Edward R. Ballister

Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient

Aurora B kinase is concentrated and activated at centromeres before release and diffusion to reach spatially distributed substrates necessary for cell division.

Localized light-induced protein dimerization in living cells using a photocaged dimerizer

Regulated protein localization is critical for many cellular processes. Several techniques have been developed for experimental control over protein localization, including chemically induced and light-induced dimerization, which both provide temporal control. Light-induced dimerization offers the distinct advantage of spatial precision within subcellular length scales. A number of elegant systems have been reported that utilize natural light-sensitive proteins to induce dimerization via direct protein-protein binding interactions, but the application of these systems at cellular locations beyond the plasma membrane has been limited. Here we present a new technique to rapidly and reversibly control protein localization in living cells with subcellular spatial resolution using a cell-permeable, photoactivatable chemical inducer of dimerization. We demonstrate light-induced recruitment of a cytosolic protein to individual centromeres, kinetochores, mitochondria and centrosomes in human cells, indicating that our system is widely applicable to many cellular locations.

Optogenetic control of organelle transport using a photocaged chemical inducer of dimerization

Summary Cell polarity, growth and signaling require organelle transport by cytoskeletal motor proteins that are precisely regulated in time and space. Probing these complex, dynamic processes requires experimental techniques with comparable temporal and spatial precision. Inducible dimerization offers the ability to recruit motor proteins to organelles in living cells. Approaches include rapamycin-induced dimerization of motors and cargo-bound binding partners [1] or the recent application of the TULIP light-inducible dimerization system [2,3]. In the latter system, motor recruitment is activated by blue light, and relaxes to an OFF state in the dark within seconds. While rapid relaxation is desirable for some applications, many experiments require sustained motor recruitment. Here, we use a photocaged chemical dimerizer to achieve sustained, spatially-defined motor recruitment to individual organelles with a single pulse of light. We demonstrate the general applicability of the system by recruiting microtubule plus end-directed kinesin-1 and minus end-directed dynein motors to peroxisomes and mitochondria in HeLa cells and primary neurons, leading to alterations in organelle transport on timescales from 10 minutes after photoactivation.

Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

Mad1 recruitment to metaphase kinetochores reactivates the mitotic checkpoint, which requires the C terminus of Mad1 in addition to its Mad2-binding domain.

Bistability of a coupled Aurora B kinase-phosphatase system in cell division

Aurora B kinase, a key regulator of cell division, localizes to specific cellular locations, but the regulatory mechanisms responsible for phosphorylation of substrates located remotely from kinase enrichment sites are unclear. Here, we provide evidence that this activity at a distance depends on both sites of high kinase concentration and the bistability of a coupled kinase-phosphatase system. We reconstitute this bistable behavior and hysteresis using purified components to reveal co-existence of distinct high and low Aurora B activity states, sustained by a two-component kinase autoactivation mechanism. Furthermore, we demonstrate these non-linear regimes in live cells using a FRET-based phosphorylation sensor, and provide a mechanistic theoretical model for spatial regulation of Aurora B phosphorylation. We propose that bistability of an Aurora B-phosphatase system underlies formation of spatial phosphorylation patterns, which are generated and spread from sites of kinase autoactivation, thereby regulating cell division. DOI: http://dx.doi.org/10.7554/eLife.10644.001

Optogenetic control of kinetochore function

Kinetochores act as hubs for multiple activities during cell division, including microtubule interactions and spindle checkpoint signaling. Each kinetochore can act autonomously, and activities change rapidly as proteins are recruited to or removed from kinetochores. Understanding this dynamic system requires tools that can manipulate kinetochores on biologically relevant temporal and spatial scales. Optogenetic approaches have the potential to provide temporal and spatial control with molecular specificity. Here we report new chemical inducers of protein dimerization that allow us to both recruit proteins to and release them from kinetochores using light. We use these dimerizers to manipulate checkpoint signaling and molecular motor activity. Our findings demonstrate specialized properties of the CENP-E (kinesin-7) motor for directional chromosome transport to the spindle equator and for maintaining metaphase alignment. This work establishes a foundation for optogenetic control of kinetochore function, which is broadly applicable to experimentally probe other dynamic cellular processes.

Minimal model for collective kinetochore–microtubule dynamics

Significance Coordinated metaphase chromosome motions are driven by microtubule (MT) dynamics. MTs stochastically switch between growing and shrinking states with rates that depend on forces and biochemical factors acting at the kinetochore–MT interface. Single-MT behavior is known from in vitro experiments, but it is unclear how many MTs cooperate to control chromosome dynamics. We construct and experimentally test a minimal model for collective MT dynamics. The force dependence of the MTs leads to bistable and hysteretic dynamics. This produces chromosome oscillations and error-correcting behavior, as observed in vivo. Our model provides a mechanistic, predictive framework in which we can incorporate further biological complexity. Chromosome segregation during cell division depends on interactions of kinetochores with dynamic microtubules (MTs). In many eukaryotes, each kinetochore binds multiple MTs, but the collective behavior of these coupled MTs is not well understood. We present a minimal model for collective kinetochore–MT dynamics, based on in vitro measurements of individual MTs and their dependence on force and kinetochore phosphorylation by Aurora B kinase. For a system of multiple MTs connected to the same kinetochore, the force–velocity relation has a bistable regime with two possible steady-state velocities: rapid shortening or slow growth. Bistability, combined with the difference between the growing and shrinking speeds, leads to center-of-mass and breathing oscillations in bioriented sister kinetochore pairs. Kinetochore phosphorylation shifts the bistable region to higher tensions, so that only the rapidly shortening state is stable at low tension. Thus, phosphorylation leads to error correction for kinetochores that are not under tension. We challenged the model with new experiments, using chemically induced dimerization to enhance Aurora B activity at metaphase kinetochores. The model suggests that the experimentally observed disordering of the metaphase plate occurs because phosphorylation increases kinetochore speeds by biasing MTs to shrink. Our minimal model qualitatively captures certain characteristic features of kinetochore dynamics, illustrates how biochemical signals such as phosphorylation may regulate the dynamics, and provides a theoretical framework for understanding other factors that control the dynamics in vivo.

Two mechanisms coordinate the recruitment of the chromosomal passenger complex to the plane of cell division

Proper positioning of the chromosomal passenger complex (CPC) at the cell division plane is required for cytokinesis. We show here that CPC targeting to the equatorial cortex depends on both the kinesin MKlp2 and a direct interaction with actin. These recruitment mechanisms converge to promote successful cleavage furrow ingression.

Probing Mitosis by Manipulating the Interactions of Mitotic Regulator Proteins Using Rapamycin-Inducible Dimerization.

Inducible dimerization is a general approach to experimentally manipulate protein-protein interactions with temporal control. This chapter describes the use of rapamycin-inducible dimerization to manipulate mitotic regulatory proteins, for example to control kinetochore localization. A significant feature of this method relative to previously described protocols is the depletion of endogenous FKBP12 protein, which markedly improves dimerization efficiency.

Chromosomal Instability: Mad2 beyond the Spindle Checkpoint

What specific defects can cause chromosomal instability in cancer cells? Overexpression of the mitotic checkpoint protein Mad2 triggers chromosome missegregation but, surprisingly, Mad2 exerts this effect through a previously unknown effect on microtubule dynamics.