Spindle Checkpoint Protein Dynamics at Kinetochores in Living Cells

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores

Spindle Checkpoint Protein Dynamics at Kinetochores in Living Cells

BACKGROUND: To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells. RESULTS: Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components. CONCLUSIONS: Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores

hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells

Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends

Taxol-Stabilized Microtubules Can Position the Cytokinetic Furrow in Mammalian Cells

How microtubules act to position the plane of cell division during cytokinesis is a topic of much debate. Recently, we showed that a subpopulation of stable microtubules extends past chromosomes and interacts with the cell cortex at the site of furrowing, suggesting that these stabilized microtubules may stimulate contractility. To test the hypothesis that stable microtubules can position furrows, we used taxol to rapidly suppress microtubule dynamics during various stages of mitosis in PtK1 cells. Cells with stabilized prometaphase or metaphase microtubule arrays were able to initiate furrowing when induced into anaphase by inhibition of the spindle checkpoint. In these cells, few microtubules contacted the cortex. Furrows formed later than usual, were often aberrant, and did not progress to completion. Images showed that furrowing correlated with the presence of one or a few stable spindle microtubule plus ends at the cortex. Actin, myosin II, and anillin were all concentrated in these furrows, demonstrating that components of the contractile ring can be localized by stable microtubules. Inner centromere protein (INCENP) was not found in these ingressions, confirming that INCENP is dispensable for furrow positioning. Taxol-stabilization of the numerous microtubule-cortex interactions after anaphase onset delayed furrow initiation but did not perturb furrow positioning. We conclude that taxol-stabilized microtubules can act to position the furrow and that loss of microtubule dynamics delays the timing of furrow onset and prevents completion. We discuss our findings relative to models for cleavage stimulation

Merotelic kinetochore orientation occurs frequently during early mitosis in mammalian tissue cells and error correction is achieved by two different mechanisms

Merotelic kinetochore orientation is an error that occurs when a single kinetochore becomes attached to microtubules from two spindle poles rather than just to one pole. We obtained the first evidence that merotelic kinetochore orientation occurs very frequently during early mitosis in mammalian tissue cells and that two different correction mechanisms are critical for accurate chromosome segregation in cells possessing bipolar spindles and unperturbed chromosomes. Our data show that about 30% of prometaphase PtK1 cells possess one or more merotelically oriented kinetochores. This frequency is increased to over 90% in cells recovering from a nocodazole-induced mitotic block. A delay in establishing spindle bipolarity is responsible for the high frequency of merotelic orientations seen in cells recovering from nocodazole, but not in untreated cells. The frequency of anaphase cells with merotelically oriented lagging chromosomes is 1% in untreated cells and 18% in cells recovering from nocodazole. Prolonging metaphase by 2 hours reduced the frequency of anaphase cells with lagging chromosomes both for untreated and for nocodazole-treated cells. Surprisingly, anaphase lagging chromosomes represented a very small fraction of merotelic kinetochore orientations present in late metaphase. Our data indicate that two correction mechanisms operate to prevent chromosome missegregation due to merotelic kinetochore orientation. The first, a pre-anaphase correction mechanism increases the ratio of kinetochore microtubules attached to the correct versus incorrect pole and might eventually result in kinetochore reorientation before anaphase onset. The increase in microtubule ratio to opposite poles is the groundwork for a second mechanism, active in anaphase, that promotes the segregation of merotelically oriented chromosomes to the correct pole

CENP-C recruits M18BP1 to centromeres to promote CENP-A chromatin assembly

CENP-C provides a link between existing CENP-A chromatin and the proteins required for new CENP-A nucleosome assembly

Both tails and the centromere targeting domain of CENP-A are required for centromere establishment

The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A—is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity

The cystic fibrosis microbiome in an ecological perspective and its impact in antibiotic therapy

The recent focus on the cystic fibrosis (CF) complex microbiome has led to the recognition that the microbes can interact between them and with the host immune system, affecting the disease progression and treatment routes. Although the main focus remains on the interactions between traditional pathogens, growing evidence supports the contribution and the role of emergent species. Understanding the mechanisms and the biological effects involved in polymicrobial interactions may be the key to improve effective therapies and also to define new strategies for disease control. This review focuses on the interactions between microbe-microbe and host-microbe, from an ecological point of view, discussing their impact on CF disease progression. There are increasing indications that these interactions impact the success of antimicrobial therapy. Consequently, a new approach where therapy is personalized to patients by taking into account their individual CF microbiome is suggested.Portuguese Foundation for Science and Technology (FCT), the strategic funding of UID/BIO/04469/2013-CEB and UID/EQU/00511/2013-LEPABE units. This study was also supported by FCT and the European Community fund FEDER, through Program COMPETE, under the scope of the Projects “DNA mimics” PIC/IC/82815/2007, RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), “BioHealth—Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027 and NORTE-07-0124-FEDER-000025—RL2_ Environment and Health, co-funded by the Programa Operacional Regional do Norte (ON.2 – O Novo Norte), QREN, FEDER. The authors also acknowledge the grant of Susana P. Lopes (SFRH/BPD/95616/2013) and of the COST-Action TD1004: Theragnostics for imaging and therapy

Angular Mapping of Protein Structure Using Nonlinear Optical Measurements

Proteins are inherently dynamic, flexible molecules that execute precise conformational changes to perform their functions, but existing techniques to directly measure relevant structural changes in solution at room temperature remain limited. Here, we demonstrate a structural technique using second-harmonic generation and two-photon fluorescence under single-laser excitation to map both the mean angular orientation and the distribution width of a probe at various sites throughout the protein with high sensitivity. Our work resolves distinct dihydrofolate reductase (DHFR) ligand-protein conformations, allows interrogation of regions unresolvable by other techniques, and reveals structural differences between DHFR and a point mutant (DHFR-G121V). The technique, angular mapping of protein structure, enables direct and rapid determination of previously unseen aspects of protein structure in a benchtop optical system