Microtubule-based transport is a highly regulated process, requiring kinesin and/or dynein motors, a multitude of motor-associated regulatory proteins including activating adaptors and scaffolding proteins, and microtubule tracks that also provide regulatory cues. While in vitro studies are invaluable, fully replicating the physiological conditions under which motility occurs in cells is not yet possible. Here, we describe two methods that can be employed to study motor-based transport and motor regulation in a cellular context. Live-cell imaging of organelle transport in neurons leverages the uniform polarity of microtubules in axons to better understand the factors regulating microtubule-based motility. Peroxisome recruitment assays allow users to examine the net effect of motors and motor-regulatory proteins on organelle distribution. Together, these methods open the door to motility experiments that more fully interrogate the complex cellular environment.

The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila, and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.

Motor proteins are responsible for transport of vesicles and organelles within the cell cytoplasm. They interact with the actin cytoskeleton and with microtubules to ensure communication and supply throughout the cell. Much work has been done in vitro and in silico to unravel the key players, including the dynein motor complex, the kinesin and myosin superfamilies, and their interacting regulatory complexes, but there is a clear need for in vivo data as recent evidence suggests previous models might not recapitulate physiological conditions. The zebrafish embryo provides an excellent system to study these processes in intact animals due to the ease of genetic manipulation and the optical transparency allowing live imaging. We present here the advantages of the zebrafish embryo as a system to study live in vivo processive transport in neurons and provide technical recommendations for successful analysis.

The proper positioning of microtubule (MT) asters underlies fundamental processes such as nuclear centration, cell polarity, division positioning, and embryogenesis. In large eggs and early blastomeres, MT asters may exhibit long range motions with atypical speed and precision to target their functional position. The biophysical mechanisms regulating such motions remain however largely unknown. The centration of sperm asters in sea urchin embryos is a stereotypical example of such aster long range motion. In this chapter, we describe methods developed in this system to (1) quantify sperm aster 3-D motion with confocal microscopy and automated image analysis and (2) severe a portion of astral MTs with a UV laser. These methods may serve as a template to dissect the generic mechanisms of aster motion and force production in other embryos and cell types.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

OBJECTIVE: To examine the influence of experimentally reduced cerebrospinal fluid pressure (CSFP) as compared to elevated intraocular pressure (IOP) on axonal morphology and axonal motor proteins in retinal ganglion cells (RGCs).
METHODS: The experimental study included 39 rats which underwent cerebrospinal fluid drainage for 6 hr, 30 rats which unilaterally underwent IOP elevation for 6 hr and 30 rats in a control group. Six hours after baseline, the animals were killed and the eyes were histologically and immunohistochemically examined.
RESULTS: In experimental models in the high-IOP group and the low-CSFP group as compared to the control group, RGC axons became abnormally dilated and accumulated vesicles. Both groups as compared to the control group showed an accumulation of dynein IC (intermediate chain) at the optic nerve head and retina and a reduction in kinesin HC (heavy chain) immunoreactivity in the optic nerve fibre axons. As a corollary, Western blot analysis revealed an elevation of dynein IC protein levels in the optic nerve head and retina and a decrease in kinesin HC protein levels in the optic nerve.
CONCLUSIONS: Experimental models with an acute IOP rise or with an acute CSFP reduction showed similar morphologic changes in the retinal ganglion cell axons and similar immunohistochemical changes in the axonal motor proteins kinesin HC and dynein IC. It supports the hypothesis that an experimental model with an acute reduction in CSFP as well as an experimental model with an acute rise in IOP may share similarities in the process of optic nerve damage.

The cilia, presenting a rotational movement in the embryonic nodes, play a crucial role in the left-right specification during embryogenesis. The characteristic architecture of these cilia is based on a cylindrical arrangement of 9 doublet microtubules and the motion of the cilia is triggered by the dynein motors located between adjacent doublets by converting the chemical energy into mechanical work. Restricted by the inherent difficulties of experiments, the dynein activation patterns in moving cilia cannot be directly observed. Thus, the mechanism of nodal ciliary movement is still unclear. In this study, we present computational models of the nodal ciliary ultrastructure based on tomographic images of the ciliary body. By employing time accurate three-dimensional solid mechanics analysis, we investigate the dynein-triggered sliding between adjacent doublet microtubules and simulate the induced ciliary bending. As an exploratory study, two dynein activation patterns are proposed and their rationality is discussed. The mathematical model presented by this paper provides a platform to investigate various assumptions of dynein activity, facilitating us to propose the most possible dynein activation pattern and therefore improving our understandings regarding the protein-beating problems of cilia.