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1.
Proc Natl Acad Sci U S A ; 118(36)2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34465625

RESUMO

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

2.
Cell Rep ; 36(10): 109674, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34496236

RESUMO

Tumor-initiating stem cells (TSCs) are critical for drug resistance and immune escape. However, the mutual regulations between TSC and tumor microenvironment (TME) remain unclear. Using DNA-label retaining, single-cell RNA sequencing (scRNA-seq), and other approaches, we investigated intestinal adenoma in response to chemoradiotherapy (CRT), thus identifying therapy-resistant TSCs (TrTSCs). We find bidirectional crosstalk between TSCs and TME using CellPhoneDB analysis. An intriguing finding is that TSCs shape TME into a landscape that favors TSCs for immunosuppression and propagation. Using adenoma-organoid co-cultures, niche-cell depletion, and lineaging tracing, we characterize a functional role of cyclooxygenase-2 (Cox-2)-dependent signaling, predominantly occurring between tumor-associated monocytes and macrophages (TAMMs) and TrTSCs. We show that TAMMs promote TrTSC proliferation through prostaglandin E2 (PGE2)-PTGER4(EP4) signaling, which enhances ß-catenin activity via AKT phosphorylation. Thus, our study shows that the bidirectional crosstalk between TrTSC and TME results in a pro-tumorigenic and immunosuppressive contexture.

3.
Mol Cell ; 81(16): 3294-3309.e12, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34293321

RESUMO

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.


Assuntos
Adaptação Fisiológica/genética , Proteoma/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Aclimatação/genética , Animais , Exposição Ambiental/efeitos adversos , Regulação Fúngica da Expressão Gênica/genética , Temperatura Alta/efeitos adversos , Saccharomycetales/genética
4.
Mol Biol Cell ; 32(16): 1487-1500, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34133218

RESUMO

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB-yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.

5.
J Biol Chem ; 297(1): 100862, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34116057

RESUMO

The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Elonguina/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase II/genética , Ubiquitina-Proteína Ligases/genética , Animais , Síndrome de Cockayne/enzimologia , Síndrome de Cockayne/genética , DNA Helicases/química , DNA Helicases/ultraestrutura , Reparo do DNA/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/ultraestrutura , Elonguina/química , Elonguina/ultraestrutura , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genética
6.
Proc Natl Acad Sci U S A ; 117(50): 31861-31870, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257578

RESUMO

Streamlined characterization of protein complexes remains a challenge for the study of protein interaction networks. Here we describe serial capture affinity purification (SCAP), in which two separate proteins are tagged with either the HaloTag or the SNAP-tag, permitting a multistep affinity enrichment of specific protein complexes. The multifunctional capabilities of this protein-tagging system also permit in vivo validation of interactions using acceptor photobleaching Förster resonance energy transfer and fluorescence cross-correlation spectroscopy quantitative imaging. By coupling SCAP to cross-linking mass spectrometry, an integrative structural model of the complex of interest can be generated. We demonstrate this approach using the Spindlin1 and SPINDOC protein complex, culminating in a structural model with two SPINDOC molecules docked on one SPIN1 molecule. In this model, SPINDOC interacts with the SPIN1 interface previously shown to bind a lysine and arginine methylated sequence of histone H3. Our approach combines serial affinity purification, live cell imaging, and cross-linking mass spectrometry to build integrative structural models of protein complexes.


Assuntos
Cromatografia de Afinidade/métodos , Espectrometria de Massas/métodos , Modelos Moleculares , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/isolamento & purificação , Proteínas Correpressoras/metabolismo , Estudos de Viabilidade , Corantes Fluorescentes/química , Células HEK293 , Humanos , Microscopia Intravital , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/metabolismo , Imagem Molecular/métodos , Sondas Moleculares/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
7.
Elife ; 92020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32851976

RESUMO

Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.


Assuntos
Centrossomo/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Ciclo Celular/genética , Ciclo Celular/fisiologia , Centrossomo/química , Quinases Ciclina-Dependentes/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/genética , Fuso Acromático/metabolismo , Corpos Polares do Fuso/genética , Corpos Polares do Fuso/metabolismo
8.
Stem Cell Reports ; 15(3): 662-676, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822591

RESUMO

Mouse embryonic stem cells (ESCs) cultured in defined medium resemble the pre-implantation epiblast in the ground state, with full developmental capacity including the germline. ß-Catenin is required to maintain ground state pluripotency in mouse ESCs, but its exact role is controversial. Here, we reveal a Tcf3-independent role of ß-catenin in restraining germline and somatic lineage differentiation genes. We show that ß-catenin binds target genes with E2F6 and forms a complex with E2F6 and HMGA2 or E2F6 and HP1γ. Our data indicate that these complexes help ß-catenin restrain and fine-tune germ cell and neural developmental potential. Overall, our data reveal a previously unappreciated role of ß-catenin in preserving lineage differentiation integrity in ground state ESCs.

9.
Proc Natl Acad Sci U S A ; 116(43): 21641-21650, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31570610

RESUMO

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética , Cromossomo X/genética , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Recombinação Genética/genética , Deleção de Sequência/genética
10.
Nat Commun ; 10(1): 3993, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488837

RESUMO

Planar cell polarity (PCP) plays crucial roles in developmental processes such as gastrulation, neural tube closure and hearing. Wnt pathway mutants are often classified as PCP mutants due to similarities between their phenotypes. Here, we show that in the zebrafish lateral line, disruptions of the PCP and Wnt pathways have differential effects on hair cell orientations. While mutations in the PCP genes vangl2 and scrib cause random orientations of hair cells, mutations in wnt11f1, gpc4 and fzd7a/b induce hair cells to adopt a concentric pattern. This concentric pattern is not caused by defects in PCP but is due to misaligned support cells. The molecular basis of the support cell defect is unknown but we demonstrate that the PCP and Wnt pathways work in parallel to establish proper hair cell orientation. Consequently, hair cell orientation defects are not solely explained by defects in PCP signaling, and some hair cell phenotypes warrant re-evaluation.


Assuntos
Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Ciliadas Auditivas/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas de Heparan Sulfato/genética , Proteínas de Membrana/genética , Morfogênese/genética , Morfogênese/fisiologia , Mutação , Defeitos do Tubo Neural/genética , Neurulação/genética , Receptores de Superfície Celular/genética , Proteína Wnt1/genética , Proteínas de Peixe-Zebra/genética
11.
J Cell Biol ; 218(8): 2492-2513, 2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31270138

RESUMO

The spatial organization of the genome is enigmatic. Direct evidence of physical contacts between chromosomes and their visualization at nanoscale resolution has been limited. We used superresolution microscopy to demonstrate that ribosomal DNA (rDNA) can form linkages between chromosomes. We observed rDNA linkages in many different human cell types and demonstrated their resolution in anaphase. rDNA linkages are coated by the transcription factor UBF and their formation depends on UBF, indicating that they regularly occur between transcriptionally active loci. Overexpression of c-Myc increases rDNA transcription and the frequency of rDNA linkages, further suggesting that their formation depends on active transcription. Linkages persist in the absence of cohesion, but inhibition of topoisomerase II prevents their resolution in anaphase. We propose that linkages are topological intertwines occurring between transcriptionally active rDNA loci spatially colocated in the same nucleolar compartment. Our findings suggest that active DNA loci engage in physical interchromosomal connections that are an integral and pervasive feature of genome organization.


Assuntos
Cromossomos Humanos/metabolismo , DNA Ribossômico/metabolismo , Microscopia/métodos , Anáfase/efeitos dos fármacos , Animais , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Humanos , Células Híbridas/efeitos dos fármacos , Células Híbridas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Poliploidia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Telomerase/metabolismo , Inibidores da Topoisomerase/farmacologia
12.
J Cell Biol ; 218(7): 2124-2135, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31118239

RESUMO

The kinetochore is a large molecular machine that attaches chromosomes to microtubules and facilitates chromosome segregation. The kinetochore includes submodules that associate with the centromeric DNA and submodules that attach to microtubules. Additional copies of several submodules of the kinetochore are added during anaphase, including the microtubule binding module Ndc80. While the factors governing plasticity are not known, they could include regulation based on microtubule-kinetochore interactions. We report that Fin1 localizes to the microtubule-proximal edge of the kinetochore cluster during anaphase based on single-particle averaging of super-resolution images. Fin1 is required for the assembly of normal levels of Dam1 and Ndc80 submodules. Levels of Ndc80 further depend on the Dam1 microtubule binding complex. Our results suggest the stoichiometry of outer kinetochore submodules is strongly influenced by factors at the kinetochore-microtubule interface such as Fin1 and Dam1, and phosphorylation by cyclin-dependent kinase. Outer kinetochore stoichiometry is remarkably plastic and responsive to microtubule-proximal regulation.


Assuntos
Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/genética , Proteínas do Citoesqueleto/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Anáfase/genética , Centrômero/genética , Cromossomos/genética , Quinases Ciclina-Dependentes/genética , Cinetocoros/metabolismo , Microtúbulos/genética , Fosforilação/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/genética
13.
PLoS One ; 14(5): e0216220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31059522

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused mostly by mutations in polycystin-1 or polycystin-2. Fluid flow leads to polycystin-dependent calcium influx and nuclear export of histone deacetylase 5 (HDAC5), which facilitates the maintenance of renal epithelial architecture by de-repression of MEF2C target genes. Here, we screened a small-molecule library to find drugs that promotes nuclear export of HDAC5. We found that dopamine receptor antagonists, domperidone and loxapine succinate, stimulate export of HDAC5, even in Pkd1-/-cells. Domperidone targets Drd3 receptor to modulate the phosphorylation of HDAC5. Domperidone treatment increases HDAC5 phosphorylation likely by reducing protein phosphatase 2A (PP2A) activity, thus shifting the equilibrium towards HDAC5-P and export from the nucleus. Treating Pkd1-/-mice with domperidone showed significantly reduced cystic growth and cell proliferation. Further, treated mice displayed a reduction in glomerular cyst and increased body weight and activity. These results suggest that HDAC5 nucleocytoplasmic shuttling may be modulated to impede disease progression in ADPKD and uncovers an unexpected role for a class of dopamine receptors in renal epithelial morphogenesis.


Assuntos
Antagonistas de Dopamina/uso terapêutico , Rim Policístico Autossômico Dominante/tratamento farmacológico , Transporte Ativo do Núcleo Celular , Animais , Proliferação de Células/efeitos dos fármacos , Domperidona/farmacologia , Domperidona/uso terapêutico , Antagonistas de Dopamina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/metabolismo , Histona Desacetilases/metabolismo , Rim/patologia , Camundongos
14.
J Cell Biol ; 218(5): 1478-1490, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-30862629

RESUMO

Bipolar spindle formation in yeast requires insertion of centrosomes (known as spindle pole bodies [SPBs]) into fenestrated regions of the nuclear envelope (NE). Using structured illumination microscopy and bimolecular fluorescence complementation, we map protein distribution at SPB fenestrae and interrogate protein-protein interactions with high spatial resolution. We find that the Sad1-UNC-84 (SUN) protein Mps3 forms a ring-like structure around the SPB, similar to toroids seen for components of the SPB insertion network (SPIN). Mps3 and the SPIN component Mps2 (a Klarsicht-ANC-1-Syne-1 domain [KASH]-like protein) form a novel noncanonical linker of nucleoskeleton and cytoskeleton (LINC) complex that is connected in both luminal and extraluminal domains at the site of SPB insertion. The LINC complex also controls the distribution of a soluble SPIN component Bbp1. Taken together, our work shows that Mps3 is a fifth SPIN component and suggests both direct and indirect roles for the LINC complex in NE remodeling.


Assuntos
Centrossomo/metabolismo , Citoesqueleto/metabolismo , Membrana Nuclear/metabolismo , Matriz Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Corpos Polares do Fuso/metabolismo , Ciclo Celular , Matriz Nuclear/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
16.
Methods ; 159-160: 157-164, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30794906

RESUMO

Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.


Assuntos
Dano ao DNA , Elonguina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Culina/metabolismo , DNA/metabolismo , DNA/efeitos da radiação , Eucariotos/enzimologia , Eucariotos/metabolismo , Lasers
17.
Genetics ; 211(4): 1269-1282, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709848

RESUMO

Inner nuclear membrane (INM) protein composition regulates nuclear function, affecting processes such as gene expression, chromosome organization, nuclear shape, and stability. Mechanisms that drive changes in the INM proteome are poorly understood, in part because it is difficult to definitively assay INM composition rigorously and systematically. Using a split-GFP complementation system to detect INM access, we examined the distribution of all C-terminally tagged Saccharomyces cerevisiae membrane proteins in wild-type cells and in mutants affecting protein quality control pathways, such as INM-associated degradation (INMAD), ER-associated degradation, and vacuolar proteolysis. Deletion of the E3 ligase Asi1 had the most specific effect on the INM compared to mutants in vacuolar or ER-associated degradation pathways, consistent with a role for Asi1 in the INMAD pathway. Our data suggest that Asi1 not only removes mistargeted proteins at the INM, but also controls the levels and distribution of native INM components, such as the membrane nucleoporin Pom33 Interestingly, loss of Asi1 does not affect Pom33 protein levels but instead alters Pom33 distribution in the nuclear envelope through Pom33 ubiquitination, which drives INM redistribution. Taken together, our data demonstrate that the Asi1 E3 ligase has a novel function in INM protein regulation in addition to protein turnover.


Assuntos
Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
18.
Methods Mol Biol ; 1840: 137-161, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30141044

RESUMO

The Saccharomyces cerevisiae and Schizosaccharomyces pombe genomes encode a single SUN domain-containing protein, Mps3 and Sad1, respectively. Both localize to the yeast centrosome (known as the spindle pole body, SPB) and are essential for bipolar spindle formation. In addition, Mps3 and Sad1 play roles in chromosome organization in both mitotic and meiotic cells that are independent of their SPB function. To dissect the function of Mps3 at the nuclear envelope (NE) and SPB, we employed cell imaging methods such as scanning fluorescence cross-correlation spectroscopy (SFCCS) and single particle averaging with structured illumination microscopy (SPA-SIM) to determine the strength, nature, and location of protein-protein interactions in vivo. We describe how these same techniques can also be used in fission yeast to analyze Sad1, providing evidence of their applicability to other NE proteins and systems.


Assuntos
Citoesqueleto/metabolismo , Proteínas Fúngicas/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Análise Espectral , Imunofluorescência , Expressão Gênica , Genes Reporter , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Análise Espectral/métodos
19.
Nat Protoc ; 13(8): 1869-1895, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30072723

RESUMO

Biologists have long been fascinated with the organization and function of intricate protein complexes. Therefore, techniques for precisely imaging protein complexes and the location of proteins within these complexes are critically important and often require multidisciplinary collaboration. A challenge in these explorations is the limited resolution of conventional light microscopy. However, a new microscopic technique has circumvented this resolution limit by making the biological sample larger, thus allowing for super-resolution of the enlarged structure. This 'expansion' is accomplished by embedding the sample in a hydrogel that, when exposed to water, uniformly expands. Here, we present a protocol that transforms thick expansion microscopy (ExM) hydrogels into sections that are physically expanded four times, creating samples that are compatible with the super-resolution technique structured illumination microscopy (SIM). This super-resolution ExM method (ExM-SIM) allows the analysis of the three-dimensional (3D) organization of multiprotein complexes at ~30-nm lateral (xy) resolution. This protocol details the steps necessary for analysis of protein localization using ExM-SIM, including antibody labeling, hydrogel preparation, protease digestion, post-digestion antibody labeling, hydrogel embedding with tissue-freezing medium (TFM), cryosectioning, expansion, image alignment, and particle averaging. We have used this approach for 3D mapping of in situ protein localization in the Drosophila synaptonemal complex (SC), but it can be readily adapted to study thick tissues such as brain and organs in various model systems. This procedure can be completed in 5 d.


Assuntos
Proteínas de Drosophila/química , Microscopia/métodos , Imagem Óptica/métodos , Complexo Sinaptonêmico/química , Animais , Drosophila , Imageamento Tridimensional/métodos
20.
Mol Cell ; 71(1): 155-168.e7, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29979963

RESUMO

Protein self-assemblies modulate protein activities over biological timescales that can exceed the lifetimes of the proteins or even the cells that harbor them. We hypothesized that these timescales relate to kinetic barriers inherent to the nucleation of ordered phases. To investigate nucleation barriers in living cells, we developed distributed amphifluoric FRET (DAmFRET). DAmFRET exploits a photoconvertible fluorophore, heterogeneous expression, and large cell numbers to quantify via flow cytometry the extent of a protein's self-assembly as a function of cellular concentration. We show that kinetic barriers limit the nucleation of ordered self-assemblies and that the persistence of the barriers with respect to concentration relates to structure. Supersaturation resulting from sequence-encoded nucleation barriers gave rise to prion behavior and enabled a prion-forming protein, Sup35 PrD, to partition into dynamic intracellular condensates or to form toxic aggregates. Our results suggest that nucleation barriers govern cytoplasmic inheritance, subcellular organization, and proteotoxicity.


Assuntos
Fatores de Terminação de Peptídeos/metabolismo , Proteínas Priônicas/metabolismo , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citometria de Fluxo , Fatores de Terminação de Peptídeos/genética , Proteínas Priônicas/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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