RESUMO
Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e., all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded â¼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only â¼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by the ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.
Assuntos
Proteínas do Citoesqueleto , Cinetocoros , Microtúbulos , Mitose , Aurora Quinases/metabolismo , Proteína Quinase CDC2/metabolismo , Segregação de Cromossomos , Ciclina B1/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células HeLa , Humanos , Cinetocoros/metabolismo , Microtúbulos/metabolismo , FosforilaçãoRESUMO
The chromokinesin KIF22 (Kid, kinesin-10 family) is the primary generator of polar ejection forces, which contribute to chromosome positioning and alignment in mitotic cells. Assessment of KIF22 function requires quantitative comparison of relative polar ejection forces between experimental conditions. This is facilitated by the generation of monopolar spindles to reduce the impact of bioriented microtubule attachment at kinetochores on chromosome positions and increase the dependence of chromosome positions on chromokinesin activity. Radial profile plots measure the intensity of chromatin signal in concentric circles around the poles of monopolar cells and represent an expedient quantitative measure of relative polar ejection forces. As such, this assay can be used to measure changes in polar ejection forces resulting from chromokinesin depletion or perturbation.
Assuntos
Cromossomos , Cinesinas , Cromossomos/genética , Proteínas de Ligação a DNA/genética , Cinetocoros , Microtúbulos , Mitose , Proteínas Nucleares/genética , Fuso AcromáticoRESUMO
Chromosomal instability (CIN) is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that compromise the mitotic spindle. Thus, CIN cells may respond differently than diploid cells to treatments that target mitotic spindle regulation. Here, we test this idea by inhibiting a subset of kinesin motor proteins involved in mitotic spindle control. KIF18A is required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but is not required in near-diploid cells. Following KIF18A inhibition, CIN tumor cells exhibit mitotic delays, multipolar spindles, and increased cell death. Sensitivity to KIF18A knockdown is strongly correlated with centrosome fragmentation, which requires dynamic microtubules but does not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.
Assuntos
Instabilidade Cromossômica , Cinesinas/metabolismo , Neoplasias/genética , Neoplasias/patologia , Pontos de Checagem do Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Centrossomo/metabolismo , Humanos , Microtúbulos/metabolismo , Mitose , Modelos Biológicos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Fuso Acromático/metabolismoRESUMO
Frontotemporal dementia (FTD) is the second most common senile neurodegenerative disease. FTD is a heterogeneous disease that can be classified into several subtypes. A mutation in CHMP2B locus (CHMP2Bintron5), which encodes a component of endosomal sorting complex required for transport-III (ESCRT-III), is associated with a rare hereditary subtype of FTD linked to chromosome 3 (FTD-3). ESCRT is involved in critical cellular processes such as multivesicular body (MVB) formation during endosomalâ»lysosomal pathway and autophagy. ESCRT mutants causes diverse physiological defects primarily due to accumulation of endosomes and defective MVBs resulting in misregulation of signaling pathways. Charged multivesicular body protein 2B (CHMP2B) is important for neuronal physiology which especially rely on precise regulation of protein homeostasis due to their post-mitotic status. Drosophila has proven to be an excellent model for charaterization of mechanistic underpinning of neurodegenerative disorders including FTD. In this review, current understanding of various FTD-related mutations is discussed with a focus on Drosophila models of CHMP2Bintron5-associated FTD.