Institute of Molecular and Cellular Biology & Department of Life Sciences

 

Dr. Oliver Wagner, Associate Professor

 

Current research interests

(1) Regulation of axonal transport in the nervous system of C. elegans

(a) Regulation of molecular motors: How does cargo bind to motors and how does cargo-binding affect motor motility?

(b) Bidirectional transport: What is the basis of frequently observed directional changes of synaptic vesicles and mitochondria in axons?

(c) Obstacles on the highway: How do motors, synaptic vesicles and mitochondria find their way thru the crowded axon? Do the various microtubule-binding proteins (MAPs, tau, plectin etc.) or neurofilaments affect their motility?

(2) Cilia development and intraflagellar transport

Which kinases and phosphatases affect C. elegans cilium biogenesis and intraflagellar transport?

Approaches

(1) Regulation of axonal transport in the nervous system of C. elegans

Neuronal cells are composed of a cell body and long cytoplasmic processes termed dendrites and axons. In the neuronal cell body proteins are synthesized, packed in vesicles and transported long ranges thru the axon and dendrites to the synapses. The neuronal processes are, however, overloaded with vesicles, synaptic precursors, molecular motors and cytoskeletal elements. Thus, it is not surprising that defects in the motor-cargo transport system can lead to abnormal aggregation of disease proteins and concomitant neuronal cell death. It is remarkable that many of these disease-related proteins have been identified as motor-interacting proteins or proteins present in cargo. For example Alzheimer's disease occurs due to the accumulation of amyloid precursor proteins which is a cargo for the molecular motor KIF5A. A mutation in the motor dynein rescues axonal transport defects and extends the life span of ALS (Amyotrophic lateral sclerosis) mice. LIS1 is associated with dynein while a mutation in LIS causes lissencephaly (severe mental retardation and poor control of movement). A mutation in the molecular motor KIF1Bb causes the human peripheral neuropathy Charcot-Marie-Tooth disease type 2A (leading to severe muscle weakness, inability to perceive touch, pain and temperature changes). A single base-pair change in the p150 Glued subunit of the motor adaptor protein dynactin causes LMN (lower motor neuron disease). Last, senile dementia is based on neuronal cell death caused by defects in the transport of synaptic vesicle precursors by the molecular motor KIF1A(UNC-104).

These examples show that proper regulation of the motor-cargo transport system is critical and that many neurodegenerative diseases display accumulation of cargo based on defects in molecular motors. However, the mechanisms of cargo transport and the regulation of motors on the molecular level is only partial known. One of the basic questions is how to explain the frequent observed bidirectional movement of molecular motors and vesicles. One common hypothesis is that one vesicle binds multiple types of motors and they either act in a cooperative manner or they compete with each other in a tug-of-war. In contrast to bidirectional movement of vesicles (on which surfaces can bind multiple cargos), the frequent observed bidirectional movement of single GFP-tagged motors might be explained by direct interaction of an opposing motor. One of our hypothesis is that bidirectional movement of anterograde (from the cell body to the synapses) moving kinesins can be explained by a direct and regulating interaction with retrograde moving dynein (from the synapses to the cell body) and that this regulating interaction may be mediated by the dynein-cargo adaptor protein dynactin.

a) Regulation of molecular motors

In our previous research we investigated the interaction between a major axonal transporter (kinesin-3 UNC-104/KIF1A) and one of its cargo SYD-2/liprin-a. SYD-2/liprin-a is a multifunctional active zone protein and important for synaptic function and synaptogenesis. Interestingly, SYD-2 scaffolds the motor UNC-104 in the nervous system of C. elegans probably making active motors available along the lengthy axons. We assume that inactive motors are exchanged by active motors at the sites of clustering. Still, SYD-2 acts bifunctional: Besides its scaffolding function we found that it regulates the motor's directionality and speed (Wagner et al., PNAS, 2009). We are now investigating whether another active zone protein LIN-2/MAGUK has similar function because it shares the UNC-104 binding site. Specifically we are interested if this protein competes with SYD-2 on the UNC-104 binding site or if it regulates the association between SYD-2 and UNC-104.

Another project focus on the effect of RIM (UNC-10) on SYD-2's binding properties to the kinesin-3. It is known that RIM interacts with liprin-a and that this complex associates via RAB3 to the vesicle membrane. However, whether there is a regulating function of UNC-10 in the SYD-2/UNC-104 complex is still unknown. We are also investigating if the protein complex RAB3-UNC-10-SYD-2 acts as an additional link that would facilitate UNC-104's interaction with the synaptic vesicle membrane (besides the UNC-104 PH-domain that is known to directly associated with the lipid-bilayer).

b) Bidirectional Transport

One key question in axonal transport is how molecular motors and synaptic vesicles undergo the frequent observed directional changes. As mentioned above, an idea would be that several (opposing) molecular motors bind to the vesicle membrane and act in a tug-of-war process. The other idea is whether direct and regulative interaction occur between opposing molecular motors as kinesins (anterograde motor) and dynein (retrograde motor). Indeed, the literature has reported few hints that these interactions might occur. We are currently investigating if mutual and functional interaction between the major axonal transporter UNC-104/KIF1A and dynein (or the accessory protein dynactin) exist. Using C. elegans we are able to perform high-speed and high-resolution single motor tracking in live animals with different mutant backgrounds. We are using a confocal spinning disk microscope that is connected to a high-sensitive EMCCD camera. An example of time-lapse imaging of UNC-104 in a single neuron of a living animal is shown below:

(A) C. elegans expressing a GFP-tagged molecular motor named UNC-104/KIF1A (brightfield photograph). C. elegans eggs and larvae are evident. (B) Fluorescent photograph of the worm expressing the GFP-tagged molecular motor UNC-104/KIF1A which is specifically expressed in the nervous system. (C) Magnification of inset in (B) displaying a single neuron and molecular motors inside the living worm. (D) Motility of UNC-104/KIF1A particles was analyzed using confocal time-lapse imaging. (E) Kymograph image from (D) making traces of the molecular motor in time and space evident. (F) Single isolated (primary) neuronal cell expressing the molecular motor UNC-104/KIF1A. Scale bars: (A) 1 mm, (B) 100 microns, (C+D) 10 microns, (E) 50 microns and (F) 10 microns.

c) "Obstacles on the highway": Effect of microtubule-associated proteins and neurofilaments on axonal transport

Microtubule-associated proteins as, i.e., tau-protein play a major role in many neuropathological phenotypes (Alzheimer's, FTDP17) and it is of major interest whether accumulated cytoskeletal elements as tau (Alzheimer's) or neurofilaments (ALS) affect axonal transport. We are currently investigating if mutations or overexpression of tau, plectin (neurofilament and microtubule binding protein) as well as neurofilaments affect transport characteristics of synaptic vesicles or molecular motors. We have previously reported an interaction between the molecular motor dynein and neurofilaments (Wagner et al., MBC, 2005) and are now investigating the role of tau on the synaptic vesicle transporter UNC-104/KIF1A using tau-knockout worms.

As we have reported earlier that neurofilaments functional associate with mitochondria (Wagner et al., J. Neurosci., 2003) we are now extending the idea whether intermediate filaments are involved in mitochondria transport in general. We are in the process of screening a dozen of different intermediate filament mutants to evaluate if mitochondria movement is affected in these worms.

(2) Cilia development and intraflagellar transport

Adult polycystic kidney disease (APKD) is one of the most common diseases in Taiwan and it is supposed to be linked to defects in the highly conserved components of the cilia and IFT (intraflagellar transport) machinery. Other ciliary disorders include primary ciliary dyskinesia (PCD), Bardet-Biedl syndrome (BBS), Meckel-Gruber syndrome (MKS) and nephronophthisis. C. elegans and Chlamydomonas are the most commonly used model organism to study ciliogenesis and intraflagellar transport (IFT) while in a collaborative effort with Tsinghua University Beijing (Dr. Junmin Pan) we are specifically studying the effect of kinases and phosphatases on cilium development and IFT. Using comparative genomic approaches, we have identified a dozen of kinases and phosphatases that are expressed in C. elegans and Chlamydomonas cilia, while their effect in cilium genesis and IFT have not yet been studied.

The image shows the head ("nose") of a worm with stained amphid cilia. The transition zone, middle segment and distal segment are visible (see image below). The corresponding schematic drawing to the right shows the arrangement of the cilia - emanating from amphid dendrites - into a bundle. The diameter of the worm near the tip of the head is about 35 micrometer.

Intraflagellar transport in C. elegans (taken and modified from www.wormbook.org)