Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Mol Cell ; 84(9): 1727-1741.e12, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38547866

RESUMO

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.


Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genética
2.
Elife ; 112022 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-36346217

RESUMO

Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.


Assuntos
Cílios , Ciliopatias , Humanos , Animais , Transporte Biológico , Tomografia com Microscopia Eletrônica , Homeostase
3.
Science ; 377(6605): 543-548, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35901159

RESUMO

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.


Assuntos
Chlamydomonas , Cílios , Flagelos , Chlamydomonas/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica/métodos , Dineínas/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Flagelos/metabolismo , Flagelos/ultraestrutura , Cinesinas/metabolismo , Transporte Proteico , Transdução de Sinais
4.
J Cell Sci ; 134(12)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34137439

RESUMO

The intraflagellar transport (IFT) system is a remarkable molecular machine used by cells to assemble and maintain the cilium, a long organelle extending from eukaryotic cells that gives rise to motility, sensing and signaling. IFT plays a critical role in building the cilium by shuttling structural components and signaling receptors between the ciliary base and tip. To provide effective transport, IFT-A and IFT-B adaptor protein complexes assemble into highly repetitive polymers, called IFT trains, that are powered by the motors kinesin-2 and IFT-dynein to move bidirectionally along the microtubules. This dynamic system must be precisely regulated to shuttle different cargo proteins between the ciliary tip and base. In this Cell Science at a Glance article and the accompanying poster, we discuss the current structural and mechanistic understanding of IFT trains and how they function as macromolecular machines to assemble the structure of the cilium.


Assuntos
Cílios , Dineínas , Transporte Biológico , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Transporte Proteico
5.
Methods Cell Biol ; 152: 179-195, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31326020

RESUMO

In situ cryo-electron tomography (cryo-ET) and subtomogram averaging are powerful tools, able to provide 3D structures of biological samples at sub-nanometer resolution, while preserving information about cellular context and higher-order assembly. Best results are typically achieved, when applied to highly repetitive structures, such as viruses. Other typical examples are protein complexes that decorate long stretches along ciliary microtubules at stereotypical and precise repeats, such as axonemal dyneins. For such cases, a plethora of subtomogram averaging protocols exist. In this chapter, we show how we use cryo-ET and subtomogram averaging to study the architecture of the intraflagellar transport (IFT) machinery, a more challenging target that appears only in low copy numbers per tomogram. In the IFT trains, repeating units of IFT adaptor proteins engage two oppositely directed molecular motors to quickly shuttle ciliary building blocks and other proteins to the tip of the cilium and/or back to the base. This dynamic and sporadic nature of IFT trains poses challenges for determining the localization or precise orientation of the particles to be averaged. Solutions to these problems are described in this chapter.


Assuntos
Chlamydomonas/metabolismo , Flagelos/metabolismo , Transporte Proteico/fisiologia , Dineínas do Axonema/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica/métodos , Microtúbulos/metabolismo
6.
Nat Cell Biol ; 20(11): 1250-1255, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30323187

RESUMO

Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself1. This bidirectional transport, known as intraflagellar transport (IFT)2, is driven by the anterograde motor kinesin-23 and the retrograde motor dynein-1b (dynein-2 in mammals)4,5. However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT6. Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos7, IFT is highly processive1,2,8. Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery.


Assuntos
Movimento Celular/fisiologia , Cílios/fisiologia , Microscopia Crioeletrônica/métodos , Dineínas/metabolismo , Animais , Transporte Biológico , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/ultraestrutura , Cílios/ultraestrutura , Cinesinas/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Transdução de Sinais/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...