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1.
Cell ; 180(1): 176-187.e19, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31923394

RESUMO

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.


Assuntos
Di-Inos/metabolismo , Ácidos Graxos/biossíntese , Álcoois Graxos/metabolismo , Solanum lycopersicum/genética , Resistência à Doença/genética , Di-Inos/química , Ácidos Graxos/metabolismo , Álcoois Graxos/química , Regulação da Expressão Gênica de Plantas/genética , Metabolômica , Família Multigênica/genética , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Fisiológico/genética
2.
Proc Natl Acad Sci U S A ; 110(10): 3925-30, 2013 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-23431147

RESUMO

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Cílios/fisiologia , Transporte Biológico Ativo , Chlamydomonas reinhardtii/genética , Flagelos/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Modelos Biológicos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
3.
iScience ; 24(4): 102354, 2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33898946

RESUMO

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry.

4.
Philos Trans R Soc Lond B Biol Sci ; 375(1792): 20190159, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-31884913

RESUMO

Cilia and flagella are ideal model organelles in which to study the general question of organelle size control. Flagellar microtubules are steady-state structures whose size is set by the balance of assembly and disassembly. Assembly requires intraflagellar transport (IFT), and measurements of IFT have shown that the rate of entry of IFT particles into the flagellum is a decreasing function of length. It has been proposed that this length dependence of IFT may be the basis for flagellar length control. Here, we test this idea by showing that three different long-flagella mutations in Chlamydomonas all cause increased IFT injection, thus confirming that IFT can influence length control. However, quantitative comparisons with mathematical models suggest that the increase in injection is not sufficient to explain the full increase in length seen in these mutants; hence, some other mechanism may be at work. One alternative mechanism that has been proposed is length-regulated binding of tubulin to the IFT particles. However, we find that the apparent length dependence of tubulin loading that has previously been reported may actually reflect length-dependent organization of IFT trains. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.


Assuntos
Chlamydomonas/fisiologia , Flagelos/fisiologia , Chlamydomonas/genética , Flagelos/genética , Microtúbulos/metabolismo , Modelos Teóricos , Mutação/fisiologia , Transporte Proteico , Tubulina (Proteína)/metabolismo
5.
Int Rev Cytol ; 260: 175-212, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17482906

RESUMO

A fundamental unsolved question in cell biology is how the cell controls the size of its organelles. Cilia and flagella are an ideal test case to study the mechanism of organelle size control, because their size is easily measured and can be represented by a single number-length. Moreover, the involvement of cilia in many developmental and physiological processes suggests an understanding of their size control system is critical for understanding ciliary diseases, many of which (e.g., autosomal recessive polycystic kidney disease) are known to involve abnormally short cilia. The flagella of the model organism Chlamydomonas reinhardtii provide the best genetic and cell-biological system to study length control of cilia. Studies in this organism using genetics, biochemistry, imaging, and mathematical modeling have revealed many genes involved in length control of cilia and flagella, and have suggested several testable models for length regulation.


Assuntos
Chlamydomonas reinhardtii/citologia , Flagelos , Animais , Transporte Biológico/fisiologia , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiologia , Cílios/fisiologia , Cílios/ultraestrutura , Modelos Animais de Doenças , Retroalimentação Fisiológica , Flagelos/fisiologia , Flagelos/ultraestrutura , Genótipo , Humanos , Nanotecnologia , Fenótipo , Regeneração , Transdução de Sinais/fisiologia
6.
Curr Biol ; 14(23): R992-3, 2004 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-15589146

RESUMO

Eukaryotic flagella produce a swimming force by coordinating thousands of dynein motor proteins. Recent work provides new clues into how this coordination is achieved.


Assuntos
Dineínas/fisiologia , Flagelos/fisiologia , Proteínas Motores Moleculares/fisiologia , Transdução de Sinais/fisiologia , Flagelos/química , Microtúbulos/fisiologia , Modelos Biológicos
7.
Elife ; 3: e01566, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24596149

RESUMO

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.


Assuntos
Proteínas de Algas/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Flagelos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Algas/genética , Animais , Proteínas de Transporte/genética , Linhagem Celular , Chlamydomonas reinhardtii/genética , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Mutação , Fenótipo , Proteínas de Plantas/genética , Transporte Proteico , Transfecção , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
8.
J Cell Biol ; 199(1): 151-67, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23027906

RESUMO

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.


Assuntos
Flagelos/metabolismo , Transporte Biológico , Células Cultivadas , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Clonagem Molecular , Dineínas/genética , Dineínas/isolamento & purificação , Dineínas/metabolismo , Flagelos/genética , Cinética , Mutação , Temperatura
9.
Cytoskeleton (Hoboken) ; 67(8): 504-18, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20540087

RESUMO

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.


Assuntos
Centríolos/ultraestrutura , Chlamydomonas reinhardtii/ultraestrutura , Fuso Acromático/ultraestrutura , Centríolos/fisiologia , Fuso Acromático/fisiologia
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