Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.fascin ͉ motor navigation ͉ myosin X ͉ filopodia ͉ single-molecule fluorescence I n a dense mesh of cellular actin, if all filaments are functionally equivalent, then myosins will move in a random walk as they translate, detach, and reattach to new filaments. Alternatively, myosins may identify and walk along certain subpopulations of actin that lead to target locations, based on a set of common structural features. One possibility is that different myosin classes may partition among filaments based on actin isoforms. However, no significant difference between ␤␥-actin versus ␣-actin has been detected for myosin V, the only motor to be tested on both tracks (1). A second possibility is that actinassociated proteins, in particular tropomyosins, modulate the binding of myosin to actin. Indeed, class I myosins are excluded from tropomyosin-decorated actin in stress-fibers, whereas class II myosins are not (2, 3). In addition to this direct regulatory mechanism, tropomyosins may also direct myosin traffic by stabilizing particular actin tracks (4). However, tropomyosins are not typically found in regions of actively polymerizing, dynamic actin, such as the leading edge of the cell. Therefore, additional mechanisms likely exist for directing myosin traffic.To identify factors that could direct myosins to specific locations, we searched for myosin classes that are localized to limited populations of actin even in the presence of nearby dynamic actin. The class X myosin meets these criteria for a selective motor. Myosin X travels to and is highly concentrated at the distal tips of filopodia: long, slender projections often found at the leading edge of migrating cells (5-7). Filopodia are used for envi...

Iwanir S; Tramm N; Nagy S; Wright C; Ish D; Biron D. The microarchitecture of C. elegans behavior during lethargus: homeostatic bout dynamics, a typical body posture, and regulation by a central neuron. SLEEP 2013;36(3):385-395.

Biological homeostasis invokes modulatory responses aimed at stabilizing internal conditions. Using tunable photo- and mechano-stimulation, we identified two distinct categories of homeostatic responses during the sleep-like state of Caenorhabditis elegans (lethargus). In the presence of weak or no stimuli, extended motion caused a subsequent extension of quiescence. The neuropeptide Y receptor homolog, NPR-1, and an inhibitory neuropeptide known to activate it, FLP-18, were required for this process. In the presence of strong stimuli, the correlations between motion and quiescence were disrupted for several minutes but homeostasis manifested as an overall elevation of the time spent in quiescence. This response to strong stimuli required the function of the DAF-16/FOXO transcription factor in neurons, but not that of NPR-1. Conversely, response to weak stimuli did not require the function of DAF-16/FOXO. These findings suggest that routine homeostatic stabilization of sleep may be distinct from homeostatic compensation following a strong disturbance.DOI: http://dx.doi.org/10.7554/eLife.04380.001

The nematode Caenorhabditis (C.) elegans, a long time work horse for behavioral genetic studies of locomotion, has recently been studied for quiescent behavior. Methods previously established for the study of C. elegans locomotion are not well-suited for the study of quiescent behavior. We describe in detail two computer vision approaches to distinguish quiescent from movement bouts focusing on the behavioral quiescence that occurs during fourth larval stage lethargus, a transition stage between the larva and the adult. The first is the frame subtraction method, which consists of subtraction of temporally adjacent images as a sensitive way to detect motion. The second, which is more computationally intensive, is the posture analysis method, which consists of analysis of the rate of local angle change of the animal’s body. Quiescence measurements should be done continuously while minimizing sensory perturbation of the animal.