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1.
Structure ; 32(8): 1248-1259.e5, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38754431

RESUMO

Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes ex vivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila.


Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Domínios Proteicos , Microscopia Crioeletrônica/métodos , Tetrahymena thermophila/metabolismo , Software , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Microtúbulos/metabolismo , Microtúbulos/química
2.
Elife ; 122024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38598282

RESUMO

Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.


Assuntos
Microtúbulos , Tubulina (Proteína) , Acetilação , Microscopia Crioeletrônica , Processamento de Proteína Pós-Traducional
3.
J Cell Sci ; 137(5)2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-37667859

RESUMO

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.


Assuntos
Tetrahymena thermophila , Tetrahymena , Tetrahymena/genética , Divisão Celular/genética , Acetamidas , Tetrahymena thermophila/genética , Citoesqueleto
4.
bioRxiv ; 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38077012

RESUMO

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of macromolecular complexes, enabling high-resolution structure determination. With the paradigm shift to in situ structural biology recently driven by the ground-breaking development of cryo-focused ion beam milling and cryo-electron tomography, there are an increasing number of structures at sub-nanometer resolution of complexes solved directly within their cellular environment. These cellular complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because the side chains cannot be visualized reliably. Here, we present DomainFit, a program for automated domain-level protein identification from cryo-EM maps at resolutions lower than 4 Å. By fitting domains from artificial intelligence-predicted models such as AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the proteins forming the density. Using DomainFit, we identified two microtubule inner proteins, one of them, a CCDC81 domain-containing protein, is exclusively localized in the proximal region of the doublet microtubule from the ciliate Tetrahymena thermophila. The flexibility and capability of DomainFit makes it a valuable tool for analyzing in situ structures.

5.
J Cell Biol ; 222(11)2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37756660

RESUMO

Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show that the microtubules at the tip are highly crosslinked with each other and stabilized by luminal proteins, plugs, and cap proteins at the plus ends. In the tip region, the central pair lacks typical projections and twists significantly. By analyzing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explained the potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and the mechanisms of ciliary assembly and length regulation.


Assuntos
Cílios , Microtúbulos , Tetrahymena thermophila , Axonema , Cílios/metabolismo , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo
6.
Mol Biol Cell ; 34(8): ar82, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37163326

RESUMO

Ciliates, such as Tetrahymena thermophila, evolved complex mechanisms to determine both the location and dimensions of cortical organelles such as the oral apparatus (OA: involved in phagocytosis), cytoproct (Cyp: for eliminating wastes), and contractile vacuole pores (CVPs: involved in water expulsion). Mutations have been recovered in Tetrahymena that affect both the localization of such organelles along anterior-posterior and circumferential body axes and their dimensions. Here we describe BCD1, a ciliate pattern gene that encodes a conserved Beige-BEACH domain-containing protein a with possible protein kinase A (PKA)-anchoring activity. Similar proteins have been implicated in endosome trafficking and are linked to human Chediak-Higashi syndrome and autism. Mutations in the BCD1 gene broaden cortical organelle domains as they assemble during predivision development. The Bcd1 protein localizes to membrane pockets at the base of every cilium that are active in endocytosis. PKA activity has been shown to promote endocytosis in other organisms, so we blocked clathrin-mediated endocytosis (using "dynasore") and inhibited PKA (using H89). In both cases, treatment produced partial phenocopies of the bcd1 pattern mutant. This study supports a model in which the dimensions of diverse cortical organelle assembly-platforms may be determined by regulated balance between constitutive exocytic delivery and PKA-regulated endocytic retrieval of organelle materials and determinants.


Assuntos
Tetrahymena thermophila , Humanos , Tetrahymena thermophila/fisiologia , Endossomos , Endocitose , Fagocitose , Vacúolos
7.
J Eukaryot Microbiol ; 69(5): e12890, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35075744

RESUMO

As single cells, ciliates build, duplicate, and even regenerate complex cortical patterns by largely unknown mechanisms that precisely position organelles along two cell-wide axes: anterior-posterior and circumferential (left-right). We review our current understanding of intracellular patterning along the anterior-posterior axis in ciliates, with emphasis on how the new pattern emerges during cell division. We focus on the recent progress at the molecular level that has been driven by the discovery of genes whose mutations cause organelle positioning defects in the model ciliate Tetrahymena thermophila. These investigations have revealed a network of highly conserved kinases that are confined to either anterior or posterior domains in the cell cortex. These pattern-regulating kinases create zones of cortical inhibition that by exclusion determine the precise placement of organelles. We discuss observations and models derived from classical microsurgical experiments in large ciliates (including Stentor) and interpret them in light of recent molecular findings in Tetrahymena. In particular, we address the involvement of intracellular gradients as vehicles for positioning organelles along the anterior-posterior axis.


Assuntos
Cilióforos , Tetrahymena thermophila , Divisão Celular , Cilióforos/genética , Tetrahymena thermophila/genética
8.
EMBO Rep ; 22(9): e52911, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34338432

RESUMO

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.


Assuntos
Dineínas do Axonema , Dineínas , Dineínas do Axonema/metabolismo , Axonema/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica , Dineínas/metabolismo , Humanos , Microtúbulos/metabolismo
9.
J Cell Biol ; 219(9)2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32642758

RESUMO

Not much is known about how organelles organize into patterns. In ciliates, the cortical pattern is propagated during "tandem duplication," a cell division that remodels the parental cell into two daughter cells. A key step is the formation of the division boundary along the cell's equator. In Tetrahymena thermophila, the cdaA alleles prevent the formation of the division boundary. We find that the CDAA gene encodes a cyclin E that accumulates in the posterior cell half, concurrently with accumulation of CdaI, a Hippo/Mst kinase, in the anterior cell half. The division boundary forms between the margins of expression of CdaI and CdaA, which exclude each other from their own cortical domains. The activities of CdaA and CdaI must be balanced to initiate the division boundary and to position it along the cell's equator. CdaA and CdaI cooperate to position organelles near the new cell ends. Our data point to an intracellular positioning mechanism involving antagonistic Hippo signaling and cyclin E.


Assuntos
Ciclina E/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Divisão Celular/fisiologia , Humanos , Organelas/metabolismo , Tetrahymena thermophila/metabolismo
10.
Cells ; 9(2)2020 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991798

RESUMO

Katanin-like 2 protein (Katnal2) orthologs have a tripartite domain organization. Two highly conserved regions, an N-terminal LisH (Lis-homology) domain and a C-terminal AAA catalytic domain, are separated by a less conserved linker. The AAA domain of Katnal2 shares the highest amino acid sequence homology with the AAA domain of the canonical katanin p60. Katnal2 orthologs are present in a wide range of eukaryotes, from protists to humans. In the ciliate Tetrahymena thermophila, a Katnal2 ortholog, Kat2, co-localizes with the microtubular structures, including basal bodies and ciliary outer doublets, and this co-localization is sensitive to levels of microtubule glutamylation. The functional analysis of Kat2 domains suggests that an N-terminal fragment containing a LisH domain plays a role in the subcellular localization, dimerization, and stability of Kat2.


Assuntos
Katanina/genética , Katanina/metabolismo , Microtúbulos/metabolismo , Tetrahymena/metabolismo , Ácido Glutâmico/metabolismo , Microscopia Eletrônica de Transmissão , Microtúbulos/ultraestrutura , Mutação , Domínios Proteicos , Multimerização Proteica/genética , Estabilidade Proteica , Tetrahymena/enzimologia , Tetrahymena/genética , Tetrahymena/ultraestrutura
11.
PLoS Genet ; 15(7): e1008099, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31339880

RESUMO

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells.


Assuntos
Cílios/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Tetrahymena/citologia , Regulação da Expressão Gênica , Locomoção , Proteínas de Protozoários/metabolismo , Tetrahymena/metabolismo , Tetrahymena/fisiologia
12.
J Cell Sci ; 132(15)2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31243050

RESUMO

Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.


Assuntos
Corpos Basais/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo , Corpos Basais/ultraestrutura , Cílios/genética , Glicosilação , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mutação , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestrutura
13.
Genetics ; 211(2): 651-663, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30593491

RESUMO

In a single cell, ciliates maintain a complex pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes. The underlying molecular mechanisms of intracellular pattern formation in ciliates are largely unknown. Ciliates divide by tandem duplication, a process that remodels the parental cell into two daughters aligned head-to-tail. In the elo1-1 mutant of Tetrahymena thermophila, the segmentation boundary/division plane forms too close to the posterior end of the parental cell, producing a large anterior and a small posterior daughter cell, respectively. We show that ELO1 encodes a Lats/NDR kinase that marks the posterior segment of the cell cortex, where the division plane does not form in the wild-type. Elo1 acts independently of CdaI, a Hippo/Mst kinase that marks the anterior half of the parental cell, and whose loss shifts the division plane anteriorly. We propose that, in Tetrahymena, two antagonistic Hippo circuits focus the segmentation boundary/division plane at the equatorial position, by excluding divisional morphogenesis from the cortical areas that are too close to cell ends.


Assuntos
Divisão Celular , Polaridade Celular , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Tetrahymena/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena/citologia , Tetrahymena/metabolismo
14.
J Cell Biol ; 217(12): 4298-4313, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30217954

RESUMO

Cilia, essential motile and sensory organelles, have several compartments: the basal body, transition zone, and the middle and distal axoneme segments. The distal segment accommodates key functions, including cilium assembly and sensory activities. While the middle segment contains doublet microtubules (incomplete B-tubules fused to complete A-tubules), the distal segment contains only A-tubule extensions, and its existence requires coordination of microtubule length at the nanometer scale. We show that three conserved proteins, two of which are mutated in the ciliopathy Joubert syndrome, determine the geometry of the distal segment, by controlling the positions of specific microtubule ends. FAP256/CEP104 promotes A-tubule elongation. CHE-12/Crescerin and ARMC9 act as positive and negative regulators of B-tubule length, respectively. We show that defects in the distal segment dimensions are associated with motile and sensory deficiencies of cilia. Our observations suggest that abnormalities in distal segment organization cause a subset of Joubert syndrome cases.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Proteínas do Domínio Armadillo/genética , Proteínas de Ciclo Celular/genética , Cerebelo/anormalidades , Cerebelo/metabolismo , Cílios/genética , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Microtúbulos/genética , Proteínas de Protozoários/genética , Retina/anormalidades , Retina/metabolismo , Tetrahymena thermophila/genética
15.
Mol Biol Cell ; 29(21): 2566-2577, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30133348

RESUMO

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.


Assuntos
Cílios/metabolismo , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena/metabolismo , Axonema/metabolismo , Fluorescência , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/metabolismo , Modelos Biológicos , Mutação/genética , Fagocitose , Subunidades Proteicas/metabolismo , Gravação em Vídeo
16.
J Cell Physiol ; 233(11): 8648-8665, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29761930

RESUMO

The mechanisms that regulate γ-tubulin, including its post-translational modifications, are poorly understood. γ-Tubulin is important for the duplication of centrioles and structurally similar basal bodies (BBs), organelles which contain a ring of nine triplet microtubules. The ciliate Tetrahymena thermophila carries hundreds of cilia in a single cell and provides an excellent model to specifically address the role of γ-tubulin in the BBs assembly and maintenance. The genome of Tetrahymena contains a single γ-tubulin gene. We show here that there are multiple isoforms of γ-tubulin that are likely generated by post-translational modifications. We identified evolutionarily conserved serine and threonine residues as potential phosphosites of γ-tubulin, including S80, S129, S131, T283, and S360. Several mutations that either prevent (S80A, S131A, T283A, S360A) or mimic (T283D) phosphorylation were conditionally lethal and at a higher temperature phenocopied a loss of γ-tubulin. Cells that overproduced S360D γ-tubulin displayed phenotypes consistent with defects in the microtubule-dependent functions, including an asymmetric division of the macronucleus and abnormalities in the pattern of BB rows, including gaps, fragmentation, and misalignment. In contrast, overexpression of S129D γ-tubulin affected the orientation, docking, and structure of the BBs, including a loss of either the B- or C-subfibers or the entire triplets. We conclude that conserved potentially phosphorylated amino acids of γ-tubulin are important for either the assembly or stability of BBs.


Assuntos
Sequência de Aminoácidos/genética , Corpos Basais/metabolismo , Tetrahymena thermophila/genética , Tubulina (Proteína)/genética , Animais , Centríolos/genética , Cílios/genética , Genoma/genética , Microtúbulos/genética , Fosforilação , Serina/genética , Treonina/genética
17.
Elife ; 62017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28562242

RESUMO

Intraflagellar transport (IFT) trains, multimegadalton assemblies of IFT proteins and motors, traffic proteins in cilia. To study how trains assemble, we employed fluorescence protein-tagged IFT proteins in Chlamydomonas reinhardtii. IFT-A and motor proteins are recruited from the cell body to the basal body pool, assembled into trains, move through the cilium, and disperse back into the cell body. In contrast to this 'open' system, IFT-B proteins from retrograde trains reenter the pool and a portion is reused directly in anterograde trains indicating a 'semi-open' system. Similar IFT systems were also observed in Tetrahymena thermophila and IMCD3 cells. FRAP analysis indicated that IFT proteins and motors of a given train are sequentially recruited to the basal bodies. IFT dynein and tubulin cargoes are loaded briefly before the trains depart. We conclude that the pool contains IFT trains in multiple stages of assembly queuing for successive release into the cilium upon completion.


Assuntos
Proteínas de Transporte/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cílios/metabolismo , Substâncias Macromoleculares/metabolismo , Biogênese de Organelas , Multimerização Proteica , Recuperação de Fluorescência Após Fotodegradação
18.
Genetics ; 206(2): 873-888, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28413159

RESUMO

The mechanisms that govern pattern formation within the cell are poorly understood. Ciliates carry on their surface an elaborate pattern of cortical organelles that are arranged along the anteroposterior and circumferential axes by largely unknown mechanisms. Ciliates divide by tandem duplication: the cortex of the predivision cell is remodeled into two similarly sized and complete daughters. In the conditional cdaI-1 mutant of Tetrahymena thermophila, the division plane migrates from its initially correct equatorial position toward the cell's anterior, resulting in unequal cell division, and defects in nuclear divisions and cytokinesis. We used comparative whole genome sequencing to identify the cause of cdaI-1 as a mutation in a Hippo/Mst kinase. CdaI is a cortical protein with a cell cycle-dependent, highly polarized localization. Early in cell division, CdaI marks the anterior half of the cell, and later concentrates at the posterior end of the emerging anterior daughter. Despite the strong association of CdaI with the new posterior cell end, the cdaI-1 mutation does not affect the patterning of the new posterior cortical organelles. We conclude that, in Tetrahymena, the Hippo pathway maintains an equatorial position of the fission zone, and, by this activity, specifies the relative dimensions of the anterior and posterior daughter cell.


Assuntos
Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Morfogênese/genética , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética , Divisão Celular/genética , Citocinese/genética , Transdução de Sinais , Tetrahymena thermophila/crescimento & desenvolvimento
19.
J Eukaryot Microbiol ; 64(3): 293-307, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27595611

RESUMO

Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.


Assuntos
Segregação de Cromossomos , Cilióforos/genética , Cinesinas/genética , Cinesinas/fisiologia , Meiose , Mitose , Tetrahymena thermophila/genética , Animais , Núcleo Celular , Cilióforos/citologia , Técnicas de Inativação de Genes , Cinesinas/classificação , Cinesinas/ultraestrutura , Macronúcleo , Prófase Meiótica I , Metáfase , Microtúbulos , Mutação , Filogenia , Proteínas Recombinantes , Fuso Acromático , Polos do Fuso , Tetrahymena/genética , Tetrahymena thermophila/citologia , Tetrahymena thermophila/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-28003186

RESUMO

Tubulin undergoes several highly conserved posttranslational modifications (PTMs) including acetylation, detyrosination, glutamylation, and glycylation. These PTMs accumulate on a subset of microtubules that are long-lived, including those in the basal bodies and axonemes. Tubulin PTMs are distributed nonuniformly. In the outer doublet microtubules of the axoneme, the B-tubules are highly enriched in the detyrosinated, polyglutamylated, and polyglycylated tubulin, whereas the A-tubules contain mostly unmodified tubulin. The nonuniform patterns of tubulin PTMs may functionalize microtubules in a position-dependent manner. Recent studies indicate that tubulin PTMs contribute to the assembly, disassembly, maintenance, and motility of cilia. In particular, tubulin glutamylation has emerged as a key PTM that affects ciliary motility through regulation of axonemal dynein arms and controls the stability and length of the axoneme.


Assuntos
Cílios/química , Tetrahymena/química , Tubulina (Proteína)/química , Acetilação , Animais , Dineínas do Axonema/química , Axonema/química , Movimento Celular , Difusão , Glutamina/química , Microtúbulos/química , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , Tirosina/química
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