Sensing centromere tension: Aurora B and the regulation of kinetochore function

Maintaining genome integrity during cell division requires regulated interactions between chromosomes and spindle microtubules. To ensure that daughter cells inherit the correct chromosomes, the sister kinetochores must attach to opposite spindle poles. Tension across the centromere stabilizes correct attachments, whereas phosphorylation of kinetochore substrates by the conserved Ipl1/Aurora B kinase selectively eliminates incorrect attachments. Here, we review our current understanding of how mechanical forces acting on the kinetochore are linked to biochemical changes to control chromosome segregation. We discuss models for tension sensing and regulation of kinetochore function downstream of Aurora B, and mechanisms that specify Aurora B localization to the inner centromere and determine its interactions with substrates at distinct locations.National Institutes of Health (U.S.) (Grant GM088313)Kinship Foundation. Searle Scholars Progra

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

SummaryCell 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 seconds to >10 minutes after photoactivation

Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.Smith Family FoundationMassachusetts Life Sciences CenterKinship Foundation. Searle Scholars ProgramNational Institute of General Medical Sciences (U.S.) (Grant number GM088313

Common Representation of Information Flows for Dynamic Coalitions

We propose a formal foundation for reasoning about access control policies within a Dynamic Coalition, defining an abstraction over existing access control models and providing mechanisms for translation of those models into information-flow domain. The abstracted information-flow domain model, called a Common Representation, can then be used for defining a way to control the evolution of Dynamic Coalitions with respect to information flow

Mps1 regulates kinetochore-microtubule attachment stability via the ska complex to ensure error-free chromosome segregation

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation

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

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes