共查询到20条相似文献,搜索用时 15 毫秒
1.
Background
Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known.Methodology/Principal Findings
Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus.Conclusions/Significance
hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase. 相似文献2.
Carlos Conde Mariana Osswald João Barbosa Tatiana Moutinho‐Santos Diana Pinheiro Sofia Guimarães Helder Maiato Claudio E Sunkel 《The EMBO journal》2013,32(12):1761-1777
Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase‐promoting complex/cyclosome‐inhibitory complexes to levels that ensure long‐term SAC activity. We propose a model in which Polo controls Mps1‐dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function. 相似文献
3.
Background
Proper execution of chromosome segregation relies on tight control of attachment of chromosomes to spindle microtubules. This is monitored by the mitotic checkpoint that allows chromosome segregation only when all chromosomes are stably attached. Proper functioning of the attachment and checkpoint processes is thus important to prevent chromosomal instability. Both processes rely on the mitotic kinase Mps1.Principal Finding
We present here two cell lines in which endogenous Mps1 has been stably replaced with a mutant kinase (Mps1-as) that is specifically inhibited by bulky PP1 analogs. Mps1 inhibition in these cell lines is highly penetrant and reversible. Timed inhibition during bipolar spindle assembly shows that Mps1 is critical for attachment error-correction and confirms its role in Aurora B regulation. We furthermore show that Mps1 has multiple controls over mitotic checkpoint activity. Mps1 inhibition precludes Mad1 localization to unattached kinetochores but also accelerates mitosis. This acceleration correlates with absence of detectable mitotic checkpoint complex after Mps1 inhibition. Finally, we show that short-term inhibition of Mps1 catalytic activity is sufficient to kill cells.Conclusions/Significance
Mps1 is involved in the regulation of multiple key processes that ensure correct chromosome segregation and is a promising target for inhibition in anti-cancer strategies. We report here two cell lines that allow specific and highly penetrant inhibition of Mps1 in a reproducible manner through the use of chemical genetics. Using these cell lines we confirm previously suggested roles for Mps1 activity in mitosis, present evidence for novel functions and examine cell viability after short and prolonged Mps1 inhibition. These cell lines present the best cellular model system to date for investigations into Mps1 biology and the effects of penetrance and duration of Mps1 inhibition on cell viability. 相似文献4.
Maintenance of genomic stability during eukaryotic cell division relies on the Spindle Assembly Checkpoint (SAC), which has evolved as a surveillance mechanism that monitors kinetochore-microtubule attachment and prevents APC/C-mediated mitotic exit until all chromosomes are properly attached to the mitotic spindle. Reversible protein phosphorylation has long been accredited as a regulatory mechanism of the SAC. Nevertheless, knowledge of how several mitotic kinases act in concert within the signaling pathway to orchestrate SAC function is still emerging. In a recent study, we undertook a comprehensive dissection of the hierarchical framework controlling SAC function in Drosophila cells. We found that Polo lies at the top of the SAC pathway promoting the efficient recruitment of Mps1 to unattached kinetochores. This renders Mps1 fully active to control BubR1 phosphorylation that generates the 3F3/2 phosphoepitope at tensionless kinetochores. We have proposed that Polo is required for SAC function and that the molecular outcome of Mps1-dependent 3F3/2 formation is to promote the association of Cdc20 with BubR1 allowing proper kinetochore recruitment of Cdc20 and efficient assembly of the Mitotic Checkpoint Complex (MCC) required for a sustained SAC response. 相似文献
5.
The Drosophila gene ald encodes the fly ortholog of mps1, a conserved kinetochore-associated protein kinase required for the meiotic and mitotic spindle assembly checkpoints. Using live imaging, we demonstrate that oocytes lacking Ald/Mps1 (hereafter referred to as Ald) protein enter anaphase I immediately upon completing spindle formation, in a fashion that does not allow sufficient time for nonexchange homologs to complete their normal partitioning to opposite half spindles. This observation can explain the heightened sensitivity of nonexchange chromosomes to the meiotic effects of hypomorphic ald alleles. In one of the first studies of the female meiotic kinetochore, we show that Ald localizes to the outer edge of meiotic kinetochores after germinal vesicle breakdown, where it is often observed to be extended well away from the chromosomes. Ald also localizes to numerous filaments throughout the oocyte. These filaments, which are not observed in mitotic cells, also contain the outer kinetochore protein kinase Polo, but not the inner kinetochore proteins Incenp or Aurora-B. These filaments polymerize during early germinal vesicle breakdown, perhaps as a means of storing excess outer kinetochore kinases during early embryonic development. 相似文献
6.
Background
When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response.Results
In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization.Significance
We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes. 相似文献7.
8.
Background
Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the apical membrane domain and adherens junction during Drosophila photoreceptor morphogenesis. It has recently been found that stable microtubules in developing Drosophila photoreceptors were linked to Crb localization. Coordinated interactions between microtubule and actin cytoskeletons are involved in many polarized cellular processes. Since Spectraplakin is able to bind both microtubule and actin cytoskeletons, the role of Spectraplakin was analyzed in the regulations of apical Crb domain in developing Drosophila photoreceptors.Methodology/Principal Findings
The localization pattern of Spectraplakin in developing pupal photoreceptors showed a unique intracellular distribution. Spectraplakin localized at rhabdomere terminal web which is at the basal side of the apical Crb or rhabdomere, and in between the adherens junctions. The spectraplakin mutant photoreceptors showed dramatic mislocalizations of Crb, adherens junctions, and the stable microtubules. This role of Spectraplakin in Crb and adherens junction regulation was further supported by spectraplakin''s gain-of-function phenotype. Spectraplakin overexpression in photoreceptors caused a cell polarity defect including dramatic mislocalization of Crb, adherens junctions and the stable microtubules in the developing photoreceptors. Furthermore, a strong genetic interaction between spectraplakin and crb was found using a genetic modifier test.Conclusions/Significance
In summary, we found a unique localization of Spectraplakin in photoreceptors, and identified the role of spectraplakin in the regulation of the apical Crb domain and adherens junctions through genetic mutational analysis. Our data suggest that Spectraplakin, an actin-microtubule cross-linker, is essential in the apical and adherens junction controls during the photoreceptors morphogenesis. 相似文献9.
Nitin Udpa Roy Ronen Dan Zhou Junbin Liang Tsering Stobdan Otto Appenzeller Ye Yin Yuanping Du Lixia Guo Rui Cao Yu Wang Xin Jin Chen Huang Wenlong Jia Dandan Cao Guangwu Guo Victoria E Claydon Roger Hainsworth Jorge L Gamboa Mehila Zibenigus Guta Zenebe Jin Xue Siqi Liu Kelly A Frazer Yingrui Li Vineet Bafna Gabriel G Haddad 《Genome biology》2014,15(2):R36
Background
Although it has long been proposed that genetic factors contribute to adaptation to high altitude, such factors remain largely unverified. Recent advances in high-throughput sequencing have made it feasible to analyze genome-wide patterns of genetic variation in human populations. Since traditionally such studies surveyed only a small fraction of the genome, interpretation of the results was limited.Results
We report here the results of the first whole genome resequencing-based analysis identifying genes that likely modulate high altitude adaptation in native Ethiopians residing at 3,500 m above sea level on Bale Plateau or Chennek field in Ethiopia. Using cross-population tests of selection, we identify regions with a significant loss of diversity, indicative of a selective sweep. We focus on a 208 kbp gene-rich region on chromosome 19, which is significant in both of the Ethiopian subpopulations sampled. This region contains eight protein-coding genes and spans 135 SNPs. To elucidate its potential role in hypoxia tolerance, we experimentally tested whether individual genes from the region affect hypoxia tolerance in Drosophila. Three genes significantly impact survival rates in low oxygen: cic, an ortholog of human CIC, Hsl, an ortholog of human LIPE, and Paf-AHα, an ortholog of human PAFAH1B3.Conclusions
Our study reveals evolutionarily conserved genes that modulate hypoxia tolerance. In addition, we show that many of our results would likely be unattainable using data from exome sequencing or microarray studies. This highlights the importance of whole genome sequencing for investigating adaptation by natural selection. 相似文献10.
Background
The fundamental role of the light receptor rhodopsin in visual function and photoreceptor cell development has been widely studied. Proper trafficking of rhodopsin to the photoreceptor membrane is of great importance. In human, mutations in rhodopsin involving its intracellular mislocalization, are the most frequent cause of autosomal dominant Retinitis Pigmentosa, a degenerative retinal pathology characterized by progressive blindness. Drosophila is widely used as an animal model in visual and retinal degeneration research. So far, little is known about the requirements for proper rhodopsin targeting in Drosophila.Methodology/Principal Findings
Different truncated fly-rhodopsin Rh1 variants were expressed in the eyes of Drosophila and their localization was analyzed in vivo or by immunofluorescence. A mutant lacking the last 23 amino acids was found to properly localize in the rhabdomeres, the light-sensing organelle of the photoreceptor cells. This constitutes a major difference to trafficking in vertebrates, which involves a conserved QVxPA motif at the very C-terminus. Further truncations of Rh1 indicated that proper localization requires the last amino acid residues of a region called helix 8 following directly the last transmembrane domain. Interestingly, the very C-terminus of invertebrate visual rhodopsins is extremely variable but helix 8 shows conserved amino acid residues that are not conserved in vertebrate homologs.Conclusions/Significance
Despite impressive similarities in the folding and photoactivation of vertebrate and invertebrate visual rhodopsins, a striking difference exists between mammalian and fly rhodopsins in their requirements for proper targeting. Most importantly, the distal part of helix 8 plays a central role in invertebrates. Since the last amino acid residues of helix 8 are dispensable for rhodopsin folding and function, we propose that this domain participates in the recognition of targeting factors involved in transport to the rhabdomeres. 相似文献11.
Background
At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.Results
In Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.Conclusion
This data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton. 相似文献12.
13.
14.
Kaiyun Chen Elena O. Gracheva Szi-Chieh Yu Qi Sheng Janet Richmond David E. Featherstone 《PloS one》2010,5(6)
Background
Neurexin is a synaptic cell adhesion protein critical for synapse formation and function. Mutations in neurexin and neurexin-interacting proteins have been implicated in several neurological diseases. Previous studies have described Drosophila neurexin mutant phenotypes in third instar larvae and adults. However, the expression and function of Drosophila neurexin early in synapse development, when neurexin function is thought to be most important, has not been described.Methodology/Principal Findings
We use a variety of techniques, including immunohistochemistry, electron microscopy, in situ hybridization, and electrophysiology, to characterize neurexin expression and phenotypes in embryonic Drosophila neuromuscular junctions (NMJs). Our results surprisingly suggest that neurexin in embryos is present both pre and postsynaptically. Presynaptic neurexin promotes presynaptic active zone formation and neurotransmitter release, but along with postsynaptic neurexin, also suppresses formation of ectopic glutamate receptor clusters. Interestingly, we find that loss of neurexin only affects receptors containing the subunit GluRIIA.Conclusions/Significance
Our study extends previous results and provides important detail regarding the role of neurexin in Drosophila glutamate receptor abundance. The possibility that neurexin is present postsynaptically raises new hypotheses regarding neurexin function in synapses, and our results provide new insights into the role of neurexin in synapse development. 相似文献15.
Peter Geuking Rajesh Narasimamurthy Bruno Lemaitre Konrad Basler Fran?ois Leulier 《PloS one》2009,4(11)
Background
The JNK pathway is a mitogen-activated protein (MAP) kinase pathway involved in the regulation of numerous physiological processes during development and in response to environmental stress. JNK activity is controlled by two MAPK kinases (MAPKK), Mkk4 and Mkk7. Mkk7 plays a prominent role upon Tumor Necrosis Factor (TNF) stimulation. Eiger, the unique TNF-superfamily ligand in Drosophila, potently activates JNK signaling through the activation of the MAPKKK Tak1.Methodology/Principal Findings
In a dominant suppressor screen for new components of the Eiger/JNK-pathway in Drosophila, we have identified an allelic series of the Mkk4 gene. Our genetic and biochemical results demonstrate that Mkk4 is dispensable for normal development and host resistance to systemic bacterial infection but plays a non-redundant role as a MAPKK acting in parallel to Hemipterous/Mkk7 in dTAK1-mediated JNK activation upon Eiger and Imd pathway activation.Conclusions/Significance
In contrast to mammals, it seems that in Drosophila both MAPKKs, Hep/Mkk7 and Mkk4, are required to induce JNK upon TNF or pro-inflammatory stimulation. 相似文献16.
17.
Anne J 《PloS one》2010,5(12):e14378
Background
In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud) which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization.Methodology/Principal Findings
In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity.Conclusions/Significance
I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud. 相似文献18.
Background
Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.Methodology/Principal Findings
Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.Conclusions/Significance
In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development. 相似文献19.
Po-Hung Kuo Te-Hsuen Tzeng Yi-Chun Huang Yu-Hao Chen Yi-Chung Chang Yi-Lwun Ho June-Tai Wu Hsiu-Hsian Lee Po-Jung Lai Kwei-Yan Liu Ya-Chen Cheng Shey-Shi Lu 《PloS one》2014,9(9)
Background
Drosophila heart tube is a feasible model for cardiac physiological research. However, obtaining Drosophila electrocardiograms (ECGs) is difficult, due to the weak signals and limited contact area to apply electrodes. This paper presents a non-invasive Gallium-Indium (GaIn) based recording system for Drosophila ECG measurement, providing the heart rate and heartbeat features to be observed. This novel, high-signal-quality system prolongs the recording time of insect ECGs, and provides a feasible platform for research on the molecular mechanisms involved in cardiovascular diseases.Methods
In this study, two types of electrode, tungsten needle probes and GaIn electrodes, were used respectively to noiselessly conduct invasive and noninvasive ECG recordings of Drosophila. To further analyze electrode properties, circuit models were established and simulated. By using electromagnetic shielded heart signal acquiring system, consisted of analog amplification and digital filtering, the ECG signals of three phenotypes that have different heart functions were recorded without dissection.Results and Discussion
The ECG waveforms of different phenotypes of Drosophila recorded invasively and repeatedly with n value (n>5) performed obvious difference in heart rate. In long period ECG recordings, non-invasive method implemented by GaIn electrodes acts relatively stable in both amplitude and period. To analyze GaIn electrode, the correctness of GaIn electrode model established by this paper was validated, presenting accuracy, stability, and reliability.Conclusions
Noninvasive ECG recording by GaIn electrodes was presented for recording Drosophila pupae ECG signals within a limited contact area and signal strength. Thus, the observation of ECG changes in normal and SERCA-depleted Drosophila over an extended period is feasible. This method prolongs insect survival time while conserving major ECG features, and provides a platform for electrophysiological signal research on the molecular mechanism involved in cardiac arrhythmia, as well as research related to drug screening and development. 相似文献20.
R. Scott Cornman 《PloS one》2010,5(9)