Forschungsbericht 2003

I. Forschungsprofil der Abteilung

Übergeordnetes Ziel der Forschungsprojekte der Abteilung Molekular- und Zellphysiologie ist die molekularen Mechanismen aufzuklären, die bei Wechselwirkung von Myosinen oder Kinesinen mit den zugehörigen Zytoskelettelementen (den Aktinfilamenten bzw. Mikrotubuli) zu gerichteten motilen Kräften führen und Grundlage fast aller biologischer Bewegungsformen sind. Ein zentraler Ansatz ist die Untersuchung funktioneller Auswirkungen von Mutationen in Myosin- und Kinesin-Molekülen, um daraus die funktionelle Relevanz der einzelnen Strukturelemente der Myosin- und Kinesin-Kopfdomänen sowie Kommunikationswege zwischen den einzelnen funktionellen Domänen identifizieren zu können.

Dazu untersuchen wir zunächst funktionelle Auswirkungen "natürlicher" Mutanten der Myosinkopfdomäne, die im Rahmen der Familiären Hypertrophischen Kardiomyopathie identifiziert wurden, um erste Hinweise auf die funktionelle Relevanz einzelner Strukturelemente ableiten sowie Ansatzpunkte für korrigierende pharmakologische Beeinflussung der primären Funktionsstörungen bei Hypertrophischer Kardiomyopathie identifizieren zu können. In einem zweiten Schritt können dann diese Strukturelemente gezielt verändert und in geeigneten nicht-muskulären Systemen exprimiert werden, um genauere Struktur-Funktionsbeziehungen ableiten zu können.

Dieses Ziel erfordert nicht nur den Einsatz eines sehr breiten Methodenspektrums, sondern auch Messungen an verschiedenen Systemen, angefangen mit reduzierten zellulären Systemen, den 'skinned fibers', d.h. Muskelfasern, deren Membranen durch Behandlung mit Detergenzien entfernt wurden, um direkten Zugang zu den kontraktilen Proteinen zu haben. Damit können an ein und demselben System Kräfte und Bewegungen gemessen und mit biochemischen und strukturanalytischen Untersuchungen korreliert werden, um daraus quantitative Vorstellungen zu den molekularen Grundlagen motiler Funktionen muskulärer Myosine weiterentwickeln zu können.

Daneben kommen auch vermehrt in vitro Systeme zum Einsatz, die aus isolierten Proteinen rekonstituiert werden. Solche in vitro Assays erlauben einerseits auch nichtmuskuläre Myosine und Kinesine sowie gezielte Mutationen in diesen Proteinen nach Expression in nichtmuskulären Systemen (z.B. in Dictyostelium discoideum) funktionell zu charakterisieren . Darüber hinaus können wir inzwischen mit solchen in vitro Assays selbst am Einzelmolekül Parameter wie Kräfte und Schrittdistanzen messen und über Fluoreszenzsignale mit Bindung und Abdissoziation einzelner Substrat- und Produkt-Moleküle korrelieren. Die Weiterentwicklung solcher in vitro Systeme wird schließlich erlauben, die in früheren Projekten an skinned fibers abgeleiteten Hypothesen zu den molekularen Mechanismen der Erzeugung motiler Kräfte und Bewegungen zu verifizieren bzw. weiter zu entwickeln. Die Weiterentwicklung solcher in vitro Assays sowie die Korrelation mit skinned fiber-Experimenten sind deshalb ein weiterer Schwerpunkt der Abteilung.

II. Forschungsprojekte

(1) Analysis of processive movement of motor-proteins

Myosin V is a highly processive double-headed motor-protein 1 that moves on actin filaments and it is associated with transport of organelles inside the cell 2. Myosin V hydrolyses ATP molecules and uses the energy to produce its mechanical movement that occurs in 36-37 nm steps (this is approximately the long-pitch periodicity of the actin filament). Due to its dimeric nature, myosin V can undergo many ATP-hydrolysis cycles while remaining attached to the actin filament and, as a result, the molecule can move several microns before detaching from its track. Recent studies have addressed the question of which model better describes the Myosin V processive action 3,4. The "hand-over-hand" model is strongly supported by these studies and predicts that the molecule moves in a similar fashion as humans walk: the two heads are attached to the actin filament for most of the ATPase cycle and they alternate in front. To understand how the processivity is connected to the mechanical action of the motor-molecule and, in particular, to the synchronisation between the two heads, we are using a single optical-trap set-up equipped with a novel force feedback loop. One of our aims is to characterise the mechanical properties of Myosin V by studying its processivity under different load conditions. Furthermore, the way the molecule responds to stress can give an important insight into its stepping action, helping clarifying the processive mechanism.

Experimental Method

Figure 1 shows the experimental set-up. An infrared diode laser beam (820 nm wavelength) is focused, using a high numerical aperture objective lens, on the sample surface and it is used to trap a submicron-size bead that has been sparsely coated with myosin V molecules. The laser beam is then projected, using a condenser lens, onto a 4-sectors photo-detector that measures the x, y and z position of the bead 5. The signal recorded by the photo-detector will show free fluctuations in the 3D space when the myosin V molecule is not attached to the actin filament.

Figure 1: Schematic diagram of the experimental set-up. A polystyrene bead (350 nm in diameter) is sparsely coated with myosin V molecules and trapped by a focused infrared laser beam. Fluorescently labelled actin filaments are immobilised on the glass surface and can be positioned in the vicinity of the bead by moving the piezo-table. A 4-sectors photo-detector measures the 3D-position of the bead. A Labview interface controls the positioning of the table and records the signal form the photo-detector.

Once a polystyrene bead is trapped in the focus position, it is maintained at around one micron from the surface while a fluorescently labelled actin filament is positioned directly underneath the bead. At this point a Labview routine starts raising the sample surface. The approach is stopped as soon as a myosin molecule attaches to the actin filament and the fluctuation of the bead in the z-direction is dramatically reduced. At this point the molecule starts moving along the actin filament (y-direction in figure 1) and the signal from the photo-detector will show a variation in the corresponding direction. When the variation from the initial value becomes larger than a predetermined set-point a feedback loop is activated and the piezo-table starts moving in the opposite direction of the molecule. This movement compensates exactly for the movement of the molecule so that the signal recorded by the photo-detector remains constant and the displacement of the table can be used as an accurate measurement of the processivity of the motor molecule.

When, after a few seconds, the myosin V molecule detaches from the actin filament the bead returns to the original position (due to the restoring force of the laser trap 6) and the feedback loop is interrupted.

Experimental Results

Figure 2 shows how the signal from the photo-detector (measuring the bead position) is related to the piezo-table position at different stages of the measuring cycle. At first the bead is free, then a myosin V molecule attaches to the actin filament and starts moving, and finally the bead detaches.

Figure 2: The grey graph is the signal from the photo-detector measuring the position of the bead in the y-direction (along the actin filament). The black curve indicates the displacement applied to the piezo-table. At point (1) the bead is free and the feedback is off, at point (2) the myosin V molecule attaches to the actin filament and starts moving; the grey signal is shifted. When it reaches the set-point value (dashed horizontal line) the feedback loop is activated and the piezo-table is now moved to compensate for the molecule’s movement (3). The bead signal is now constant and the black curve shows a fairly constant translation. At point (4) the bead detaches and returns to the centre position. Point (5) the feedback loop stops.

It is important to notice that, because the myosin V molecule is attached to the bead, it will displace the bead from the centre of the optical trap as soon as it starts moving. This will cause a restoring force (load) that will pull the bead back to its original position. This force will increase as the molecule moves further out of the centre of the trap6. The reason why the feedback technique described here is called force-feedback is because, once the feedback is running, the force remains constant and the molecule can move the entire length of the filament experiencing a constant load. An example of the processive stepping of myosin V, recorded with this technique, is shown in figure 3a). A 36 nm grid is superimposed to guide the eyes and to emphasise the fairly constant size of the recorded steps.

Figure 3: (a) Curve showing the stepping action of myosin V recorded using force-feedback as describe in the text (curve low-pass filtered). The feedback was run at 0.5 kHz. The ATP concentration was 2 mM and the constant load was 0.45 pN. (b) the curve shows the stepping action of myosin V without the force-feedback at the same ATP concentration. After three steps the dwell time of the steps becomes larger and the last step is only half the size (18 nm) of the previous steps. These effects are due to the increase in load while the myosin molecule moves towards the edge of the optical trap.