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1.
Nat Rev Mol Cell Biol ; 22(3): 215-235, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33169001

RESUMO

Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.


Assuntos
Substâncias Macromoleculares , Complexos Multiproteicos/fisiologia , Animais , Fenômenos Bioquímicos , Fenômenos Fisiológicos Celulares , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Células Eucarióticas/fisiologia , Humanos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Complexos Multiproteicos/química , Organelas/química , Organelas/genética , Organelas/metabolismo , Agregados Proteicos/fisiologia
2.
Cell ; 173(3): 677-692.e20, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677512

RESUMO

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS. Karyopherin-ß2 (also called Transportin-1) engages PY-NLSs to inhibit and reverse FUS, TAF15, EWSR1, hnRNPA1, and hnRNPA2 fibrillization, whereas Importin-α plus Karyopherin-ß1 prevent and reverse TDP-43 fibrillization. Remarkably, Karyopherin-ß2 dissolves phase-separated liquids and aberrant fibrillar hydrogels formed by FUS and hnRNPA1. In vivo, Karyopherin-ß2 prevents RBPs with PY-NLSs accumulating in stress granules, restores nuclear RBP localization and function, and rescues degeneration caused by disease-linked FUS and hnRNPA2. Thus, NIRs therapeutically restore RBP homeostasis and mitigate neurodegeneration.


Assuntos
Transporte Ativo do Núcleo Celular , Príons/química , Proteínas de Ligação a RNA/química , Receptores Citoplasmáticos e Nucleares/química , Adulto , Idoso , Animais , Citoplasma/química , Proteínas de Ligação a DNA/química , Drosophila melanogaster , Feminino , Proteínas de Fluorescência Verde/química , Células HEK293 , Células HeLa , Homeostase , Humanos , Carioferinas/química , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/química , Mutação , Doenças Neurodegenerativas/patologia , Domínios Proteicos , Proteína EWS de Ligação a RNA/química , Fatores Associados à Proteína de Ligação a TATA/química , beta Carioferinas/química
3.
Annu Rev Cell Dev Biol ; 34: 1-28, 2018 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-30059630

RESUMO

Intermediate filaments (IFs) are one of the three major elements of the cytoskeleton. Their stability, intrinsic mechanical properties, and cell type-specific expression patterns distinguish them from actin and microtubules. By providing mechanical support, IFs protect cells from external forces and participate in cell adhesion and tissue integrity. IFs form an extensive and elaborate network that connects the cell cortex to intracellular organelles. They act as a molecular scaffold that controls intracellular organization. However, IFs have been revealed as much more than just rigid structures. Their dynamics is regulated by multiple signaling cascades and appears to contribute to signaling events in response to cell stress and to dynamic cellular functions such as mitosis, apoptosis, and migration.


Assuntos
Biologia Celular/tendências , Citoplasma/genética , Filamentos Intermediários/genética , Microtúbulos/genética , Actinas/química , Actinas/genética , Citoplasma/química , Citoesqueleto/química , Citoesqueleto/genética , Proteína Glial Fibrilar Ácida/genética , Humanos , Filamentos Intermediários/química , Microtúbulos/química , Mitose/genética , Transdução de Sinais/genética
4.
Cell ; 165(5): 1067-1079, 2016 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-27203111

RESUMO

Over a century ago, colloidal phase separation of matter into non-membranous bodies was recognized as a fundamental organizing principal of cell "protoplasm." Recent insights into the molecular properties of such phase-separated bodies present challenges to our understanding of cellular protein interaction networks, as well as opportunities for interpreting and understanding of native and pathological genetic and molecular interactions. Here, we briefly review examples of and discuss physical principles of phase-separated cellular bodies and then reflect on how knowledge of these principles may direct future research on their functions.


Assuntos
Proteínas/química , Animais , Coloides/química , Citoplasma/química , Dequalínio/química , Humanos , Organelas/química , Mapeamento de Interação de Proteínas
5.
Cell ; 162(5): 1066-77, 2015 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-26317470

RESUMO

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases.


Assuntos
Envelhecimento/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Mutação , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética , Envelhecimento/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Núcleo Celular/química , Citoplasma/química , Humanos , Príons/química , Agregados Proteicos , Estrutura Terciária de Proteína , Proteína FUS de Ligação a RNA/metabolismo
6.
Cell ; 158(4): 822-832, 2014 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-25126787

RESUMO

Molecular motors in cells typically produce highly directed motion; however, the aggregate, incoherent effect of all active processes also creates randomly fluctuating forces, which drive diffusive-like, nonthermal motion. Here, we introduce force-spectrum-microscopy (FSM) to directly quantify random forces within the cytoplasm of cells and thereby probe stochastic motor activity. This technique combines measurements of the random motion of probe particles with independent micromechanical measurements of the cytoplasm to quantify the spectrum of force fluctuations. Using FSM, we show that force fluctuations substantially enhance intracellular movement of small and large components. The fluctuations are three times larger in malignant cells than in their benign counterparts. We further demonstrate that vimentin acts globally to anchor organelles against randomly fluctuating forces in the cytoplasm, with no effect on their magnitude. Thus, FSM has broad applications for understanding the cytoplasm and its intracellular processes in relation to cell physiology in healthy and diseased states.


Assuntos
Citoplasma/química , Microscopia de Força Atômica/métodos , Animais , Fenômenos Biomecânicos , Embrião de Mamíferos/citologia , Fibroblastos/química , Camundongos , Proteínas/química , Vimentina/química
7.
Cell ; 156(1-2): 183-94, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24361104

RESUMO

The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the cytoplasm impacts all intracellular processes involving large components.


Assuntos
Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Escherichia coli/citologia , Fenômenos Biofísicos , Caulobacter crescentus/química , Cromossomos Bacterianos/metabolismo , Citoplasma/química , Escherichia coli/química , Escherichia coli/metabolismo , Plasmídeos/metabolismo
8.
Mol Cell ; 81(15): 3145-3159.e7, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34214465

RESUMO

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.


Assuntos
Bacteriófago T7/genética , DNA Viral/química , Periplasma/química , Proteínas do Core Viral/química , Biologia Computacional , Microscopia Crioeletrônica , Citoplasma/química , DNA Viral/metabolismo , Bicamadas Lipídicas/metabolismo , Periplasma/genética , Periplasma/metabolismo , Podoviridae/química , Podoviridae/genética , Proteínas do Core Viral/metabolismo
9.
Annu Rev Cell Dev Biol ; 30: 39-58, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25288112

RESUMO

Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.


Assuntos
Compartimento Celular , Líquido Intracelular/química , Animais , Compartimento Celular/fisiologia , Citoplasma/química , Difusão , Entropia , Géis , Origem da Vida , Transição de Fase , Solubilidade , Terminologia como Assunto
10.
Nature ; 598(7882): 667-671, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34646014

RESUMO

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1-3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4-6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.


Assuntos
Modelos Estruturais , Poro Nuclear/química , Linhagem Celular Tumoral , Núcleo Celular/química , Citoplasma/química , Tomografia com Microscopia Eletrônica , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/química
11.
Nature ; 595(7866): 303-308, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34108682

RESUMO

Liquid-liquid phase separation is a major mechanism of subcellular compartmentalization1,2. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism3,4, whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins5-8. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM-NORAD and PUM-PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs9-11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.


Assuntos
Instabilidade Genômica , Transição de Fase , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Linhagem Celular , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Humanos , RNA/química , RNA/genética , RNA/metabolismo , RNA Longo não Codificante/química
12.
Chem Rev ; 124(4): 1899-1949, 2024 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-38331392

RESUMO

Macromolecular crowding affects the activity of proteins and functional macromolecular complexes in all cells, including bacteria. Crowding, together with physicochemical parameters such as pH, ionic strength, and the energy status, influences the structure of the cytoplasm and thereby indirectly macromolecular function. Notably, crowding also promotes the formation of biomolecular condensates by phase separation, initially identified in eukaryotic cells but more recently discovered to play key functions in bacteria. Bacterial cells require a variety of mechanisms to maintain physicochemical homeostasis, in particular in environments with fluctuating conditions, and the formation of biomolecular condensates is emerging as one such mechanism. In this work, we connect physicochemical homeostasis and macromolecular crowding with the formation and function of biomolecular condensates in the bacterial cell and compare the supramolecular structures found in bacteria with those of eukaryotic cells. We focus on the effects of crowding and phase separation on the control of bacterial chromosome replication, segregation, and cell division, and we discuss the contribution of biomolecular condensates to bacterial cell fitness and adaptation to environmental stress.


Assuntos
Bactérias , Separação de Fases , Substâncias Macromoleculares/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Bactérias/metabolismo , Homeostase
13.
Cell ; 147(6): 1295-308, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22153074

RESUMO

As nascent polypeptides exit ribosomes, they are engaged by a series of processing, targeting, and folding factors. Here, we present a selective ribosome profiling strategy that enables global monitoring of when these factors engage polypeptides in the complex cellular environment. Studies of the Escherichia coli chaperone trigger factor (TF) reveal that, though TF can interact with many polypeptides, ß-barrel outer-membrane proteins are the most prominent substrates. Loss of TF leads to broad outer-membrane defects and premature, cotranslational protein translocation. Whereas in vitro studies suggested that TF is prebound to ribosomes waiting for polypeptides to emerge from the exit channel, we find that in vivo TF engages ribosomes only after ~100 amino acids are translated. Moreover, excess TF interferes with cotranslational removal of the N-terminal formyl methionine. Our studies support a triaging model in which proper protein biogenesis relies on the fine-tuned, sequential engagement of processing, targeting, and folding factors.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptidilprolil Isomerase/metabolismo , Ribossomos/metabolismo , Citoplasma/química , Escherichia coli/citologia , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Biossíntese de Proteínas , Transporte Proteico
14.
RNA ; 28(1): 76-87, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34706978

RESUMO

Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.


Assuntos
Condensados Biomoleculares/química , Chaperonas Moleculares/química , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/química , Sítios de Ligação , Condensados Biomoleculares/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Eucariotos , Células Eucarióticas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Agregados Proteicos , Ligação Proteica , Biossíntese de Proteínas , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
15.
Nature ; 557(7706): 558-563, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29743672

RESUMO

In Lewy body diseases-including Parkinson's disease, without or with dementia, dementia with Lewy bodies, and Alzheimer's disease with Lewy body co-pathology 1 -α-synuclein (α-Syn) aggregates in neurons as Lewy bodies and Lewy neurites 2 . By contrast, in multiple system atrophy α-Syn accumulates mainly in oligodendrocytes as glial cytoplasmic inclusions (GCIs) 3 . Here we report that pathological α-Syn in GCIs and Lewy bodies (GCI-α-Syn and LB-α-Syn, respectively) is conformationally and biologically distinct. GCI-α-Syn forms structures that are more compact and it is about 1,000-fold more potent than LB-α-Syn in seeding α-Syn aggregation, consistent with the highly aggressive nature of multiple system atrophy. GCI-α-Syn and LB-α-Syn show no cell-type preference in seeding α-Syn pathology, which raises the question of why they demonstrate different cell-type distributions in Lewy body disease versus multiple system atrophy. We found that oligodendrocytes but not neurons transform misfolded α-Syn into a GCI-like strain, highlighting the fact that distinct α-Syn strains are generated by different intracellular milieus. Moreover, GCI-α-Syn maintains its high seeding activity when propagated in neurons. Thus, α-Syn strains are determined by both misfolded seeds and intracellular environments.


Assuntos
Citoplasma/metabolismo , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Neurônios/metabolismo , alfa-Sinucleína/classificação , alfa-Sinucleína/metabolismo , Animais , Citoplasma/química , Citoplasma/patologia , Feminino , Humanos , Corpos de Lewy/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Neurônios/patologia , Oligodendroglia/química , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Especificidade de Órgãos , Dobramento de Proteína , alfa-Sinucleína/química
16.
Mol Cell ; 64(2): 282-293, 2016 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-27720645

RESUMO

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.


Assuntos
Algoritmos , Anotação de Sequência Molecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/classificação , RNA/química , Animais , Sítios de Ligação , Núcleo Celular/química , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Expressão Gênica , Ontologia Genética , Células HEK293 , Humanos , Motivos de Nucleotídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Software , Dedos de Zinco
17.
Curr Microbiol ; 81(9): 265, 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39003318

RESUMO

Protists, including ciliates retain crystals in their cytoplasm. However, their functions and properties remain unclear. To comparatively analyze the crystals of Paramecium bursaria, a ciliate, associated with and without the endosymbiotic Chlorella variabilis, we investigated the isolated crystals using a light microscope and analyzed their length and solubility. A negligible number of crystals was found in P. bursaria cells harboring symbiotic algae. The average crystal length in alga-free and algae-reduced cells was about 6.8 µm and 14.4 µm, respectively. The crystals of alga-free cells were spherical, whereas those of algae-reduced cells were angular in shape. The crystals of alga-free cells immediately dissolved in acids and bases, but not in water or organic solvents, and were stable at - 20 °C for more than 3 weeks. This study, for the first time, reveals that the characteristics of crystals present in the cytoplasm of P. bursaria vary greatly depending on the amount of symbiotic algae.


Assuntos
Chlorella , Paramecium , Simbiose , Chlorella/química , Chlorella/metabolismo , Paramecium/metabolismo , Cristalização , Citoplasma/química
18.
Anal Chem ; 95(26): 9739-9745, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37347195

RESUMO

The accumulation and spatial distribution of intracellular nanoplastic particles provide useful information about their spatiotemporal toxicological effects mediated by the physicochemical parameters of nanoplastics in living cells. In this study, a sample injection-transfer method was designed with an accuracy of up to femtoliters to attoliters to match the volume required for ultranarrow-bore open-tubular liquid chromatography. The separation and concentration quantification of mixed polystyrenes in different regions in living cells were achieved by directly transferring picoliter/femtoliter volumes of intracellular cytoplasm to an ultranarrow-bore open-tubular chromatographic column. The measurement of pollutant concentration in different areas of a small-volume target (single cell) was realized. This method is expected to be used in the qualitative and quantitative analyses of complex, mixed, and label-free nanoplastics (a few nm in size) in the subregions of living cells.


Assuntos
Microplásticos , Poliestirenos , Microplásticos/análise , Cromatografia Líquida/métodos , Poliestirenos/análise , Citoplasma/química
19.
Annu Rev Microbiol ; 72: 255-271, 2018 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-30200855

RESUMO

RNA localization mechanisms have been intensively studied and include localized protection of mRNA from degradation, diffusion-coupled local entrapment of mRNA, and directed transport of mRNAs along the cytoskeleton. While it is well understood how cells utilize these three mechanisms to organize mRNAs within the cytoplasm, a newly appreciated mechanism of RNA localization has emerged in recent years in which mRNAs phase-separate and form liquid-like droplets. mRNAs both contribute to condensation of proteins into liquid-like structures and are themselves regulated by being incorporated into membraneless organelles. This ability to condense into droplets is in many instances contributing to previously appreciated mRNA localization phenomena. Here we review how phase separation enables mRNAs to selectively and efficiently colocalize and be coregulated, allowing control of gene expression in time and space.


Assuntos
Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Células Eucarióticas/metabolismo , Células Procarióticas/metabolismo , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Citoplasma/química , Grânulos Citoplasmáticos/química , Células Eucarióticas/química , Células Procarióticas/química , RNA Mensageiro/análise
20.
J Microsc ; 290(2): 125-133, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36864642

RESUMO

A secondary ion mass spectrometry (SIMS)-based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC-PK1 cells. The CAMECA IMS-3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high-vacuum requirements of the instrument the cells were prepared cryogenically with a freeze-fracture method and frozen freeze-dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to 44 Ca stable isotope in the extracellular buffer for 10 min. The 44 Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 (40 Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post-metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of 44 Ca in 10 min was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the 44 Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division.


Assuntos
Cálcio , Espectrometria de Massa de Íon Secundário , Metáfase , Cálcio/análise , Espectrometria de Massa de Íon Secundário/métodos , Divisão Celular , Citoplasma/química
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