We discovered that many proteins located in the kinetochore outer domain, but not the inner core, are depleted from kinetochores and accumulate at spindle poles when ATP production is suppressed in PtK1 cells, and that microtubule depolymerization inhibits this process. These proteins include the microtubule motors CENP-E and cytoplasmic dynein, and proteins involved with the mitotic spindle checkpoint, Mad2, Bub1R, and the 3F3/2 phosphoantigen. Depletion of these components did not disrupt kinetochore outer domain structure or alter metaphase kinetochore microtubule number. Inhibition of dynein/dynactin activity by microinjection in prometaphase with purified p50 “dynamitin” protein or concentrated 70.1 anti-dynein antibody blocked outer domain protein transport to the spindle poles, prevented Mad2 depletion from kinetochores despite normal kinetochore microtubule numbers, reduced metaphase kinetochore tension by 40%, and induced a mitotic block at metaphase. Dynein/dynactin inhibition did not block chromosome congression to the spindle equator in prometaphase, or segregation to the poles in anaphase when the spindle checkpoint was inactivated by microinjection with Mad2 antibodies. Thus, a major function of dynein/dynactin in mitosis is in a kinetochore disassembly pathway that contributes to inactivation of the spindle checkpoint.

The ability of kinetochores to recruit microtubules, generate force, and activate the mitotic spindle checkpoint may all depend on microtubule-and/or tension-dependent changes in kinetochore assembly. With the use of quantitative digital imaging and immunofluorescence microscopy of PtK1 tissue cells, we find that the outer domain of the kinetochore, but not the CREST-stained inner core, exhibits three microtubule-dependent assembly states, not directly dependent on tension. First, prometaphase kinetochores with few or no kinetochore microtubules have abundant punctate or oblate fluorescence morphology when stained for outer domain motor proteins CENP-E and cytoplasmic dynein and checkpoint proteins BubR1 and Mad2. Second, microtubule depolymerization induces expansion of the kinetochore outer domain into crescent and ring morphologies around the centromere. This expansion may enhance recruitment of kinetochore microtubules, and occurs with more than a 20-to 100-fold increase in dynein and relatively little change in CENP-E, BubR1, and Mad2 in comparison to prometaphase kinetochores. Crescents disappear and dynein decreases substantially upon microtubule reassembly. Third, when kinetochores acquire their full metaphase complement of kinetochore microtubules, levels of CENP-E, dynein, and BubR1 decrease by three-to sixfold in comparison to unattached prometaphase kinetochores, but remain detectable. In contrast, Mad2 decreases by 100-fold and becomes undetectable, consistent with Mad2 being a key factor for the "wait-anaphase" signal produced by unattached kinetochores. Like previously found for Mad2, the average amounts of CENP-E, dynein, or BubR1 at metaphase kinetochores did not change with the loss of tension induced by taxol stabilization of microtubules.

The spindle checkpoint prevents errors in chromosome segregation by inhibiting anaphase onset until all chromosomes have aligned at the spindle equator through attachment of their sister kinetochores to microtubules from opposite spindle poles. A key checkpoint component is the mitotic arrest–deficient protein 2 (Mad2), which localizes to unattached kinetochores and inhibits activation of the anaphase-promoting complex (APC) through an interaction with Cdc20. Recent studies have suggested a catalytic model for kinetochore function where unattached kinetochores provide sites for assembling and releasing Mad2–Cdc20 complexes, which sequester Cdc20 and prevent it from activating the APC. To test this model, we examined Mad2 dynamics in living PtK1 cells that were either injected with fluorescently labeled Alexa 488-XMad2 or transfected with GFP-hMAD2. Real-time, digital imaging revealed fluorescent Mad2 localized to unattached kinetochores, spindle poles, and spindle fibers depending on the stage of mitosis. FRAP measurements showed that Mad2 is a transient component of unattached kinetochores, as predicted by the catalytic model, with a t 1/2 of ∼24–28 s. Cells entered anaphase ∼10 min after Mad2 was no longer detectable on the kinetochores of the last chromosome to congress to the metaphase plate. Several observations indicate that Mad2 binding sites are translocated from kinetochores to spindle poles along microtubules. First, Mad2 that bound to sites on a kinetochore was dynamically stretched in both directions upon microtubule interactions, and Mad2 particles moved from kinetochores toward the poles. Second, spindle fiber and pole fluorescence disappeared upon Mad2 disappearance at the kinetochores. Third, ATP depletion resulted in microtubule-dependent depletion of Mad2 fluorescence at kinetochores and increased fluorescence at spindle poles. Finally, in normal cells, the half-life of Mad2 turnover at poles, 23 s, was similar to kinetochores. Thus, kinetochore-derived sites along spindle fibers and at spindle poles may also catalyze Mad2 inhibitory complex formation.

The cytokinesis phase, or C phase, of the cell cycle results in the separation of one cell into two daughter cells after the completion of mitosis. Although it is known that microtubules are required for proper positioning of the cytokinetic furrow [1] [2], the role of pre-anaphase microtubules in cytokinesis has not been clearly defined for three key reasons. First, inducing microtubule depolymerization or stabilization before the onset of anaphase blocks entry into anaphase and cytokinesis via the spindle checkpoint [3]. Second, microtubule organization changes rapidly at anaphase onset as the mitotic kinase, Cdc2-cyclin B, is inactivated [4]. Third, the time between the onset of anaphase and the initiation of cytokinesis is very short, making it difficult to unambiguously alter microtubule polymer levels before cytokinesis, but after inactivation of the spindle checkpoint. Here, we have taken advantage of the discovery that microinjection of antibodies to the spindle checkpoint protein Mad2 (mitotic arrest deficient) in prometaphase abrogates the spindle checkpoint, producing premature chromosome separation, segregation, and normal cytokinesis [5] [6]. To test the role of pre-anaphase microtubules in cytokinesis, microtubules were disassembled in prophase and prometaphase cells, the cells were then injected with anti-Mad2 antibodies and recorded through C phase. The results show that exit from mitosis in the absence of microtubules triggered a 50 minute period of cortical contractility that was independent of microtubules. Furthermore, upon microtubule reassembly during this contractile C-phase period, approximately 30% of the cells underwent chromosome poleward movement, formed a midzone microtubule complex, and completed cytokinesis.

After decades on the margins of primary health care, surgical and anaesthesia care is gaining increasing priority within the global development arena. The 2015 publications of the Disease Control Priorities third edition on Essential Surgery and the Lancet Commission on Global Surgery created a compelling evidenced-based argument for the fundamental role of surgery and anaesthesia within cost-effective health systems strengthening global strategy. The launch of the Global Alliance for Surgical, Obstetric, Trauma, and Anaesthesia Care in 2015 has further coordinated efforts to build priority for surgical care and anaesthesia. These combined efforts culminated in the approval of a World Health Assembly resolution recognizing the role of surgical care and anaesthesia as part of universal health coverage. Momentum gained from these milestones highlights the need to identify consensus goals, targets and indicators to guide policy implementation and track progress at the national level. Through an open consultative process that incorporated input from stakeholders from around the globe, a global target calling for safe surgical and anaesthesia care for 80% of the world by 2030 was proposed. In order to achieve this target, we also propose 15 consensus indicators that build on existing surgical systems metrics and expand the ability to prioritize surgical systems strengthening around the world.