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1.
EMBO Rep ; 24(8): e56100, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37291955

RESUMO

GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.


Assuntos
Mitose , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fosforilação , Cromossomos/metabolismo
2.
Nat Commun ; 12(1): 6577, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34772942

RESUMO

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.


Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Fosfatidilinositóis/metabolismo , Pinocitose/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Endocitose/fisiologia , Humanos , Fosfatos de Fosfatidilinositol , Salmonella , Transcriptoma , Proteínas de Transporte Vesicular/genética
3.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34109410

RESUMO

Macropinocytosis allows cells to take up extracellular material in a non-selective manner into large vesicles called macropinosomes. After internalization, macropinosomes acquire phosphatidylinositol 3-phosphate (PtdIns3P) on their limiting membrane as they mature into endosomal-like vesicles. The molecular mechanisms that underlie recycling of membranes and transmembrane proteins from these macropinosomes still need to be defined. Here, we report that JIP4 (officially known as SPAG9), a protein previously described to bind to microtubule motors, is recruited to tubulating subdomains on macropinosomes by the PtdIns3P-binding protein Phafin2 (officially known as PLEKHF2). These JIP4-positive tubulating subdomains on macropinosomes contain F-actin, the retromer recycling complex and the retromer cargo VAMP3. Disruption of the JIP4-Phafin2 interaction, deletion of Phafin2 or inhibition of PtdIns3P production by VPS34 impairs JIP4 recruitment to macropinosomes. Whereas knockout of JIP4 suppresses tubulation, its overexpression enhances tubulation from macropinosomes. JIP4-knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Fosfatidilinositóis , Proteínas de Transporte Vesicular , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Humanos , Fosfatidilinositóis/metabolismo , Pinocitose , Ligação Proteica , Transporte Proteico , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
4.
Cell Rep ; 33(9): 108469, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33264625

RESUMO

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.


Assuntos
Cromatina/efeitos dos fármacos , Proteínas Cromossômicas não Histona/uso terapêutico , RNA Polimerase II/metabolismo , Proteínas Cromossômicas não Histona/farmacologia , Humanos , Transfecção
5.
Nat Cell Biol ; 22(7): 856-867, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601372

RESUMO

The ESCRT-III membrane fission machinery maintains the integrity of the nuclear envelope. Although primary nuclei resealing takes minutes, micronuclear envelope ruptures seem to be irreversible. Instead, micronuclear ruptures result in catastrophic membrane collapse and are associated with chromosome fragmentation and chromothripsis, complex chromosome rearrangements thought to be a major driving force in cancer development. Here we use a combination of live microscopy and electron tomography, as well as computer simulations, to uncover the mechanism underlying micronuclear collapse. We show that, due to their small size, micronuclei inherently lack the capacity of primary nuclei to restrict the accumulation of CHMP7-LEMD2, a compartmentalization sensor that detects loss of nuclear integrity. This causes unrestrained ESCRT-III accumulation, which drives extensive membrane deformation, DNA damage and chromosome fragmentation. Thus, the nuclear-integrity surveillance machinery is a double-edged sword, as its sensitivity ensures rapid repair at primary nuclei while causing unrestrained activity at ruptured micronuclei, with catastrophic consequences for genome stability.


Assuntos
Núcleo Celular/patologia , Cromatina/metabolismo , Aberrações Cromossômicas , Dano ao DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Instabilidade Genômica , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos
6.
Cell Cycle ; 18(17): 2006-2025, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31306061

RESUMO

A single inner centromere protein (INCENP) found throughout eukaryotes modulates Aurora B kinase activity and chromosomal passenger complex (CPC) localization, which is essential for timely mitotic progression. It has been proposed that INCENP might act as a rheostat to regulate Aurora B activity through mitosis, with successively higher activity threshold levels for chromosome alignment, the spindle checkpoint, anaphase spindle transfer and finally spindle elongation and cytokinesis. It remains mechanistically unclear how this would be achieved. Here, we reveal that the urochordate, Oikopleura dioica, possesses two INCENP paralogs, which display distinct localizations and subfunctionalization in order to complete M-phase. INCENPa was localized on chromosome arms and centromeres by prometaphase, and modulated Aurora B activity to mediate H3S10/S28 phosphorylation, chromosome condensation, spindle assembly and transfer of the CPC to the central spindle. Polo-like kinase (Plk1) recruitment to CDK1 phosphorylated INCENPa was crucial for INCENPa-Aurora B enrichment on centromeres. The second paralog, INCENPb was enriched on centromeres from prometaphase, and relocated to the central spindle at anaphase onset. In the absence of INCENPa, meiotic spindles failed to form, and homologous chromosomes did not segregate. INCENPb was not required for early to mid M-phase events but became essential for the activity and localization of Aurora B on the central spindle and midbody during cytokinesis in order to allow abscission to occur. Together, our results demonstrate that INCENP paralog switching on centromeres modulates Aurora B kinase localization, thus chronologically regulating CPC functions during fast embryonic divisions in the urochordate O. dioica. Abbreviations: CCAN: constitutive centromere-associated network; CENPs: centromere proteins; cmRNA: capped messenger RNA; CPC: chromosomal passenger complex; INCENP: inner centromere protein; Plk1: polo-like kinase 1; PP1: protein phosphatase 1; PP2A: protein phosphatase 2A; SAC: spindle assembly checkpoint; SAH: single α-helix domain.


Assuntos
Aurora Quinase B/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos/genética , Mitose/genética , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Citocinese/genética , Humanos , Cinetocoros/metabolismo , Fosforilação/genética , Plâncton/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Fuso Acromático/genética , Quinase 1 Polo-Like
7.
Nat Commun ; 10(1): 2850, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253801

RESUMO

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Membrana Celular , Exocitose/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Microtúbulos , Fosfatos de Fosfatidilinositol/fisiologia , Transporte Proteico , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
8.
ACS Nano ; 12(8): 7791-7802, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30001106

RESUMO

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications.


Assuntos
Núcleo Celular/metabolismo , Nanopartículas/metabolismo , Membrana Nuclear/metabolismo , Núcleo Celular/química , Sobrevivência Celular , Células HeLa , Humanos , Nanopartículas/química , Membrana Nuclear/química , Imagem Óptica , Processos Fotoquímicos , Células Tumorais Cultivadas , Volatilização
9.
Trends Biochem Sci ; 42(1): 42-56, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27669649

RESUMO

The endosomal sorting complex required for transport (ESCRT) machinery is an assembly of protein subcomplexes (ESCRT I-III) that cooperate with the ATPase VPS4 to mediate scission of membrane necks from the inside. The ESCRT machinery has evolved as a multipurpose toolbox for mediating receptor sorting, membrane remodeling, and membrane scission, with ESCRT-III as the major membrane-remodeling component. Cellular membrane scission processes mediated by ESCRT-III include biogenesis of multivesicular endosomes, budding of enveloped viruses, cytokinetic abscission, neuron pruning, plasma membrane wound repair, nuclear pore quality control, nuclear envelope reformation, and nuclear envelope repair. We describe here the involvement of the ESCRT machinery in these processes and review current models for how ESCRT-III-containing multimeric filaments serve to mediate membrane remodeling and scission.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Adenosina Trifosfatases/metabolismo , Humanos
10.
Trends Cell Biol ; 27(1): 1-11, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27810282

RESUMO

Cytokinesis is the final stage of cell division and is concluded by abscission of the intercellular bridge to physically separate the daughter cells. Timing of cytokinetic abscission is monitored by a molecular machinery termed the abscission checkpoint. This machinery delays abscission in cells with persistent chromatin in the intercellular bridge. Recent work has also uncovered its response to high membrane tension, nuclear pore defects, and DNA replication stress. Although it is known that the abscission checkpoint depends on persistent activity of the Aurora B protein kinase, we have only recently begun to understand its molecular basis. We propose here a molecular framework for abscission checkpoint signaling and we discuss outstanding questions relating to its function and physiological relevance.


Assuntos
Pontos de Checagem do Ciclo Celular , Animais , Citocinese , Replicação do DNA , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Modelos Biológicos , Transdução de Sinais
11.
Curr Opin Cell Biol ; 41: 1-8, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27031044

RESUMO

The endosomal sorting complex required for transport (ESCRT), originally identified for its role in endosomal protein sorting and biogenesis of multivesicular endosomes (MVEs), has proven to be a versatile machinery for involution and scission of narrow membrane invaginations filled with cytosol. Budding of enveloped viruses and cytokinetic abscission were early described functions for the ESCRT machinery, and recently a number of new ESCRT functions have emerged. These include cytokinetic abscission checkpoint control, plasma membrane repair, exovesicle release, quality control of nuclear pore complexes, neuron pruning, and sealing of the newly formed nuclear envelope. Here we review these novel ESCRT mechanisms and discuss similarities and differences between the various ESCRT-dependent activities.


Assuntos
Células/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Animais , Citocinese , Endossomos/metabolismo , Humanos , Corpos Multivesiculares/metabolismo , Plasticidade Neuronal , Membrana Nuclear/metabolismo
12.
Curr Opin Cell Biol ; 40: 90-97, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27016712

RESUMO

The nuclear envelope (NE) ensures nucleo-cytoplasmic compartmentalization, with trafficking of macromolecules across this double membrane controlled by embedded nuclear pore complexes (NPCs). The NE and associated proteins are dismantled during open mitosis and reestablishment of this barrier during mitotic exit requires dynamic remodeling of endoplasmic reticulum (ER) membranes and coordination with NPC reformation, with NPC deposition continuing during subsequent interphase. In this review, we discuss recent progress in our understanding of NE reformation and nuclear pore complex generation, with special focus on work implicating the endosomal sorting complex required for transport (ESCRT) membrane remodeling machinery in these events.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Membrana Nuclear/metabolismo , Animais , Divisão Celular , Cromossomos/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Mitose , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico
13.
J Cell Biol ; 212(5): 499-513, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26929449

RESUMO

Cytokinetic abscission, the final stage of cell division where the two daughter cells are separated, is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. The ESCRT-III subunit CHMP4B is a key effector in abscission, whereas its paralogue, CHMP4C, is a component in the abscission checkpoint that delays abscission until chromatin is cleared from the intercellular bridge. How recruitment of these components is mediated during cytokinesis remains poorly understood, although the ESCRT-binding protein ALIX has been implicated. Here, we show that ESCRT-II and the ESCRT-II-binding ESCRT-III subunit CHMP6 cooperate with ESCRT-I to recruit CHMP4B, with ALIX providing a parallel recruitment arm. In contrast to CHMP4B, we find that recruitment of CHMP4C relies predominantly on ALIX. Accordingly, ALIX depletion leads to furrow regression in cells with chromosome bridges, a phenotype associated with abscission checkpoint signaling failure. Collectively, our work reveals a two-pronged recruitment of ESCRT-III to the cytokinetic bridge and implicates ALIX in abscission checkpoint signaling.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Células HeLa , Humanos , Células Tumorais Cultivadas
14.
Nature ; 522(7555): 231-5, 2015 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-26040712

RESUMO

At the onset of metazoan cell division the nuclear envelope breaks down to enable capture of chromosomes by the microtubule-containing spindle apparatus. During anaphase, when chromosomes have separated, the nuclear envelope is reassembled around the forming daughter nuclei. How the nuclear envelope is sealed, and how this is coordinated with spindle disassembly, is largely unknown. Here we show that endosomal sorting complex required for transport (ESCRT)-III, previously found to promote membrane constriction and sealing during receptor sorting, virus budding, cytokinesis and plasma membrane repair, is transiently recruited to the reassembling nuclear envelope during late anaphase. ESCRT-III and its regulatory AAA (ATPase associated with diverse cellular activities) ATPase VPS4 are specifically recruited by the ESCRT-III-like protein CHMP7 to sites where the reforming nuclear envelope engulfs spindle microtubules. Subsequent association of another ESCRT-III-like protein, IST1, directly recruits the AAA ATPase spastin to sever microtubules. Disrupting spastin function impairs spindle disassembly and results in extended localization of ESCRT-III at the nuclear envelope. Interference with ESCRT-III functions in anaphase is accompanied by delayed microtubule disassembly, compromised nuclear integrity and the appearance of DNA damage foci in subsequent interphase. We propose that ESCRT-III, VPS4 and spastin cooperate to coordinate nuclear envelope sealing and spindle disassembly at nuclear envelope-microtubule intersection sites during mitotic exit to ensure nuclear integrity and genome safeguarding, with a striking mechanistic parallel to cytokinetic abscission.


Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fusão de Membrana , Membrana Nuclear/metabolismo , Fuso Acromático/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Anáfase , Pontos de Checagem do Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Humanos , Microtúbulos/metabolismo , Espastina , ATPases Vacuolares Próton-Translocadoras/metabolismo
15.
Cell Cycle ; 14(13): 2129-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25928155

RESUMO

The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbß splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbß-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.


Assuntos
Núcleo Celular/metabolismo , Proliferação de Células/fisiologia , Ciclina D/biossíntese , Variação Genética/fisiologia , Células Germinativas/metabolismo , Células Gigantes/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/genética , Cordados não Vertebrados , Ciclina D/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular
16.
Cell Cycle ; 14(6): 880-93, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25714331

RESUMO

Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program.


Assuntos
Proteína Quinase CDC2/metabolismo , Cordados/metabolismo , Meiose , Oogênese , Homologia de Sequência de Aminoácidos , Animais , Técnicas de Silenciamento de Genes , Centro Organizador dos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Membrana Nuclear/metabolismo , Fenótipo , RNA de Cadeia Dupla/metabolismo
17.
Mol Biol Evol ; 32(3): 585-99, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25525214

RESUMO

Polycistronic mRNAs transcribed from operons are resolved via the trans-splicing of a spliced-leader (SL) RNA. Trans-splicing also occurs at monocistronic transcripts. The phlyogenetically sporadic appearance of trans-splicing and operons has made the driving force(s) for their evolution in metazoans unclear. Previous work has proposed that germline expression drives operon organization in Caenorhabditis elegans, and a recent hypothesis proposes that operons provide an evolutionary advantage via the conservation of transcriptional machinery during recovery from growth arrested states. Using a modified cap analysis of gene expression protocol we mapped sites of SL trans-splicing genome-wide in the marine chordate Oikopleura dioica. Tiled microarrays revealed the expression dynamics of trans-spliced genes across development and during recovery from growth arrest. Operons did not facilitate recovery from growth arrest in O. dioica. Instead, we found that trans-spliced transcripts were predominantly maternal. We then analyzed data from C. elegans and Ciona intestinalis and found that an enrichment of trans-splicing and operon gene expression in maternal mRNA is shared between all three species, suggesting that this may be a driving force for operon evolution in metazoans. Furthermore, we found that the majority of known terminal oligopyrimidine (TOP) mRNAs are trans-spliced in O. dioica and that the SL contains a TOP-like motif. This suggests that the SL in O. dioica confers nutrient-dependent translational control to trans-spliced mRNAs via the TOR-signaling pathway. We hypothesize that SL-trans-splicing provides an evolutionary advantage in species that depend on translational control for regulating early embryogenesis, growth and oocyte production in response to nutrient levels.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Óperon/genética , Trans-Splicing/genética , Animais , Caenorhabditis elegans/genética , Ciona intestinalis/genética , Feminino , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Urocordados/genética
18.
Nat Cell Biol ; 16(6): 550-60, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24814515

RESUMO

During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring.


Assuntos
Aurora Quinase B/metabolismo , Pontos de Checagem do Ciclo Celular , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Oncogênicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Aurora Quinase B/genética , Cromatina/metabolismo , Aberrações Cromossômicas , Segregação de Cromossomos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Oncogênicas/genética , Fosforilação , Transporte Proteico , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , ATPases Vacuolares Próton-Translocadoras/genética
19.
PLoS One ; 9(4): e93787, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24695788

RESUMO

It is proposed that the ageing process is linked to signaling from the germline such that the rate of ageing can be adjusted to the state of the reproductive system, allowing these two processes to co-evolve. Mechanistic insight into this link has been primarily derived from iteroparous reproductive models, the nematode C. elegans, and the arthropod Drosophila. Here, we examined to what extent these mechanisms are evolutionarily conserved in a semelparous chordate, Oikopleura dioica, where we identify a developmental growth arrest (GA) in response to crowded, diet-restricted conditions, which can extend its lifespan at least three-fold. Under nutritional stress, the iteroparative models sacrifice germ cells that have entered meiosis, while maintaining a reduced pool of active germline stem cells (GSCs). In contrast, O. dioica only entered GA prior to meiotic entry. Stress conditions encountered after this point led to maturation in a normal time frame but with reduced reproductive output. During GA, TOR signaling was inhibited, whereas MAPK, ERK1/2 and p38 pathways were activated, and under such conditions, activation of these pathways was shown to be critical for survival. Direct inhibition of TOR signaling alone was sufficient to prevent meiotic entry and germline differentiation. This inhibition activated the p38 pathway, but did not activate the ERK1/2 pathway. Thus, the link between reproductive status and lifespan extension in response to nutrient-limited conditions is interpreted in a significantly different manner in these iteroparative versus semelparous models. In the latter case, meiotic entry is a definitive signal that lifespan extension can no longer occur, whereas in the former, meiotic entry is not a unique chronological event, and can be largely erased during lifespan extension in response to nutrient stress, and reactivated from a pool of maintained GSCs when conditions improve.


Assuntos
Envelhecimento/fisiologia , Longevidade/fisiologia , Meiose/fisiologia , Urocordados/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Urocordados/efeitos dos fármacos
20.
Arch Biochem Biophys ; 545: 83-91, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24440309

RESUMO

The multivesicular body (MVB) sorting pathway is a mechanism for delivering transmembrane proteins into the lumen of the lysosome for degradation. ESCRT-III is the final complex in the pathway that assembles on endosomes and executes membrane scission of intraluminal vesicles. In addition, proteins of this complex are involved in other topologically similar processes such as cytokinesis, virus egress and autophagy. Here we show that protein kinase CK2α is involved in the phosphorylation of the ESCRT-III subunits CHMP3 and CHMP2B, as well as of VPS4B/SKD1, an ATPase that mediates ESCRT-III disassembly. This phosphorylation is observed both in vitro and in cells. While we do not observe recruitment of CK2α to endosomes, we demonstrate the localization of CK2α to midbodies during cytokinesis. Phosphomimetic and non-phosphorylatable mutants of ESCRT-III proteins can still bind endosomes and localize to midbodies, indicating that CK2α does not regulate ESCRT-III localization. Finally, we analyzed two cellular functions where CHMP3, CHMP2B and VPS4 are known to be involved, epidermal growth factor degradation and cytokinetic abscission. We demonstrate that the former is impaired by CK2α downregulation whereas the latter is not affected. Taken together, our results indicate that CK2α regulates the function of ESCRT-III proteins in MVB sorting.


Assuntos
Adenosina Trifosfatases/metabolismo , Caseína Quinase II/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/análise , Caseína Quinase II/análise , Caseína Quinase II/genética , Regulação para Baixo , Complexos Endossomais de Distribuição Requeridos para Transporte/análise , Endossomos/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilação
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