Session L12: Invited Session: The Physics of Cell Division

Microtubule Depolymerization as a Driver for Chromosome Motion

Microtubules (MTs) are rigid polymers of the protein, tubulin, which function as intracellular struts. They are also tracks along which motor enzymes can run, carrying cargo to specific cellular locations. Most MTs are dynamic; they assemble and disassemble rapidly, particularly during cell division when the cell forms the ``mitotic spindle,'' a machine that organizes the duplicated chromosomes into a planar disk, then pulls the duplicate copies apart, moving them to opposite ends of the cell. This process is necessary for the daughter cells to have a full complement of DNA. The mitotic spindle is a labile framework that exerts several kinds of forces on the chromosomes to move them in well organized ways. It contains many motor enzymes that contribute to spindle formation, but genetic evidence shows that the motors that attach to chromosomes and might contribute to chromosome motion are dispensable for normal mitosis. Apparently MT dynamics can also serve as a motor and is an important source of force for chromosome motion. We have studied this process and find that MTs can be coupled to a load by specific spindle proteins so that MT depolymerization can exert substantial force. With the yeast protein, Dam1, a single MT can generate 30 pN, about 5-fold more than is generated by a motor enzyme like kinesin or myosin. The resulting motions are processive, so a depolymerizing MT can carry its load for many micrometers. However, Dam1 is found only in fungi. We have therefore sought other proteins that can serve as analogous couplers. Several MT-dependent motor enzymes can do the job in ways that do not require ATP, their normal source of energy. Some non-motor MT-associated proteins will also work, e.g., the kinetochore proteins NDC80 and CENP-F. Data will be presented that show the strengths and weaknesses of each coupler, allowing some generalization about how the mitotic machinery works.   

Microtubules search for chromosomes by pivoting around the spindle pole

During cell division, proper segregation of genetic material between the two daughter cells requires that the spindle microtubules attach to the chromosomes via kinetochores, protein complexes on the chromosome. The central question, how microtubules find kinetochores, is still under debate. We observed in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole body, instead of growing towards the kinetochores. By introducing a theoretical model, we show that the observed angular movement of microtubules is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of the capture process depends mainly on how fast microtubules pivot. We confirmed this prediction experimentally by speeding up and slowing down microtubule pivoting. Thus, microtubules explore space by pivoting, as they search for intracellular targets such as kinetochores.\\[4pt] In collaboration with Iva Tolic-Norrelykke, Max Planck Institute of Molecular Cell Biology and Genetics.   

A minimal model for kinetochore-microtubule dynamics

During mitosis, chromosome pairs align at the center of a bipolar microtubule (MT) spindle and oscillate as MTs attaching them to the cell poles polymerize and depolymerize. The cell fixes misaligned pairs by a tension-sensing mechanism. Pairs later separate as shrinking MTs pull each chromosome toward its respective cell pole. We present a minimal model for these processes based on properties of MT kinetics. We apply the measured tension-dependence of single MT kinetics [1] to a stochastic many MT model, which we solve numerically and with master equations. We find that the force-velocity curve for the single chromosome system is bistable and hysteretic. Above some threshold load, tension fluctuations induce MTs to spontaneously switch from a pulling state into a growing, pushing state. To recover pulling from the pushing state, the load must be reduced far below the threshold. This leads to oscillations in the two-chromosome system. Our minimal model quantitatively captures several aspects of kinetochore dynamics observed experimentally. \\[4pt] [1] Akiyoshi et al. (2010) Nature 468, 576.   

An engineer's understanding of kinetochore motility and signaling

The kinetochore is a macromolecular motor that couples chromosome movement to microtubule polymerization and depolymerization. It is also a mechanochemical signaling hub. A kinetochore that lacks microtubule attachment generates a biochemical signal to arrest the cell cycle. Both kinetochore functions require numerous copies of $\sim$ 8 proteins and protein complexes. A cohesive explanation of how multiple kinetochore components cooperate to achieve motility and signaling remains elusive. I will describe on-going ``architecture-function'' analysis in my lab that applies an engineer's perspective to study the machine that is the kinetochore. This analysis is based on the definition of the protein architecture of the kinetochore using known protein structures, copy numbers in the kinetochore, average positions, and distributions. This architecture enables us to assign specific functions to each kinetochore component in generating movement. The architecture also reveals the molecular mechanism of kinetochore signaling embedded within the kinetochore.   

Positioning of Microtubule organizing centers (MTOC) in 3D confinement

Important functions of eucaryotic cells, like motility or division, depend sensitively on cytoskeletal mechanics and organization. In particular, microtubules (MTs) are dynamic polymers that can move and position organelles such as their MTOC by pushing, pulling or sliding [1]. How the shape and size of cells, as well as the presence of pushing and/or pulling forces influence this positioning is in many cases still unclear. To assess the influence of confinement on MTOC positioning, we reconstruct a dynamic microtubule cytoskeleton in vitro, inside 3D water in oil emulsion droplets. We study the positioning of centrosomes, from which microtubules are nucleated, that exert pushing and/or dynein- mediated-pulling forces when reaching the cortex. We show that the central position of one centrosome confined in a spherical droplet is drastically destabilized by pushing forces, while pulling forces tend to center the centrosome. Interestingly, when two centrosomes are present, pushing forces will lead the centrosomes to take a stable position at opposite sides of the droplet. When both pushing and pulling forces are present, two centrosomes adopt an equilibrium position balancing the centering effect of the cortical pulling forces and the repulsion effect of the two centrosomes. Summarizing, we show that very simple systems, involving only microtubule dynamics, confinement, pushing and pulling forces can lead to self-organized patterns that are biologically relevant. In particular, we reproduce a ‘mitotic spindle’ like organization with just these components. This sets the stage for a better understanding of the function of additional components of natural mitotic spindles such as mitotic motors and crosslinkers that we plan to add to our system. \\[4pt] [1] Tolic-Norrelykke I, \textbf{2008} Push-me-pull-you: how microtubules organize the cell interior \textit{Eur Biophys J} 37: 1271-1278