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How is Networking of the Cytoskeleton Achieved?
Elucidating the unconventional interaction between Myosin V and microtubules

by Dennis Zimmermann (10.04.2012)

Inside cells, molecular motors such as myosins and kinesins are known to be involved in a number of transport processes. Among many others, such processes include the transport of pigment granules from the center to the periphery of the cell and vice versa. This process called pigmentation, displays a useful tool for fish and amphibians to quickly change the color of their skin in order to perform camouflage. In addition, it protects the human skin from UV-irradiation.

The efficient transport of thousands of such cargo molecules throughout the cell can only be achieved through the interplay between long-range (kinesin-2) and short-range (myosin V) transporters, which in turn requires a handoff mechanism between both transport systems. To date, the mechanistic nature and regulation of this crosstalk still remains elusive.

Recent in vitro studies by Ali et al. (1) have revealed that myosin V, normally interacting with the short-range track system (actin), is able to also use the long-range microtubule track system, on which typically kinesins travel along. This interaction was proposed of being mediated by an electrostatic attraction between a positively charged surface loop (loop 2) on myosin V and negatively charged portions (E-hooks) on the microtubule surface. Furthermore, it was shown that the initial interaction of myosin V with the microtubule is followed by a non-energy consuming scanning motion (one-dimensional diffusion) of the transporter along the microtubule track.

I hypothesize, that the unconventional interaction of myosin V with microtubules could play an essential role in aiding the networking between both transport systems.

Therefore a central part of my work as a PhD-student was to understand the molecular basis of this interaction (2). For this, three major questions have been addressed: Does indeed the charge of loop 2 contribute to microtubule binding? Or does rather the amino acid composition of loop 2 make the difference? And most importantly, after the initial binding to the microtubule, what biophysical feature enables myosin V to start diffusing along the filaments?

I used single molecule Total Internal Reflection Fluorescence Microscopy (TIRFM) to characterize the binding and diffusion behavior of myosin V mutants containing negatively instead of positively (as it is the case for Wildtype myosin V) charged loop 2 motifs on microtubules.

Figure 1. One-dimensional diffusion of myosin V loop 2 constructs on microtubules.[Bildunterschrift / Subline]: Figure 1. One-dimensional diffusion of myosin V loop 2 constructs on microtubules. (A) Schematic view of untreated microtubules containing E-hooks. (B) Single-molecule kymographs depicting diffusive movement of fluorescently labeled myosin V Wildtype as well as net negatively charged loop 2 mutants (Minus4 and Minus13). Control represents a stationary, non-diffusing motor molecule on the microtubule. (For further details see reference 2 below).

Based on the previously proposed electrostatic model by Ali et al. (1), one would expect that myosin V mutants containing a net negative charge on their loops would cease to interact with the negatively charged microtubules. Surprisingly, my data now suggests that myosin V diffusion on microtubules is neither determined nor limited by the charge of loop 2 as both, the positively charged Wildtype myosin V and the negatively charged loop 2 mutants, bind to and diffuse along microtubules (Figure 1A+B). Most strikingly, neither for the initial association nor for the subsequent diffusion of myosin V along microtubules E-hooks are required (Figure 2A+B).

Figure 2. One-dimensional diffusive motion of myosin V on microtubules lacking the E-hook.[Bildunterschrift / Subline]: Figure 2. One-dimensional diffusive motion of myosin V on microtubules lacking the E-hook. (A) Schematic view of microtubules, which after proteolytic treatment lack the E-hooks. (B) Kymograph depicting the diffusive motion of a single fluorescently labeled Wildtype myosin V molecule. Control represents a stationary, non-diffusing motor molecule on the microtubule. (For further details see reference 2 below).

Further analyses concerning the microtubule binding and diffusion behavior of those oppositely charged myosin V constructs assayed, now suggest that in addition to charge-charge interactions between myo V and the microtubule also non-ionic (e.g. van-der-Waals) attraction co-determines the interaction between myo V and microtubules, while hydrophilic effects by loop 2 merely play a subordinate role in facilitating diffusion on microtubules (Figure 3).

Figure 3. The balance between attraction forces determines the diffusive state of myosin V on microtubules.[Bildunterschrift / Subline]: Figure 3. The balance between attraction forces determines the diffusive state of myosin V on microtubules. (Left part) Strong attraction forces prevent microtubule-bound myosin V molecules from advancing to the diffusive state. (Middle part) Diffusion takes place if for myosin V the attraction toward the microtubule is of moderate strength. (Right part) Weak attraction toward microtubules prevents myosin V from binding effectively to the filament, and hence diffusion becomes unlikely. Red and blue colors indicate strong and weak attraction forces toward the microtubule surface, respectively. (For further details see reference 2 below).

In consistence with the observation that myosin V is able to assist kinesin-driven transport in vitro (3), our results point to a synergism between E-hook-independent tethering by myosin V to microtubules that in turn enhances the E-hook-dependent processive movement of kinesin (Figure 4). This biophysical trick could eventually enable cells to exploit both tracks for the same transport process without switching motors.

Figure 4. A model of how myosin V could assist kinesin-driven transport on microtubules.[Bildunterschrift / Subline]: Figure 4. A model of how myosin V could assist kinesin-driven transport on microtubules. Without the help of myosin V, kinesin falls of the filament after a rather short travel distance (runlength) and hence cargo transport would end before having reached the final destination (left part). The fact that myosin V is capable of interacting with microtubules, makes tethering of kinesin to the filament possible. Thereby facilitated kinesin-based transport would take place, which subsequently leads to enhanced runlengths. The ability of myosin V to scan long distances along microtubules might even increase the likelihood of meeting up with a kinesin that is in need of assistance.

References:

1. Ali MY, Krementsova EB, Kennedy GG, Mahaffy R, Pollard TD, et al. (2007) Myosin Va maneuvers through actin intersections and diffuses along microtubules. Proc Natl Acad Sci U S A 104: 4332-4336.

2. Zimmermann D, Abdel Motaal B, Voith von Voithenberg L, Schliwa M, Ökten Z (2011) Diffusion of Myosin V on Microtubules: A Fine-Tuned Interaction for Which E-Hooks Are Dispensable. PLoS ONE 6(9): e25473. doi:10.1371/journal.pone.0025473.

3. Ali MY, Lu H, Bookwalter CS, Warshaw DM, Trybus KM (2008) Myosin V and Kinesin act as tethers to enhance each others' processivity. Proc Natl Acad Sci U S A 105: 4691-4696.


Stationen
  • since 07/2008
  • PhD Student: Ludwig-Maximilians-University, Munich, Germany, Institute for Cell Biology, Prof. Manfred Schliwa
  • 09/2007 bis 07/2008
  • Diplom Thesis: Ludwig-Maximilians-University, Munich, Germany, Institute for Cell Biology, Prof. Manfred Schliwa
  • 04 - 05/2007
  • Research Intern: Max-Planck-Institute for Biochemistry, Martinsried, Germany, Department of Molecular Medicine, Prof. Reinhard Fässler
  • 09 - 10/2006
  • Research Intern: Woods Hole Oceanographic Institute, Woods Hole, U.S.A., Department of Marine Biochemistry, Prof. John Stegeman, PhD
  • 10/2003-09/2008
  • Biology (Dipl. Biol. univ.): Ludwig-Maximilians-University, Munich, Germany

Talks and Publications

Scientific Prizes and Awards
  • 10/2010
  • ASCB Predoctoral Travel Award 2010, Award by American Society for Cell Biology (ASCB), Philadelphia, U.S.A.
  • 09/2010
  • GlaxoSmithKline Travel Fellowship 2010, Funded by the GlaxoSmithKline Foundation, Barcelona, Spain