much insight into how molecular motors work. The kinesin-microtubule system is also a strong candidate for the job of nanoparticle transporter in nanotechnology devices. In most cases, several motors transport each filament. Each motor serves both to bind the microtubule to a stationary surface and to propel the microtubule along the surface. By applying a uniform transverse force of 4-19 pN to a superparamagnetic bead attached to the trailing end of the microtubule, we have measured the distance d between binding points (motors). The average value of d was determined as a function of motor surface density σ. The measurements agree well with the scaling model of Duke, Holy, and Liebler, which predicts that (d)~σ(-2/5) if 0.05≤σ≤20 µm(-2) [Phys. Rev. Lett. 74, 330 (1995)]. The distribution of d fits an extension of the model. The radius of curvature of a microtubule bent at a binding point by the force of the magnetic bead was ≈1 µm, 5000-fold smaller than the radius of curvature of microtubules subjected only to thermal forces. This is evidence that at these points of high bending stress, generated by the force on the magnetic bead, the microtubule is in the more flexible state of a two-state model of microtubule bending proposed by Heussinger, Schüller, and Frey [Phys. Rev. E 81, 021904 (2010)].