Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Leake, Mark C.

doi:10.1080/21541264.2018.1475806

Cited by 8 publications

(5 citation statements)

References 81 publications

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“…The corresponding Slimfield images and histogram for complete strain sets are shown in Supplemental Figure 3. involved in bacterial DNA replication and remodeling (Reyes-Lamothe et al, 2010;Badrinarayanan et al, 2012), gene regulation in budding yeast cells (Wollman et al, 2017;Leake, 2018), bacterial cell division (Lund et al, 2018), and chemokine signaling in lymph nodes (Miller et al, 2018). Our prior measurements using relatively fast-maturing fluorescent proteins such as YFP suggest that less than 15% of fluorescent proteins are likely to be in a nonfluorescent immature state (Leake et al, 2008;Shashkova et al, 2018).…”

Section: Protein Stoichiometry Of Functional Carboxysomes At the Singmentioning

confidence: 99%

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

et al. 2019

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The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO 2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single b-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO 2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.

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Section: Protein Stoichiometry Of Functional Carboxysomes At the Singmentioning

confidence: 99%

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

et al. 2019

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“…We used single-molecule Slimfield microscopy (Plank et al, 2009) to visualize individual carboxysomes that were fused with YFP ( Figure 1, Supplemental Figure 3). This technique allows detection of fluorescently-labelled proteins with millisecond sampling, enabling real-time tracking of rapid protein dynamics inside living cells, exploited previously to study functional proteins involved in bacterial DNA replication and remodeling (Reyes-Lamothe et al, 2010;Badrinarayanan et al, 2012), gene regulation in budding yeast cells Leake, 2018), bacterial cell division (Lund et al, 2018), and chemokine signaling in lymph nodes (Miller et al, 2018). Our prior measurements using relatively fast maturing fluorescent proteins such as YFP suggest that less than 15% of fluorescent proteins are likely to be in a non-fluorescent immature state Shashkova et al, 2018).…”

Section: Protein Stoichiometry Of Functional Carboxysomes At the Singmentioning

confidence: 99%

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

Sun

Wollman

Huang

et al. 2019

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26The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon 27 assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that 28 self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO 2 29 fixation and potentials in bioengineering applications, the formation of carboxysomes and their 30 structural composition, stoichiometry and adaptation to cope with environmental changes remain 31unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and 32 electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of 33 single β-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We 34 determine the physiological abundance of individual building blocks within the icosahedral 35 carboxysome. We further find that the protein stoichiometry, diameter, localization and mobility 36 patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO 2 and 37 light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, 38 also applicable to other bacterial microcompartments and macromolecular self-assembling systems, 39advance our knowledge of the principles that mediate carboxysome formation and structural 40 modulation. It will empower rational design and construction of entire functional metabolic factories in 41 heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity. 42 43Keywords 44Bacterial microcompartment, carboxysome, protein stoichiometry, self-assembly, single-molecule 45 fluorescence imaging, structural flexibility 46 47 48 Organelle formation and compartmentalization within eukaryotic and prokaryotic cells provide 49 the structural foundation for segmentation and modulation of metabolic reactions in space and 50 time. Bacterial microcompartments (BMCs) are self-assembling organelles widespread 51 among bacterial phyla (Axen et al., 2014). By physically sequestering specific enzymes key 52 for metabolic processes from the cytosol, these organelles play important roles in CO 2 fixation, 53 pathogenesis, and microbial ecology (Yeates et al., 2010; Bobik et al., 2015). According to 54 their physiological roles, three types of BMCs have been characterized: the carboxysomes for 55 CO 2 fixation, the PDU microcompartments for 1,2−propanediol utilization, and the EUT 56 microcompartments for ethanolamine utilization. 57 58 The common features of various BMCs are that they are ensembles composed of purely 59 protein constituents and comprise an icosahedral single-layer shell that encases the catalytic 60 enzyme core. This proteinaceous shell, structurally resembling virus capsids, is self-61 assembled from several thousand polypeptides of multiple protein paralogs that form 62 hexagons, pentagons and trimers (Kerfeld and Erbilgin, 2015; Sutter et al., 2016; Faulkner et 63 al., 2017). The highly-ordered shell ...

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“…By investigating its nuclear organisation, dynamics, interactome and biochemical characteristics, we have been able to link its function to transcription and DNA regulation. To enhance their activity and functional efficiency, many nuclear proteins involved in transcription and other nuclear processes form molecular clusters 17,18,[29][30][31] . Here, we have determined that NDP52 clusters at regions of transcription initiation with RNAPII, and that its overexpression can increase the number of transcriptional clusters available in the nucleus.…”

Section: Discussionmentioning

confidence: 99%

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

Santos

Rollins

Hari-Gupta

et al. 2022

Preprint

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NDP52 is an autophagy receptor involved in the recognition and degradation of invading pathogens and damaged organelles. Although NDP52 was first identified in the nucleus and is expressed throughout the cell, to date, there is no clear nuclear function for NDP52. Here, we use a multidisciplinary approach to characterise the biochemical properties and nuclear roles of NDP52. We found that NDP52 clusters with RNA Polymerase II (RNAPII) at transcription initiation sites and that its overexpression promotes the formation of additional transcriptional clusters. We also show that depletion of NDP52 impacts overall gene-expression levels in two model mammalian cells, and that transcription inhibition affects the spatial organisation and molecular dynamics of NDP52 in the nucleus. This directly links NDP52 to a role in RNAPII-dependent transcription. Furthermore, we also show that NDP52 binds specifically and with high affinity to double-stranded DNA (dsDNA) and that this interaction leads to changes in DNA structure in vitro. This, together with our proteomics data indicating enrichment for interactions with nucleosome remodelling proteins and DNA structure regulators, suggests a possible function for NDP52 in chromatin regulation. Overall, here we uncover novel nuclear roles for NDP52 in gene expression and DNA structure regulation.

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Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

Cited by 8 publications

References 81 publications

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment

Autophagy receptor NDP52 alters DNA conformation to modulate RNA Polymerase II transcription

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