Cytoskeletal motor proteins are essential to the function of a wide range of intra-cellular mechano-systems. The biophysical characterization of the movement of motor proteins along their filamentous tracks is therefore of large importance. Towards this end, in vitro stepping motility assays are commonly used to determine the motor’s velocities and runlengths. However, comparing results from such experiments has proved difficult due to influences from variations in the experimental setups, the experimental conditions and the data analysis methods. This work describes a novel unified method to evaluate traces of fluorescently-labeled, processive dimeric motor proteins and proposes an algorithm to correct the measurements for finite filament length as well as photobleaching. Statistical errors of the proposed evaluation method are estimated by a bootstrap method. Numerical simulation and experimental data from GFP-labeled kinesin-1 motors stepping along immobilized microtubules was used to verify the proposed approach and it was shown (i) that the velocity distribution should be fitted by a t location-scale probability density function rather than a normal distribution, (ii) that the temperature during the experiments should be controlled with a precision well below 1 K, (iii) that the impossibility to measure events shorter than the image acquisition time needs to be accounted for, (iv) that the motor’s runlength can be estimated independent of the filament length distribution, and (v) that the dimeric nature of the motors needs to be considered when correcting for photobleaching. This allows for a better statistical comparison of motor proteins influenced by other external factors e.g. ionic strength, ATP concentration, or post-translational modifications of the filaments. In this context, the described method was then applied to experimental data to investigate the influence of the nucleotide state of the microtubule on the motility behavior of the kinesin-1 motor proteins. Here, a small but significant difference in the velocity measurements was found, but no significant difference in the runlength and interaction time measurements. Consequently, this work provides a framework for the evaluation of a wide range of experiments with single fluorescently-labeled motor proteins.

The cytoskeletal motor protein kinesin-1 (conventional kinesin) is the fast carrier for intracellular cargo transport along microtubules. So far most studies aimed at investigating the transport properties of individual motor molecules. However, the transport in cells usually involves the collective work of more than one motor. In the present work, we have studied the movement of beads as artificial loads/organelles pulled by several kinesin-1 motors in vitro. For a wide range of motor coverage of the beads and different bead (cargo) sizes the transport parameters walking distance or run length, velocity and force generation are measured. The results indicate that the transport parameters are influenced by the number of motors carrying the bead. While the transport velocity slightly decreases, an increase in the run length was measured and higher forces are determined, when more motors are involved. The effective number of motors pulling a bead is estimated by measuring the change in the hydrodynamic diameter of kinesin-coated beads using dynamic light scattering. The geometrical constraints imposed by the transport system have been taken into account. Thus, results for beads of different size and motor-surface coverage could be compared. In addition, run length-distributions obtained for the smallest bead size were matched to theoretically calculated distributions. The latter yielded an average number of pulling motors, which is in agreement with the effective motor numbers determined experimentally. / Kinesin-1 (konventionelles Kinesin) ist ein Motorprotein des Zytoskeletts, das für den schnellen intrazellulären Lastentransport auf Mikrotubuli verantwortlich ist. Das Hauptinteresse vieler Studien lag bisher auf der Erforschung der Transporteigenschaften von Einzelmotormolekülen. Der Transport in der Zelle erfordert aber gewöhnlich kollektive Arbeit von mehreren Motoren. In dieser Arbeit wurde die Bewegung von Kugeln als Modell für Zellorganellen, die von Kinesin-1 Molekülen gezogen werden, in Anhängigkeit von der Motorendichte auf der Kugeloberfläche und unterschiedlichen Kugeldurchmessern in vitro untersuchten. Die Transportparameter Weglänge, Geschwindigkeit und die erzeugte Kraft wurden gemessen. Die Ergebnisse zeigen, dass die Transportgeschwindigkeit leicht abnimmt, wohingegen die Weglänge und die erzeugten Kräfte mit steigender Molekülkonzentration zunehmen. Die tatsächliche Anzahl der Motoren, die aktiv am Transport der Kugeln beteiligt sind, wurde bestimmt, indem die Änderung des hydrodynamischen Durchmessers der mit Kinesin bedeckten Kugeln mittels dynamischer Lichtstreuung gemessen wurde. Außerdem wurden sterische Effekte des verwendeten Transportsystems in die Berechnung einbezogen. Damit werden Ergebnisse vergleichbar, die für unterschiedliche Kugeldurchmesser und Motorkonzentrationen ermittelt wurden. Zusätzlich wurden die Verteilungen der Weglängen für die kleinste Kugelgröße mit theoretisch ermittelten Verteilungen verglichen. Letzteres ergab durchschnittliche Anzahlen der aktiv am Transport beteiligten Motormoleküle, die mit den experimentell bestimmten Ergebnissen übereinstimmen.

Conventional kinesin (kinesin-1) transports membrane-bounded cargos such as mitochondria and vesicles along microtubules. In vivo it is likely that several kinesins move a single organelle and it is important that they operate in a coordinated fashion so that they do not interfere with each other. Evidence for coordination comes from in vitro assays, which show that the gliding speed of a microtubule driven by many kinesins is as high as one driven by just a single kinesin molecule. Coordination is thought to be facilitated by flexible domains so that when one motor is bound another can work irrespectively of their orientations. The tail of kinesin-1 is predicted to be composed of a coiled-coil with two main breaks, the “swivel” (380-442 Dm numbering) and the hinge (560-624). The rotational Brownian motion of microtubules attached to a glass surface by single kinesin molecules was analyzed and measured the torsion elasticity constant. The deletion of the hinge and subsequent tail domains increase the stiffness of the motor (8±1 kBT/rad) compared to the full length (0.06±0.01 kBT/rad measured previously), but does not impair motor cooperation (700±16nm/s vs. full length 756±55nm/s - speed in high motor density motility assays). Removal of the swivel domain generates a stiff construct (7±1 kBT/rad), which is fully functional at single molecule (657±63nm/s), but it cannot work in large numbers (151±46nm/s). Due to the similar value of flexibility for both short construct (8±11 kBT/rad vs 7±1 1 kBT/rad) and their different behavior at high density (700±16 nm/s vs. 151±46 nm/s) a new hypothesis is presented, the swivel might have a strain dependent conformation. Using Circular Dichroism and Fluorescence the secondary structure of this tail region was studied. The central part of the swivel is dimeric α-helical and it is surrounded by random coils, thereby named helix-coil (HC) region. Furthermore, an experimental set-up is developed to exert a torque on individual kinesin molecules using hydrodynamic flow. The results obtained suggest for the first time the possibility that a structural element within the kinesin tail (HC region) has a force-dependent conformation and that this allows motor cooperation.

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

Microtubule-based mitochondrial transport into dendrites and axons is vital for sustaining neuronal function. Transport along microtubules proceeds in a series of plus- and minus-end directed movements facilitated by kinesin and dynein motors. How the opposing movements are controlled to achieve effective long distance transport remains unclear. Previous studies showed that the conserved mitochondrial GTPase Miro is required for mitochondrial transport into axons and dendrites. To directly examine Miro's significance for kinesin- and/or dynein-mediated mitochondrial motility, we live imaged movements of GFP-tagged mitochondria in larval Drosophila motor axons upon genetic manipulations of Miro. Loss of Drosophila Miro (dMiro) reduced the effectiveness of either antero- or retrograde mitochondrial transport by selectively impairing kinesin- or dynein-mediated movements, depending on the direction of net transport. In both cases, the duration of short stationary phases increased proportionally. Overexpression (OE) of dMiro also impaired the effectiveness of mitochondrial transport. Finally, loss and OE of dMiro altered the length of mitochondria in axons through a mechanistically separate pathway. We concluded that dMiro promotes effective antero- and retrograde mitochondrial transport by extending the processivity of kinesin and dynein motors according to a mitochondrion's programmed direction of transport. To determine how Miro achieves this control mechanistically, we introduced point mutations that render each GTPase either constitutively active or inactive. Expression of either first GTPase mutant impaired antero- (inactive) or retrograde motor movements (active) in a direction dependent manner. The active state of the second GTPase domain up-regulated the number of consecutive kinesin motions during anterograde transport but impaired kinesin transport biases while the inactive second GTPase state impaired transport in either direction. Together, these data suggest that Miro's first GTPase domain is major factor that controls the execution of either the antero- or retrograde directional program while Miro's second GTPase may provide a signal that supports or disfavors transport. In addition, the active state of the first and the second GTPase domain increased the length of stationary mitochondria but only the first GTPase domain modified motile mitochondrial lengths. Overexpression of these mutations generated opposing effects. We conclude that both domains control antero- and retrograde transport in a switch-like manner.

Peroxisomes are ubiquitous organelles found in almost all eukaryotes. They are sensitive to changes in cellular homeostasis and involved in various metabolic processes. Deficiencies in peroxisome function cause severe neurological problems. Here I report, investigation of peroxisome motility and its relation to peroxisomal functions in the fungal model system Ustilago maydis. Peroxisomes are mostly motile in Ustilago maydis. Motile peroxisomes show different motility patterns: short-range pulse type movements and long range bidirectional motility. Motility behaviour is not static as oscillating peroxisomes may start long-range motility. Here, I present evidence that long-range bidirectional peroxisome motility is an energy driven process and is essential for homogeneous distribution of peroxisomes. Similar to early endosomes and endoplasmic reticulum, microtubule motors kinesin-3 and dynein are responsible for long-range peroxisome transport. In addition to using the same molecular motors for transport, early endosomes, endoplasmic reticulum and peroxisomes have the same transport velocity. Interestingly, motile peroxisomes and endoplasmic reticulum tubules co-localize with early endosomes. Functional investigation of early endosome mutants, Δrab5a and Yup1ts has revealed a novel transport mechanism where endoplasmic reticulum and peroxisomes hitch hike on early endosomes. Additionally, I report functional characterization of an AAA-ATPase, um05592, which has high homology to human protein NP_055873. Altogether these results reveal molecular mechanism of peroxisome transport in Ustilago maydis. Similarities in transport machinery illustrate Ustilago maydis as a model system to study peroxisome function in mammalian cells.

No description available.

About 5% to 10% of epileptic patients suffer from Juvenile Myoclonic Epilepsy (JME), which is characterized by spasms of the arms, ataxia (uncoordinated movements), and general tonic-clonic seizures. In a recent study, a group of patients with myoclonic epilepsy was found to harbor mutations in the PRICKLE1 and PRICKLE2 genes. This suggested that PRICKLE genes might be linked to epilepsy, and given that PRICKLE is highly evolutionarily conserved (including in fruit flies), we decided to use Drosophila in order to determine, first, whether flies with prickle mutations were seizure-prone, and if so, to then use the powerful genetic tools of Drosophila to elucidate the underlying mechanism of the prickle-associated epilepsy. In this work, we show that mutation of the pksple isoform (one of the two adult prickle isoforms in flies) lowers the seizure threshold in the mutant flies (resulting in seizure activity), while mutation of the other adult isoform, pkpk, had no effect. This was demonstrated through both behavioral assays (where the pksple mutant flies showed a reduction in recovery of climbing behavior after being subjected to mechanical stimulation while the pkpk mutant flies did not) as well as electrophysiological analysis (where pksple mutants were shown to be hyperexcitable after electrical stimulation, while the pkpk allele showed no change in spiking activity). We demonstrated that the underlying mechanism of the hyperexcitability seen in the pksple flies was due to enhanced anterograde transport on microtubule (MT) tracks in neurons, the main route for transport in neurons, which could be suppressed by reducing the dose of either of two Kinesin motor proteins, the motors involved in anterograde transport in neurons. On the other hand, the pkpk mutants showed the reverse effect, exhibiting a significant reduction in vesicle transport dynamics. We showed that microtubule polarity could be partially reversed by tipping the balance of the pk isoforms similar to what is seen in the pkpk mutants (such that a large percentage of MTs now had their plus ends oriented towards the cell body, which is extremely rare in axons), suggesting that the vesicle transport defects seen in the pkpk mutants might be due to mixed polarity of MTs. Next, we showed that the seizure-prone pksple mutants, but not the pkpk mutants, exhibited a myoclonic form of epilepsy, as well as abnormal walking patterns and uncoordinated movements, paralleling the ataxia phenotype seen in the epileptic patients with PRICKLE mutations. These data suggest that the primary aspects of the epilepsy-ataxia syndrome seen in patients with PRICKLE mutations are recapitulated in flies, which underscores the utility of using the fruit fly genetic system to model this disorder. Finally, our preliminary results suggest that the pk alleles have different effects on neuronal morphology due to changes in sizes of terminal boutons at the neuromuscular junction (NMJ) in larvae. These data suggest that pk is having a direct effect on synaptic formation and likely function. In conclusion, by using our Drosophila model system, we were able to link prickle mutations to epilepsy as well as identify the cellular mechanism of the prickle-associated epilepsy, a novel epilepsy mechanism previously associated with neurodegeneration. To our knowledge, this is the first example of a gene that, when mutated, will cause seizures in flies, zebrafish, mice, and humans, indicating that the role of prickle in controlling seizure activity is remarkably conserved in animals. Significantly, since about one third of patients with epilepsy do not respond to current AEDs, our fly model and the techniques we have developed will enable us to conduct drug screens for testing potential chemical compounds as new AEDs.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Utilizing various systems such as cell-based assays but also multicellular organisms such as Drosophila melanogaster and C.elegans, for example, the endocytic system has been shown to consist of a network of biochemically and morphologically distinct organelles that carry out specialized tasks in the uptake, recycling and catabolism of growth factors and nutrients, serving a plethora of key biological functions (Mellman, 1996). Different classes of endosomes were found to exhibit a characteristic intracellular steady state distribution. This distribution pattern observed at steady state results from a dynamic interaction of endosomes with the actin and the microtubule cytoskeleton. It remains unclear, however, which microtubule-based motors besides Dynein control the intracellular distribution and motility of early endosomes and how their function is integrated with the sorting and transport of cargo. The first part of this thesis research outlines the search for such motor. I describe the identification of KIF16B which functions as a novel endocytic motor protein. This molecular motor, a kinesin-3, transports early endosomes to the plus end of microtubules, in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the peri-nuclear region, delayed receptor recycling to the plasma membrane and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. In displaying Rab5 and PI(3)P-containing cargo selectivity, a remarkable property of KIF16B is that it is subjected to the same regulatory principles governing the membrane tethering and fusion machinery (Zerial and McBride, 2001). Since KIF16B can modulate growth factor degradation, we propose that this motor could have also important implications for signaling. Importantly, KIF16B has provided novel insight into how intracellular localization of endosomes governs the transport activity of these organelles. The second part of this thesis describes the proof-of-principle of a genome-wide screening strategy aimed at gaining insights into the next level of understanding: How the spatial distribution of organelles is linked to their function in an experimental system which features cellular polarity, for example, a tissue or organ. The suitability of C. elegans as a model organism to identify genes functioning in endocytosis has been demonstrated by previous genetic screens (Grant and Hirsh 1999; Fares and Greenwald, 2001). Offering excellent morphological resolution and polarization, the nematode intestine represents a good system to study the apical sorting of a transmembrane marker. The steady state localization of such a marker is likely the result of a dynamic process that depends on biosynthetic trafficking to the apical surface, apical endocytosis and recycling occurring through apical recycling endosomes. Therefore, mis-sorting of this marker upon RNA-mediated interference will be indicative of a failure in one of the aforementioned processes. Furthermore, since it is still largely unclear why apical endosomes maintain their polarized localization, this screen will also monitor the morphology of this endocytic compartment using a second marker. Following image acquisition based on an automated confocal microscope, data can be analyzed using custom-built software allowing objective phenotypic analysis. The successful establishment of the proof-of-principle marks the current state-of-the-art of this large-scale screening project.