AG Scholz

Copyright MHH, MZP, AG Scholz
Copyright MHH, MZP, AG Scholz

MAPs Single Molecule Motility Group / Single Molecule Motility of Motor Proteins

 

 

Research focus

One focus of the group is the functional characterization of kinesins, myosins and dynein at the single molecule level. The intracellular transport e.g. in nerve cells, the separation of replicated chromosomes during cell division, the beating of flagella and cilia as well as the muscle contraction of multicellular organisms are examples of movements of and in living organisms. They are based on the cyclic interaction of the motor proteins kinesin, myosin and dynein with structural proteins of the cytoskeleton such as actin and tubulin.
Motor proteins of the kinesin family are microtubule-activated ATPases. They move along microtubules, tubular structures of the cytoskeleton. These are formed from protofilaments created by polymerization of α/β-tubulin heterodimers. Conventional kinesin (also kinesin-1, Figure 1) was originally discovered in giant axons of squid, where it is responsible for vesicle transport into the cell periphery of neurons. In contrast to muscular myosin-2, conventional kinesin is a processive motor molecule. This means that it can take hundreds of 8 nm long steps, corresponding to the size of an α/β-tubulin heterodimer, along a microtubule without dissociating from it. Presumably, each step is coupled to the hydrolysis of a molecule of ATP. The ability of kinesin to travel long distances along microtubules without dissociating from them is extremely important for its biological function as a long-distance transporter. However, it has not yet been clarified in detail how kinesin molecules coordinate their two motor domains for this purpose or how the direction of travel of kinesin molecules is determined.


Copyright: MuZP / MHH

Fig 1: Conventional kinesin-1 (after Kozielski, Mandelkow, Cell, 1997)


Measurements at the level of individual molecules offer possibilities for investigating these questions. TIRF (Total Internal Reflection Fluorescence) or evanescent wave microscopy is a very suitable technique for this. It can be used, for example, to detect individual fluorescently labeled ATP and motor molecules and track their movements. In this way, the movement of individual motor molecules can be correlated with the binding and hydrolysis of fluorescently labeled ATP. These assays also allow us to characterize the functional effects of specifically introduced mutations in the motor proteins. This allows us to deduce the functional relevance of individual structural elements as well as the structural basis of Communications between the individual domains of the motor proteins.

 

Copyright: MuZP / MHH

Movie 1: Single molecule assay kinesin-1 on a microtubule

Movie 1 shows an example of the ATP-driven processive movements of single, fluorescently labeled kinesin molecules (green) on an immobilized, also fluorescently labeled microtubule (red).


Copyright: MuZP / MHH

Fig. 2: (A) Snapshot of single moving kinesin-1 molecules (green) along an immobilized labeled microtubule (length 34 µm, red) in the presence of ATP. (B) A single kinesin molecule (green) moves processively along the immobilized microtubule. (C) Legal movements of the individual kinesin molecules can be recognized in a position-time plot (kymograph) as lines deviating to the right (green) (running speed 500-600 nm/s).


Copyright: MuZP / MHH

Movie 2: Microtubule sliding assay

Movie 2 shows a microtubule sliding assay in which fluorescently labelled microtubules (red) are moved on a lawn of immobilized kinesin molecules (not visible) while consuming ATP.