Force- and kinesin-8-dependent effects in the spatial regulation of fission yeast microtubule dynamics

• Published in: Molecular systems biology Vol. 5; no. 1; pp. 250 - n/a
• Main Authors: Dogterom, Marileen, Tischer, Christian, Brunner, Damian
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.03.2009; John Wiley & Sons, Ltd; EMBO Press; Nature Publishing Group; Springer Nature
• Subjects: Biomechanical Phenomena; Catastrophes; Cell morphology; Cell Polarity; Cytology; Dependence; Depolymerization; Dynamics; EMBO05; EMBO10; Fission; fission yeast; forces; Gene Deletion; Image analysis; Image processing; Intracellular; Investigations; Kinesin; Kinesin - deficiency; Kinesin - metabolism; kinesin‐8; Mathematical models; Mathematical morphology; Microtubule-Associated Proteins - deficiency; Microtubule-Associated Proteins - metabolism; Microtubules; Microtubules - metabolism; Polymerization; Protein Transport; Proteins; Schizosaccharomyces - cytology; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - metabolism; Switches; Yeast; Yeasts; fission yeast; microtubules; catastrophes; kinesin‐8; forces
• ISSN: 1744-4292; 1744-4292
• DOI: 10.1038/msb.2009.5

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin‐8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells. Synopsis Microtubules (MTs) are dynamic protein polymers that change their length by switching between growing and shrinking states in a process termed ‘dynamic instability’ (Mitchison and Kirschner, 1984 ; Desai and Mitchison, 1997 ). MTs are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology (Kirschner and Mitchison, 1986 ; Hayles and Nurse, 2001 ). To better understand how MTs control the organisation of the intracellular space, it is important to quantitatively understand how dynamic instability is regulated, because this affects MT length (Verde et al , 1992 ; Dogterom and Leibler, 1993 ) as well as the ability of MTs to exert pushing and pulling forces (Inoue and Salmon, 1995 ; Dogterom et al , 2005 ). Several proteins have been characterised that globally affect MT growth, shrinkage, catastrophes (switches from growth to shrinkage) and rescues (switches from shrinkage to growth) (Howard and Hyman, 2007 ), but it is a largely open question how such regulation is achieved locally, in response to spatially varying biochemical cues and/or mechanical effects induced by the shape and size of cells. In fact, the precise and spatially resolved measurement of MT catastrophe and rescue frequencies is a challenging task, because those appear to be stochastic events that are governed by an average rate (Odde, 1995 ; Howell et al , 1997 ). This has two consequences: statistical accuracy is a serious issue when investigating catastrophe frequencies, and, the stochastic nature of the process makes it very difficult to avoid picking a subset of events when examining data by visual inspection. In this article, we present quantitative investigations of spatial MT catastrophe regulation in interphase fission yeast cells ( Schizosaccharomyces pombe ) with high statistical accuracy (Hayles and Nurse, 2001 ). Fission yeast is an excellent model system, because MTs are well organised and the rigid cylindrical cell wall makes it possible to accurately assign catastrophes to specific locations within the cell (Figure 1A and B ) (Hagan, 1998 ). MT minus ends are generally found close to the nucleus within the central overlap zone of the MTs, whereas dynamic plus tips grow and shrink between the nucleus and the cell poles (Drummond and Cross, 2000 ; Tran et al , 2001 ). Catastrophe events are mainly restricted to the regions of the two cell poles by an unknown mechanism. This local regulation of MT catastrophes is, however, crucial for the maintenance of correct fission yeast morphology and intracellular organisation (Beinhauer et al , 1997 ; Mata and Nurse, 1997 ; Browning et al , 2000 ; Brunner and Nurse, 2000 ; Hayles and Nurse, 2001 ; Tran et al , 2001 ; Sawin and Snaith, 2004 ; Tolic‐Norrelykke et al , 2005 ; Daga et al , 2006). To obtain good statistics and to ensure unbiased observations, we developed fully automated image analysis software that generates spatially resolved maps of MT dynamics from movies of GFP‐labelled MTs in fission yeast. Our spatially resolved measurements show that there is both local enhancement of the catastrophe frequency specifically at cell poles as well as long‐range modulation before the cell pole is reached (Figure 2 ). We find several indications that the local regulation at the cell pole is (at least in part) due to compressive forces that build up when bundle tips hit the cell pole (Figure 4 ). In addition, we find evidence that the long‐range catastrophe regulation is an MT length‐dependent effect mediated by the kinesin‐8 proteins Klp5/6. As physical boundaries and kinesin‐8 proteins are also present in other eukaryotic systems, we think that the relevance of our findings reaches beyond the fission yeast model system. Specifically, it is interesting to note that f cat is also enhanced at the boundaries of animal cells (Komarova et al , 2002 ; Mimori‐Kiyosue et al , 2005 ) by a yet unknown mechanism. Our findings in fission yeast, taken together with earlier in vitro observations (Dogterom and Yurke, 1997 ; Janson et al , 2003 ; Janson and Dogterom, 2004 ), suggest that there are intrinsic relations between polymerisation force, v g and f cat that help terminate MT growth at physical boundaries. In a living cell, the spatial extent of the cell and the length of its MT cytoskeleton must be well adapted to each other. There has been evidence that MT dynamics play a role in establishing cell shape (Kirschner and Mitchison, 1986 ; Hayles and Nurse, 2001 ). Our data indicate that, vice versa, the shape of a cell also influences MT dynamics. Thus, cell shape and MT organisation may not be separable components but should be viewed as one system. Moreover, Klp5/Klp6 are part of the kinesin‐8 family, comprising Kip3 ( Saccharomyces cerevisiae ), KLP67A ( Drosophila melanogaster ), Kif18A ( Homo sapiens ) and KipB ( Aspergillus nidulans ), which have in common that mutants show defects in mitosis (Garcia et al , 2002 ; West et al , 2002 ; Rischitor et al , 2004 ; Tytell and Sorger, 2006 ; Mayr et al , 2007 ; Stumpff et al , 2008 ). In this context, it has been speculated that a kinesin‐8‐mediated increase in f cat with MT length could contribute to proper chromosome centring and spindle length regulation (Gardner et al , 2008 ; Stumpff et al , 2008 ). Our data provide good experimental evidence that kinesin‐8 proteins indeed specifically enhance f cat of long MTs. Finally, we would like to point out a related article in Molecular Systems Biology by Foethke et al . In this article, the authors perform a 3D simulation of the self‐organisation of dynamic MTs within the physical confinement of a fission yeast cell. Specifically, following results of earlier in vitro experiments (Dogterom and Yurke, 1997 ; Janson et al , 2003 ; Janson and Dogterom, 2004 ), the authors investigate a model in which MT growth and catastrophe frequencies depend on compressive physical forces. The authors find that such force dependence is sufficient to reproduce most, but not all, of the known experimental data on the spatiotemporal organisation of MTs inside interphase fission yeast cells. Interestingly, evoking in addition an MT length dependence of the catastrophe frequency, as provided evidence for by our measurements, allows the model to reproduce further observations on MT dynamics in fission yeast that were so far considered to be signatures of more complicated processes. This indicates that a susceptibility of MT dynamics to physical forces and to MT length are simple yet very efficient mechanisms for adapting MT dynamics to cell size and shape. We developed fully automated image analysis software to investigate the spatial regulation of microtubule dynamics in interphase fission yeast cells with high statistical accuracy. Our data provide evidence that compressive forces generated by microtubules growing against the cell pole locally reduce microtubule growth velocities and enhance catastrophes frequencies. We also find evidence for a microtubule length‐dependent increase in the catastrophe frequency that is mediated by kinesin‐8 motor proteins (Klp5/6). The inclusion of these experimental findings in a 3‐D simulation of interphase fission yeast cells by Foethke et al. allows the authors to reproduce many observations on MT dynamics in fission yeast that were so far considered to be signatures of more complicated processes.