RESUMO
Membraneless compartments, also known as condensates, provide chemically distinct environments and thus spatially organize the cell. A well-studied example of condensates is P granules in the roundworm Caenorhabditis elegans that play an important role in the development of the germline. P granules are RNA-rich protein condensates that share the key properties of liquid droplets such as a spherical shape, the ability to fuse, and fast diffusion of their molecular components. An outstanding question is to what extent phase separation at thermodynamic equilibrium is appropriate to describe the formation of condensates in an active cellular environment. To address this question, we investigate the response of P granule condensates in living cells to temperature changes. We observe that P granules dissolve upon increasing the temperature and recondense upon lowering the temperature in a reversible manner. Strikingly, this temperature response can be captured by in vivo phase diagrams that are well described by a Flory-Huggins model at thermodynamic equilibrium. This finding is surprising due to active processes in a living cell. To address the impact of such active processes on intracellular phase separation, we discuss temperature heterogeneities. We show that, for typical estimates of the density of active processes, temperature represents a well-defined variable and that mesoscopic volume elements are at local thermodynamic equilibrium. Our findings provide strong evidence that P granule assembly and disassembly are governed by phase separation based on local thermal equilibria where the nonequilibrium nature of the cytoplasm is manifested on larger scales.
Assuntos
Condensados Biomoleculares/fisiologia , Grânulos de Ribonucleoproteínas de Células Germinativas/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Entropia , Grânulos de Ribonucleoproteínas de Células Germinativas/metabolismo , Células Germinativas/metabolismo , Solubilidade , Temperatura , TermodinâmicaRESUMO
Spontaneous pattern formation in Turing systems relies on feedback. Patterns in cells and tissues however often do not form spontaneously, but are under control of upstream pathways that provide molecular guiding cues. The relationship between guiding cues and feedback in controlled biological pattern formation remains unclear. We explored this relationship during cell polarity establishment in the one-cell-stage C. elegans embryo. We quantified the strength of two feedback systems that operate during polarity establishment, feedback between polarity proteins and the actomyosin cortex, and mutual antagonism amongst polarity proteins. We characterized how these feedback systems are modulated by guiding cues from the centrosome. By coupling a mass-conserved Turing-like reaction-diffusion system for polarity proteins to an active gel description of the actomyosin cortex, we reveal a transition point beyond which feedback ensures self-organized polarization even when cues are removed. Notably, the baton is passed from a guide-dominated to a feedback-dominated regime significantly beyond this transition point, which ensures robustness. Together, this reveals a general criterion for controlling biological pattern forming systems: feedback remains subcritical to avoid unstable behaviour, and molecular guiding cues drive the system beyond a transition point for pattern formation.
RESUMO
Cell polarity arises from a combination of interactions between biological molecules, such as activation, inhibition, and positive or negative feedback between specific polarity units. Activation and inhibition often take place in the form of a membrane binding switch. Lethal giant larvae (LGL), a conserved regulator of cell polarity in animals, was suggested to function as such a switch. LGL localizes to both the cytoplasm and, asymmetrically, the membrane. However, the spatial regulation mechanism of LGL membrane localization has remained unclear. For systematic elucidation, we set out to reconstitute a minimal polarity unit using a model membrane, Caenorhabditis elegans LGL (LGL-1), and atypical protein kinase C (aPKC) supposed to activate the membrane switch. We identified a membrane binding sequence (MBS) in LGL-1 by a screen in vivo, reconstituted LGL-1 membrane binding in vitro, and successfully implemented the membrane switch by aPKC phosphorylation activity, detaching LGL from membranes. Upon membrane binding, LGL-1 MBS folds into an alpha-helix in which three regions can be identified: a positively charged patch, a switch area containing the three aPKC phosphorylation sites, and a hydrophobic area probably buried in the membrane. Phosphorylation by aPKC dramatically reduces the binding affinity of the LGL-1 MBS to negatively charged model membranes, inducing its detachment. Specific residues in the MBS are critical for LGL-1 function in C. elegans.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Membrana Celular/fisiologia , Polaridade Celular , Animais , Análise Mutacional de DNA , Fosforilação , Ligação Proteica , Conformação Proteica , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
P granules are non-membrane-bound RNA-protein compartments that are involved in germline development in C. elegans. They are liquids that condense at one end of the embryo by localized phase separation, driven by gradients of polarity proteins such as the mRNA-binding protein MEX-5. To probe how polarity proteins regulate phase separation, we combined biochemistry and theoretical modeling. We reconstitute P granule-like droplets in vitro using a single protein PGL-3. By combining in vitro reconstitution with measurements of intracellular concentrations, we show that competition between PGL-3 and MEX-5 for mRNA can regulate the formation of PGL-3 droplets. Using theory, we show that, in a MEX-5 gradient, this mRNA competition mechanism can drive a gradient of P granule assembly with similar spatial and temporal characteristics to P granule assembly in vivo. We conclude that gradients of polarity proteins can position RNP granules during development by using RNA competition to regulate local phase separation.
Assuntos
Caenorhabditis elegans/metabolismo , RNA Mensageiro/metabolismo , Animais , Proteínas de Caenorhabditis elegans/análise , Proteínas de Caenorhabditis elegans/metabolismo , Polaridade Celular , Embrião não Mamífero , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Modelos Teóricos , Ligação Proteica , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/metabolismoRESUMO
A hallmark of cell polarity in metazoans is the distribution of partitioning defective (PAR) proteins into two domains on the membrane. Domain boundaries are set by the collective integration of mechanical, biochemical and biophysical signals, and the resulting PAR domains define areas of cytosol specialization. However, the complexity of the signals acting on PAR proteins has been a barrier to uncovering the general principles of PAR polarity. We propose that physical studies, when combined with genetic data, provide new understanding of the mechanisms of polarity establishment in the Caenorhabditis elegans embryo and other organisms.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Polaridade Celular/fisiologia , Animais , Caenorhabditis elegans/metabolismo , Embrião não Mamífero , HumanosRESUMO
Polarization of cells by PAR proteins requires the segregation of antagonistic sets of proteins into two mutually exclusive membrane-associated domains. Understanding how nanometer scale interactions between individual PAR proteins allow spatial organization across cellular length scales requires determining the kinetic properties of PAR proteins and how they are modified in space. We find that PAR-2 and PAR-6, which localize to opposing PAR domains, undergo exchange between well mixed cytoplasmic populations and laterally diffusing membrane-associated states. Domain maintenance does not involve diffusion barriers, lateral sorting, or active transport. Rather, both PAR proteins are free to diffuse between domains, giving rise to a continuous boundary flux because of lateral diffusion of molecules down the concentration gradients that exist across the embryo. Our results suggest that the equalizing effects of lateral diffusion are countered by actin-independent differences in the effective membrane affinities of PAR proteins between the two domains, which likely depend on the ability of each PAR species to locally modulate the membrane affinity of opposing PAR species within its domain. We propose that the stably polarized embryo reflects a dynamic steady state in which molecules undergo continuous diffusion between regions of net association and dissociation.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Actinas/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Difusão , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Cinética , Modelos Biológicos , Estrutura Terciária de ProteínaRESUMO
Many metazoan cell types are polarized by asymmetric partitioning of the conserved PAR (PAR-3/PAR-6/PKC-3) complex. Cortical domains containing this PAR complex are counterbalanced by opposing domains of varying composition. The tumor-suppressor protein LGL facilitates asymmetric localization of cell fate determinants, in part through modulating the activity of the PAR complex. However, the mechanisms by which LGL acts to maintain a cortical domain remain unclear. Here we identify Caenorhabditis elegans LGL in a biochemical complex with PAR proteins, which localize to the anterior cortex. But LGL itself localizes to the posterior cortex. We show that increasing the amounts of LGL can restrict localization of the PAR complex to an anterior cortical domain, even in the absence of PAR-2. Importantly, LGL must be phosphorylated on conserved residues to exert this function. LGL and the PAR complex can maintain two cortical domains that are sufficient to partition cell fate determinants. Our data suggest a mechanism of "mutual elimination" in which an LGL phosphorylation cycle regulates association of the PAR complex with the cortex: binding of LGL to the PAR complex at the interface of the two domains stimulates its phosphorylation by PKC-3, and the whole complex leaves the cortex.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Polaridade Celular/fisiologia , Fase de Clivagem do Zigoto/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Fase de Clivagem do Zigoto/metabolismo , Biologia Computacional , Proteínas de Fluorescência Verde/metabolismo , Imunoprecipitação , Espectrometria de Massas , Fosforilação , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina QuinasesRESUMO
In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.
Assuntos
Caenorhabditis elegans/embriologia , Grânulos Citoplasmáticos/fisiologia , Embrião não Mamífero/citologia , Células Germinativas/ultraestrutura , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Fenômenos Químicos , Citoplasma/metabolismo , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Transição de Fase , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA de Helmintos/química , Solubilidade , Tensão Superficial , ViscosidadeRESUMO
The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Animais , Caenorhabditis elegans/embriologia , Divisão Celular , Citosol/metabolismo , Difusão , Movimento , Cadeias Pesadas de Miosina/metabolismo , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Since cdc48 mutants were isolated by the first genetic screens for cell division cycle (cdc) mutants in yeast, the requirement of the chaperone-like ATPase Cdc48/p97 during cell division has remained unclear. Here, we discover an unanticipated function for Caenorhabditis elegans CDC-48 in DNA replication linked to cell cycle control. Our analysis of the CDC-48(UFD-1/NPL-4) complex identified a general role in S phase progression of mitotic cells essential for embryonic cell division and germline development of adult worms. These developmental defects result from activation of the DNA replication checkpoint caused by replication stress. Similar to loss of replication licensing factors, DNA content is strongly reduced in worms depleted for CDC-48, UFD-1, and NPL-4. In addition, these worms show decreased DNA synthesis and hypersensitivity toward replication blocking agents. Our findings identified a role for CDC-48(UFD-1/NPL-4) in DNA replication, which is important for cell cycle progression and genome stability.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Replicação do DNA , Proteínas Nucleares/metabolismo , Animais , Caenorhabditis elegans/embriologia , Cromatina/patologia , Regulação para Baixo , Embrião não Mamífero/citologia , Fase S , Proteína com ValosinaRESUMO
Caenorhabditis elegans embryos establish cortical domains of PAR proteins of reproducible size before asymmetric cell division. The ways in which the size of these domains is set remain unknown. Here we identify the GTPase-activating proteins (GAPs) RGA-3 and RGA-4, which regulate the activity of the small GTPase RHO-1. rga-3/4(RNAi) embryos have a hypercontractile cortex, and the initial relative size of their anterior and posterior PAR domains is altered. Thus, RHO-1 activity appears to control the level of cortical contractility and concomitantly the size of cortical domains. These data support the idea that in C. elegans embryos the initial size of the PAR domains is set by regulating the contractile activity of the acto-myosin cytoskeleton through the activity of RHO-1. RGA-3/4 have functions different from CYK-4, the other known GAP required for the first cell division, showing that different GAPs cooperate to control the activity of the acto-myosin cytoskeleton in the first cell division of C. elegans embryos.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Polaridade Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Actomiosina/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ciclo Celular , Embrião não Mamífero/metabolismo , Imunofluorescência , Proteínas Ativadoras de GTPase/genética , Miosinas/metabolismo , Interferência de RNARESUMO
Homologous recombination is essential for genetic exchange, meiosis and error-free repair of double-strand breaks. Central to this process is Rad52, a conserved homo-oligomeric ring-shaped protein, which mediates the exchange of the early recombination factor RPA by Rad51 and promotes strand annealing. Here, we report that Rad52 of Saccharomyces cerevisiae is modified by the ubiquitin-like protein SUMO, primarily at two sites that flank the conserved Rad52 domain. Sumoylation is induced on DNA damage and triggered by Mre11-Rad50-Xrs2 (MRX) complex-governed double-strand breaks (DSBs). Although sumoylation-defective Rad52 is largely recombination proficient, mutant analysis revealed that the SUMO modification sustains Rad52 activity and concomitantly shelters the protein from accelerated proteasomal degradation. Furthermore, our data indicate that sumoylation becomes particularly relevant for those Rad52 molecules that are engaged in recombination.
Assuntos
Dano ao DNA , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Proteína SUMO-1/metabolismo , Western Blotting , Linhagem Celular , Reparo do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Mutação/genética , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína SUMO-1/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , TransfecçãoRESUMO
Damaged DNA, if not repaired before replication, can lead to replication fork stalling and genomic instability; however, cells can switch to different damage bypass modes that permit replication across lesions. Two main bypasses are controlled by ubiquitin modification of proliferating cell nuclear antigen (PCNA), a homotrimeric DNA-encircling protein that functions as a polymerase processivity factor and regulator of replication-linked functions. Upon DNA damage, PCNA is modified at the conserved lysine residue 164 by either mono-ubiquitin or a lysine-63-linked multi-ubiquitin chain, which induce error-prone or error-free replication bypasses of the lesions. In S phase, even in the absence of exogenous DNA damage, yeast PCNA can be alternatively modified by the small ubiquitin-related modifier protein SUMO; however the consequences of this remain controversial. Here we show by genetic analysis that SUMO-modified PCNA functionally cooperates with Srs2, a helicase that blocks recombinational repair by disrupting Rad51 nucleoprotein filaments. Moreover, Srs2 displays a preference for interacting directly with the SUMO-modified form of PCNA, owing to a specific binding site in its carboxy-terminal tail. Our finding suggests a model in which SUMO-modified PCNA recruits Srs2 in S phase in order to prevent unwanted recombination events of replicating chromosomes.
Assuntos
DNA Helicases/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Recombinação Genética , Fase S , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Dano ao DNA , DNA Helicases/química , Replicação do DNA , Epistasia Genética , Mutagênese/genética , Mutação/genética , Fenótipo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Protein degradation in eukaryotes usually requires multiubiquitylation and subsequent delivery of the tagged substrates to the proteasome. Recent studies suggest the involvement of the AAA ATPase CDC48, its cofactors, and other ubiquitin binding factors in protein degradation, but how these proteins work together is unclear. Here we show that these factors cooperate sequentially through protein-protein interactions and thereby escort ubiquitin-protein conjugates to the proteasome. Central to this pathway is the chaperone CDC48/p97, which coordinates substrate recruitment, E4-catalyzed multiubiquitin chain assembly, and proteasomal targeting. Concomitantly, CDC48 prevents the formation of excessive multiubiquitin chain sizes that are surplus to requirements for degradation. In yeast, this escort pathway guides a transcription factor from its activation in the cytosol to its final degradation and also mediates proteolysis at the endoplasmic reticulum by the ERAD pathway.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/metabolismo , Adenosina Trifosfatases , Animais , Retículo Endoplasmático/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Dobramento de Proteína , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteína com ValosinaRESUMO
The RAD6 pathway is central to post-replicative DNA repair in eukaryotic cells; however, the machinery and its regulation remain poorly understood. Two principal elements of this pathway are the ubiquitin-conjugating enzymes RAD6 and the MMS2-UBC13 heterodimer, which are recruited to chromatin by the RING-finger proteins RAD18 and RAD5, respectively. Here we show that UBC9, a small ubiquitin-related modifier (SUMO)-conjugating enzyme, is also affiliated with this pathway and that proliferating cell nuclear antigen (PCNA) -- a DNA-polymerase sliding clamp involved in DNA synthesis and repair -- is a substrate. PCNA is mono-ubiquitinated through RAD6 and RAD18, modified by lysine-63-linked multi-ubiquitination--which additionally requires MMS2, UBC13 and RAD5--and is conjugated to SUMO by UBC9. All three modifications affect the same lysine residue of PCNA, suggesting that they label PCNA for alternative functions. We demonstrate that these modifications differentially affect resistance to DNA damage, and that damage-induced PCNA ubiquitination is elementary for DNA repair and occurs at the same conserved residue in yeast and humans.