Molecular motors in neuronal development, intracellular transport and diseases

Molecular motors such as kinesin superfamily proteins (KIFs), dynein superfamily proteins and myosin superfamily proteins have diverse and fundamental roles in many cellular processes, including neuronal development and the pathogenesis of neuronal diseases. During neuronal development, KIFs take significant roles in the regulation of axon-collateral branch extension, which is essential for brain wiring. Cytoplasmic dynein together with LIS1 takes pivotal roles in neocortical layer formation. In axons, anterograde transport is mediated by KIFs, whereas retrograde transport is mediated mainly by cytoplasmic dynein, and dysfunction of motors results in neurodegenerative diseases. In dendrites, the transport of NMDA and AMPA receptors is mediated by KIFs, and the motor has been shown to play a significant part in establishing learning and memory.     

Coordination of opposite-polarity microtubule motors

Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.     

Rab GTPases and microtubule motors

Rab proteins are a family of small GTPases which, since their initial identification in the late 1980s, have emerged as master regulators of all stages of intracellular trafficking processes in eukaryotic cells. Rabs cycle between distinct conformations that are dependent on their guanine-nucleotide-bound status. When active (GTP-bound), Rabs are distributed to the cytosolic face of specific membranous compartments where they recruit downstream effector proteins. Rab-effector complexes then execute precise intracellular trafficking steps, which, in many cases, include vesicle motility. Microtubule-based kinesin and cytoplasmic dynein motor complexes are prominent among the classes of known Rab effector proteins. Additionally, many Rabs associate with microtubule-based motors via effectors that act as adaptor molecules that can simultaneously associate with the GTP-bound Rab and specific motor complexes. Thus, through association with motor complexes, Rab proteins can allow for membrane association and directional movement of various vesicular cargos along the microtubule cytoskeleton. In this mini-review, we highlight the expanding repertoire of Rab/microtubule motor protein interactions, and, in doing so, present an outline of the multiplicity of transport processes which result from such interactions.     

Meiosis,mitosis and microtubule motors

A framework for understanding the complex movements of mitosis and meiosis has been provided by the recent discovery of microtubule motor proteins, required for the proper distribution of chromosomes or the structural integrity of the mitotic or meiotic spindle. Although overall features of mitosis and meiosis are often assumed to be similar in mechanism, it is now clear that they differ in several important aspects. These include spindle structure and assembly, and timing of chromosome segregation to opposite poles. Here we review progress in the functional characterization of several newly identified microtubule motor proteins, emphasizing their possible roles in spindle structure and function.     

Crowding of molecular motors determines microtubule depolymerization

The assembly and disassembly dynamics of microtubules (MTs) is tightly controlled by MT-associated proteins. Here, we investigate how plus-end-directed depolymerases of the kinesin-8 family regulate MT depolymerization dynamics. Using an individual-based model, we reproduce experimental findings. Moreover, crowding is identified as the key regulatory mechanism of depolymerization dynamics. Our analysis reveals two qualitatively distinct regimes. For motor densities above a particular threshold, a macroscopic traffic jam emerges at the plus-end and the MT dynamics become independent of the motor concentration. Below this threshold, microscopic traffic jams at the tip arise that cancel out the effect of the depolymerization kinetics such that the depolymerization speed is solely determined by the motor density. Because this density changes over the MT length, length-dependent regulation is possible. Remarkably, motor cooperativity affects only the end-residence time of depolymerases and not the depolymerization speed.     

Intracellular cargo transport by kinesin-3 motors

Intracellular transport along microtubules enables cellular cargoes to efficiently reach the extremities of large, eukaryotic cells. While it would take more than 200 years for a small vesicle to diffuse from the cell body to the growing tip of a one-meter long axon, transport by a kinesin allows delivery in one week. It is clear from this example that the evolution of intracellular transport was tightly linked to the development of complex and macroscopic life forms. The human genome encodes 45 kinesins, 8 of those belonging to the family of kinesin-3 organelle transporters that are known to transport a variety of cargoes towards the plus end of microtubules. However, their mode of action, their tertiary structure, and regulation are controversial. In this review, we summarize the latest developments in our understanding of these fascinating molecular motors.     

Direct observation of microtubule treadmilling by electron microscopy

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.     

Fluorescence microscopy

Although fluorescence microscopy permeates all of cell and molecular biology, most biologists have little experience with the underlying photophysical phenomena. Understanding the principles underlying fluorescence microscopy is useful when attempting to solve imaging problems. Additionally, fluorescence microscopy is in a state of rapid evolution, with new techniques, probes and equipment appearing almost daily. Familiarity with fluorescence is a prerequisite for taking advantage of many of these developments. This review attempts to provide a framework for understanding excitation of and emission by fluorophores, the way fluorescence microscopes work, and some of the ways fluorescence can be optimized.     

Fluorescence polarization applied to cellular microrheology

The fluorescence polarization of probe molecules gives information on the "fluidity" of probe environment. Although the data cannot be related with absolute values of microviscosity, the method is largely used for probing the "fluidity" of lipid regions of biological membranes. Therefore, fluorescence polarization is of interest in clinical research, for membrane alterations are associated with either pathological processes of red cells, platelets, leukocytes or important cell functions.     

Yeast kinetochore microtubule dynamics analyzed by high-resolution three-dimensional microscopy

We have probed single kinetochore microtubule (k-MT) dynamics in budding yeast in the G1 phase of the cell cycle by automated tracking of a green fluorescent protein tag placed proximal to the centromere on chromosome IV and of a green fluorescent protein tag fused to the spindle pole body protein Spc42p. Our method reliably distinguishes between different dynamics in wild-type and mutant strains and under different experimental conditions. Using our methods we established that in budding yeast, unlike in metazoans, chromosomes make dynamic attachments to microtubules in G1. This makes it possible to interpret measurements of centromere tag dynamics as reflecting k-MT dynamics. We have examined the sensitivity of our assay by studying the effect of temperature, exposure to benomyl, and a tubulin mutation on k-MT dynamics. We have found that lowering the temperature and exposing cells to benomyl attenuate k-MT dynamics in a similar manner. We further observe that, in contrast to previous reports, the mutant tub2-150 forms k-MTs that depolymerize faster than wild type. Based on these findings, we propose high-resolution light microscopy of centromere dynamics in G1 yeast cells as a sensitive assay for the regulation of single k-MT dynamics.     

Fluorescence microscopy today

Fluorescence microscopy has undergone a renaissance in the last decade. The introduction of green fluorescent protein (GFP) and two-photon microscopy has allowed systematic imaging studies of protein localization in living cells and of the structure and function of living tissues. The impact of these and other new imaging methods in biophysics, neuroscience, and developmental and cell biology has been remarkable. Further advances in fluorophore design, molecular biological tools and nonlinear and hyper-resolution microscopies are poised to profoundly transform many fields of biological research.     

Preparing the way: fungal motors in microtubule organization

Fungal growth, development and pathogenicity require hyphal tip growth, which is supported by polar exocytosis at the expanding growth region. It is assumed that molecular motors transport growth supplies along the fibrous elements of the cytoskeleton, such as microtubules, to the hyphal apex. Recent advances in live-cell imaging of fungi revealed additional roles for motors in organizing their own tracks. These unexpected roles of the molecular motors are modifying microtubule dynamics directly, targeting stability-determining factors to microtubule plus ends, and transporting and arranging already-assembled microtubules.     

Quantitative fluorescence microscopy to probe intracellular microenvironments

Some intracellular pathogens avoid killing within phagosomes--which are specialized microbicidal organelles in cells of the innate immune system--by altering phagosomal maturation or by entering a different subcellular compartment. The fate of the microorganisms is ultimately dictated by the composition of the surrounding environment. The unique problems associated with in situ measurements of intracellular microenvironments within intact cells and the advantages of quantitative fluorescence microscopy have recently been investigated. Of particular interest are the various techniques and reagents used in analysis of the pH and reactive oxygen intermediates in phagosomes and invasion vacuoles.     

Nonlinear vibrational microscopy applied to lipid biology

Optical microscopy is an indispensable tool that is driving progress in cell biology. It still is the only practical means of obtaining spatial and temporal resolution within living cells and tissues. Most prominently, fluorescence microscopy based on dye-labeling or protein fusions with fluorescent tags is a highly sensitive and specific method of visualizing biomolecules within sub-cellular structures. It is however severely limited by labeling artifacts, photo-bleaching and cytotoxicity of the labels. Coherent Raman Scattering (CRS) has emerged in the last decade as a new multiphoton microscopy technique suited for imaging unlabeled living cells in real time with high three-dimensional spatial resolution and chemical specificity. This technique has proven to be particularly successful in imaging unstained lipids from artificial membrane model systems, to living cells and tissues to whole organisms. In this article, we will review the experimental implementations of CRS microscopy and their application to imaging lipids. We will cover the theoretical background of linear and non-linear vibrational micro-spectroscopy necessary for the understanding of CRS microscopy. The different experimental implementations of CRS will be compared in terms of sensitivity limits and excitation and detection methods. Finally, we will provide an overview of the applications of CRS microscopy to lipid biology.     

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).     

Fluorescence flow cytometry and immunology applied to cancer

Antibodies against cell-surface determinants present on limited types of normal cells, such as differentiation antigens, offer an approach to detecting and characterizing tumor cells that is well-suited to fluorescence flow cytometry. This paper reviews the technology involved and its application in the study of hematologic malignancies, especially in the detection and characterization of tumor cells in leukemias and lymphomas.