Architecture-Function Analysis of the Kinetochore Reveals the Mechanism of Spindle AssemblyCheckpoint Signaling.

[摘要] In a dividing cell, equal segregation of chromosomes between the two daughters is necessary for accurate genome inheritance. For successful segregation, chromosomes must attach to and move along microtubule tracks provided by the division machinery. A complex, multi-protein machine called the kinetochore establishes this attachment and generates force to move chromosomes. It also signals the absence of attachment by triggering a biochemical cascade called the Spindle Assembly Checkpoint (SAC), which in turn stalls the cell cycle. My thesis work provides mechanistic insights into this attachment-sensitive execution of the SAC by the kinetochore. These findings have significant implications in understanding how the checkpoint fails in conditions like cancer and Down;;s syndrome.Functionality of kinetochore emerges from its ;;architecture;;, defined by the spatial arrangement of multiple copies of > 60 proteins. I first reconstructed this architecture in the presence of microtubule attachment by combining high resolution imaging with FRET microscopy in budding yeast. This allowed me to then probe this architecture for changes that directly controlled SAC signaling. Using novel methods, I discovered a specific attachment-sensitive change in kinetochore architecture that regulates SAC signaling. Attachment controls the spatial separation between two conserved kinetochore proteins, Ndc80 and Spc105, like a mechanical toggle-switch. This nanoscale separation of ~ 30 nm in turn controls a key phosphorylation event, to turn the SAC on or off. I then investigated the biochemical reaction cascade that acts downstream from the triggering phosphorylation event to produce the final SAC signal. To understand the operational characteristics of individual steps in this cascade, I perturbed key parameters that govern these reactions and measured the effects on the steady-state concentration of the reaction intermediates in vivo, using quantitative fluorescence microscopy. This revealed novel mechanisms that tune the maximal signaling capacity of unattached kinetochores in the cell. Two commonly occurring regulatory themes, substrate limitation and modulation of binding affinities through negative cooperativity, tune the maximal SAC signal output from unattached kinetochores. These regulations likely enable sensitive detection of unattached kinetochores, while ensuring rapid reversibility of the SAC cascade following microtubule attachment.

[发布机构] University of Michigan

[效力级别] Developmental of biophysical tools to probe and characterize macromolecular structures

[关键词] Mechanism of mitotic checkpoint that ensures accurate chromosome segregation during cell division;Developmental of biophysical tools to probe and characterize macromolecular structures;Macromolecular protein architecture of kinetochore shapes emergent functions;Biochemical parameters provide handles to modulate checkpoint signaling;High resolution and fluorescence microscopy approaches to address biological problems;Quantitative approaches to define checkpoint signaling to predict implications in diseased conditions like cancer;Biological Chemistry;Molecular;Cellular and Developmental Biology;Science (General);Health Sciences;Science;Biophysics