Biophysics of contractile ring assembly

Transcription

1 Biophysics of contractile ring assembly Dimitrios Vavylonis Department of Physics, Lehigh University October 1, 2007

2 Physical biology of the cell Physical processes in cell organization and function: Transport of biomolecules e.g. transport of cargo molecules along neurons by motor proteins diffusion of proteins across the cell Assembly of proteins into structures e.g. polymerization of actin and tubulin packing of DNA in the nucleus Chemical energy work, pattern/order efficiency, robustness important Response to mechanical signals e.g. mechanical forces in the mitotic spindle small scales (~ individual molecules) molecular biology molecular biophysics/biochemistry large scales (> thousands of molecules) cell biology/biophysics

3 Collaborators Jian-Qiu Wu Department of Molecular Genetics, Ohio State University Thomas Pollard Department of Molecular, Cellular and Developmental Biology, Yale University Ben O Shaughnessy Department of Chemical Engineering, Columbia University

4 Cell Division of Animal Cells 5 nm actin monomers ~50 μm Pollard and Earnshaw, Cell Biology (2002) actin filament Ring assembly involves ~ 50 or more protein components. How do protein molecules assemble in a ring? What is the mechanistic pathway? How is it coordinated and controlled? How does the ring exert the required force?

5 Fission yeast (Schizosaccharomyces pombe, a fungus originally from East Africa) is a model organism to study cytokinesis during cell division 10 μm Why? time We know almost all the proteins/genes involved (a lot of work on genetics) It is much easier to make cells expressing GFP-fusion proteins Cytokinesis is rather similar to animal cells

6 Imaging Cell Dynamics with Green Fluorescent Protein green fluorescent spots Aequorea Victoria

7 GFP can be expressed in a different species and remains fluorescent gene gene fused to GFP gene Live cells become fluorescent The cell makes its own fluorophore many more colors: Tsien lab, UCSD 1994

8 Cytokinesis in fission yeast Actin (GFP-CHD) spindle poles Actin (GFP-CHD) spindle poles Jian-Qiu Wu (Pollard lab, Yale Univ) 2007

9 Contractile ring assembles from ~ 65 nodes Rlc1p-3GFP (myosin light chain) spinning disk confocal microscopy Vavylonis, Wu, Hao, O Shaughnessy, Pollard

10 Resolution limited by the wavelength of light ~ 500 nm typical protein size ~ 5 nm Molecules per node: Motor protein: Myo2p myosin-ii heavy chain 43 Rlc1p myosin light chain 35 Actin nucleator: Cdc12p formin dimers 2 Wu and Pollard, Science 2005 Wu et al. J.Cell Biol. 2006

11 Formins nucleate actin filaments and remain associated with the growing end: Kovar, et al. Cell, (2006) actin formin dimer formins are molecular polymerization machines FH2 FH1 profilin actin purified fluorescent actin + formin mdia1 Vavylonis, Kovar, O Shaughnessy, Pollard Mol. Cell 2006

12 Lateral contraction model node Wu et al. J. Cell Biol. 2006

13 Model with static connections condenses nodes into clumps! numerical simulation time pattern and dynamics of connections is important

14 Each node forms within ~ 50 sec 15x time lapse 3 minutes single focal plane near top of cell pre-existent node new nodes

15 Start-stop motion of nodes 15x time lapse 10 minutes each frame: average of 6

16 Classes of event during condensation intermittent directed motions coalescence and splitting speed duration of movements

17 Diffusive motion of stationary nodes estimate of force 15x, 200s total ImageJ Spottracker2D plugin mean square displacement diffusion coefficients (nm 2 ) ~ 4-5 orders of magnitude smaller than single membrane proteins

18 diffusive stage thermal (?) motion without force D 2 30 nm /sec condensation stage velocity in response to force v 30 nm/sec F v Einstein relation for Brownian motion: kt F v 4 pn D = (lower bound) consistent with force exerted by few molecular motors

19 Onset of node motion correlates with actin arrival in broad band (+2 min after spindle pole separation) 30x time lapse, 100 sec binds F-actin nodes cdc25-22 cells v pol ~ 200 nm/s ~ 75 subunits/s

20 Search, capture, pull and release model actin filament polymerization actin filament capture ~ 0.1 μm/sec r c ~ 100 nm lifetime of connections ~ 20 sec lifetime of filaments F 4 pn traction on filaments between nodes ~ 20 sec (?) Dynamic reestablishment of connections plasticity of network

21 Model results Simulated image 30x time lapse, 20min red: nodes green: actin 0 2πR experiment:

22 Results of model v pol = 0.2 μm/sec v pol = 0.04 μm/sec red: nodes green: actin filaments

23 Long connection lifetimes lead to clumps sec Too wide broad bands form clumps Dependence on number of nodes

24 Summary of required parameter values: >60 nodes 2-4 formins per node zone width < 2.8 µm actin polymerization rate > 0.1 µm/sec filament lifetime >10 sec connection breakage time <45 sec Is this an optimized process?

25 Leading actin cable model: Kamasaki, Osumi, Mabuchi J. Cell Biol. August 2007 What is the role of actin bundles? Perhaps progressive stabilization? There is still much more to learn!