In vitro reconstitution of motor-driven nuclear oscillations

• Antragstellerinnen / Antragsteller: Professor Dr. Stefan Diez; Professorin Iva Marija Tolic, Ph.D.
• Fachliche Zuordnung: Biophysik
• Förderung: Förderung von 2009 bis 2012
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 107847528

• Erstellungsjahr: 2016

Zusammenfassung der Projektergebnisse

Molecular motors play a crucial role in fundamental cell functions such as cell division and intracellular transport. Although much is known about the behavior of individual motors, the interplay of a multitude of motors, which leads to concerted movements in the cell, is not yet well understood. Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe have been used as an easily accessible model process to study intracellular movements driven by dynein motors operating on microtubules (MTs). In this project, a number of steps towards an “artificial cell” system which will allow for investigating nuclear oscillations in a controlled and simplified environment in vitro were made. (i) To mimic the bidirectional motion of MTs during nuclear oscillations in S. pombe, a novel MT-doublet gliding assay was developed. In particular, anti-parallel doublets of polarity-marked MTs were prepared via crosslinking by anti-tubulin antibodies. When studying the motility of these doublets on surfaces coated by motor proteins distinct modes of slow and fast movements, as well as sharp transitions between these modes and regions of coexistence were found. In combination with a quantitative theory based on the physical properties of individual motors, the obtained results show that mechanical interactions between motors can collectively generate coexisting transport regimes with distinct velocities. (ii) To explore the effects of confined geometries on the movement of MTs, microchambers that mimic the geometry of S. pombe cells were fabricated and oscillations of artificial MT-organizing centers were studied in chambers of various sizes and shapes, with high throughput. This setup is suitable for studies of the role of chamber size and shape, as well as of motor proteins and other MT-associated proteins on the positioning and oscillations of asters. (iii) To compare our in vitro results with the behavior of MTs in vivo, two studies on fission yeast cells were performed. In the first study, dynein-driven oscillations of the nucleus that occur during meiotic prophase were investigated. Single dyneins during meiotic nuclear oscillations were observed and the steps of the dynein binding process: from the cytoplasm to the microtubule and from the microtubule to cortical anchors, were identified. It was observed that dyneins on the microtubule moved either in a diffusive or directed manner, with the switch from diffusion to directed movement occurring upon binding of dynein to cortical anchors. This dual behavior of dynein on the microtubule, together with the two steps of binding, enables dyneins to self-organize into a spatial pattern needed for them to generate large collective forces. In a second study, we explored oscillations of the nucleus during interphase driven by MT pushing, representing an in vivo equivalent of the oscillations of the artificial MT-organizing centers studied in vitro in the confined chambers. Taking together, the project results help to elucidate the fundamentals of the collective behavior of motors leading to large-scale movements in living cells.

Projektbezogene Publikationen (Auswahl)

• Axial Nanometer Distances Measured by Fluorescence Lifetime Imaging Microscopy. Nano Lett. 10(4), 1497-1500 (2010)
  M. Berndt, M. Lorenz, J. Enderlein, S. Diez
  
• Collective Behavior of Antagonistically Acting Kinesin-1 Motors. Phys. Rev. Lett. 105(12), (2010)
  C. Leduc, N. Pavin, F. Jülicher, S. Diez
  
• Tracking single particles and elongated filaments with nanometer precision. Biophys. J. 100(11), 2820-2828 (2011)
  F. Ruhnow, D. Zwicker, S. Diez
  
• Dynein motion switches from diffusive to directed upon cortical anchoring. Cell 153(7): 1526- 1536 (2013)
  V. Ananthanarayanan, M. Schattat, S.K. Vogel, A. Krull, N. Pavin, and I.M. Tolić-Nørrelykke
  
• Multi-motor transport in a system of active and inactive kinesin-1 motors. Biophys. J. 107(2), 365-372 (2014)
  L. Scharrel, R. Ma, R. Schneider, F. Jülicher, S. Diez
  
• Kinesin-8 motors improve nuclear centering by promoting microtubule catastrophe. Phys. Rev. Lett. 114(7): 078103 (2015)
  M. Glunčić, N. Maghelli, A. Krull, V. Krstić, D. Ramunno-Johnson, N. Pavin, and I.M. Tolić
  
• Single-molecule imaging of cytoplasmic dynein in vivo. Methods Cell Biol. 125: 1-12 (2015)
  V. Ananthanarayanan, I.M. Tolić