RESUMEN
The microtubule (MT)-depolymerizing activity of MCAK/Kif2C can be quantified by expressing the motor in cultured cells and measuring tubulin fluorescence levels after enough hours have passed to allow tubulin autoregulation to proceed. This method allows us to score the impact of point mutations within the motor domain. We found that, despite their distinctly different activities, many mutations that impact transport kinesins also impair MCAK/Kif2C's depolymerizing activity. We improved our workflow using CellProfiler to significantly speed up the imaging and analysis of transfected cells. This allowed us to rapidly interrogate a number of MCAK/Kif2C motor domain mutations documented in the cancer database cBioPortal. We found that a large proportion of these mutations adversely impact the motor. Using green fluorescent protein-FKBP-MCAK CRISPR cells we found that one deleterious hot-spot mutation increased chromosome instability in a wild-type (WT) background, suggesting that such mutants have the potential to promote tumor karyotype evolution. We also found that increasing WT MCAK/Kif2C protein levels over that of endogenous MCAK/Kif2C similarly increased chromosome instability. Thus, endogenous MCAK/Kif2C activity in normal cells is tuned to a mean level to achieve maximal suppression of chromosome instability.
Asunto(s)
Cinesinas/genética , Microscopía , Mutación/genética , Neoplasias/genética , Secuencia de Aminoácidos , Inestabilidad Cromosómica , Células HCT116 , Humanos , Cinesinas/química , Cinesinas/metabolismo , Modelos Moleculares , Mutación Puntual/genética , Dominios ProteicosRESUMEN
Cellular microtubules are rigid in comparison to other cytoskeletal elements (1,2). To facilitate cytoplasmic remodeling and timely responses to cell signaling events, microtubules depolymerize and repolymerize rapidly at their ends (3). These dynamic properties are critically important for many cellular functions, such as spindle assembly, the capture and segregation of chromosomes during cell division and cell motility. Microtubule dynamics are spatially and temporally controlled in the cell by accessory proteins. Molecular motor proteins of the kinesin superfamily that act to destabilize microtubules play important roles in this regulation (4).
Asunto(s)
Bioquímica/métodos , Regulación de la Expresión Génica de las Plantas , Cinesinas/fisiología , Microtúbulos/química , Animales , Sitios de Unión , Células CHO , División Celular , Movimiento Celular , Cricetinae , Cricetulus , Citoesqueleto/metabolismo , Técnicas In Vitro , Cinesinas/química , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/metabolismo , Transducción de Señal , Tubulina (Proteína)/químicaRESUMEN
Water-mediated hydrogen exchange (HX) processes involving the protein main chain are sensitive to structural dynamics and molecular interactions. Measuring deuterium uptake in amide bonds provides information on conformational states, structural transitions and binding events. Increasingly, deuterium levels are measured by mass spectrometry (MS) from proteolytically generated peptide fragments of large molecular systems. However, this bottom-up method has limited spectral capacity and requires a burdensome manual validation exercise, both of which restrict analysis of protein systems to generally less than 150 kDa. In this study, we present a bottom-up HX-MS(2) method that improves peptide identification rates, localizes high-quality HX data and simplifies validation. The method combines a new peptide scoring algorithm (WUF, weighted unique fragment) with data-independent acquisition of peptide fragmentation data. Scoring incorporates the validation process and emphasizes identification accuracy. The HX-MS(2) method is illustrated using data from a conformational analysis of microtubules treated with dimeric kinesin MCAK. When compared to a conventional Mascot-driven HX-MS method, HX-MS(2) produces two-fold higher α/ß-tubulin sequence depth at a peptide utilization rate of 74%. A Mascot approach delivers a utilization rate of 44%. The WUF score can be constrained by false utilization rate (FUR) calculations to return utilization values exceeding 90% without serious data loss, indicating that automated validation should be possible. The HX-MS(2) data confirm that N-terminal MCAK domains anchor kinesin force generation in kinesin-mediated depolymerization, while the C-terminal tails regulate MCAK-tubulin interactions.
Asunto(s)
Medición de Intercambio de Deuterio/métodos , Cinesinas/química , Espectrometría de Masas/métodos , Microtúbulos/química , Tubulina (Proteína)/química , Algoritmos , Animales , Bovinos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Modelos Moleculares , Conformación Proteica , Programas Informáticos , Tubulina (Proteína)/metabolismoRESUMEN
Members of the kinesin-13 subfamily use motor domains in an unconventional fashion to initiate microtubule (MT) depolymerization at MT ends, suggesting unique conformational transitions for lattice engagement, end adaptation, or both. Using hydrogen-deuterium exchange and electron microscopy, we explored conformational changes in free dimeric mitotic centromere-associated kinesin (MCAK) and when bound to a depolymerization intermediate. ATP hydrolysis relaxes the conformation of the dimer, notably in the neck and N-terminal domain. Exchanging ADP in dimeric MCAK with ATP at the MT plus end induces outward curvature in α/ß-tubulin, accompanied by a restructuring of the MCAK neck and N terminus, as it returns to a closed state. Reestablishing a closed dimer induces lateral separation of paired tubulin dimers, which may assist in depolymerization. Thus, full-length ADP-MCAK transitions from an open diffusion-competent configuration to a closed state upon plus end-mediated nucleotide exchange, which is mediated by conformational changes in the N-terminal domains of the dimer.
Asunto(s)
Cinesinas/química , Cinesinas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Moleculares , Animales , Fenómenos Biomecánicos , Línea Celular , Cromatografía Liquida , Dimerización , Hidrólisis , Cinesinas/ultraestructura , Espectrometría de Masas , Microscopía Electrónica , Microtúbulos/ultraestructura , Mariposas Nocturnas , Nefelometría y Turbidimetría , Polimerizacion , Conformación ProteicaRESUMEN
To ensure genetic integrity, replicated chromosomes must be accurately distributed to daughter cells-a process that is accomplished on the microtubule spindle. Kinesin-13 motors play an essential role in this process by performing regulated microtubule depolymerization. We set out to dissect the depolymerization mechanism of these kinesins, and in particular, the role of their conserved neck sequence. We used a monomeric kinesin-13 MCAK, consisting of the neck and motor core, which has strong depolymerizing activity. In the presence of a non-hydrolysable ATP analogue, this construct induced formation of rings around microtubules. The rings are built from tubulin protofilaments that are bent by the kinesin-13 motor engaged at the ATP-binding step of its ATPase cycle. Our data suggest that the ring-microtubule interaction is mediated by the neck and support the idea of a role for the kinesin-13 neck in depolymerization efficiency, acting by optimizing release of tubulin from microtubule ends.