A multi-layered structure of the interphase chromocenter revealed by proximity-based biotinylation

Abstract During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.

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Super-Resolution Microscopy of Chromatin

Genes ◽

10.3390/genes10070493 ◽

2019 ◽

Vol 10 (7) ◽

pp. 493 ◽

Cited By ~ 1

Author(s):

Birk

Keyword(s):

Gene Regulation ◽

Data Analysis ◽

Super Resolution ◽

Fluorescence Labeling ◽

Cellular Processes ◽

Direct Assessment ◽

Chromatin Interactions ◽

Resolution Imaging ◽

Super Resolution Microscopy ◽

Insight Into

Since the advent of super-resolution microscopy, countless approaches and studies have been published contributing significantly to our understanding of cellular processes. With the aid of chromatin-specific fluorescence labeling techniques, we are gaining increasing insight into gene regulation and chromatin organization. Combined with super-resolution imaging and data analysis, these labeling techniques enable direct assessment not only of chromatin interactions but also of the function of specific chromatin conformational states.

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FIP200 organizes the autophagy machinery at p62-ubiquitin condensates beyond activation of the ULK1 kinase

10.1101/2020.07.07.191189 ◽

2020 ◽

Author(s):

Eleonora Turco ◽

Irmgard Fischer ◽

Sascha Martens

Keyword(s):

Kinase Activity ◽

De Novo ◽

Super Resolution ◽

Membrane Vesicles ◽

Degradation Pathway ◽

Autophagosome Formation ◽

Ubiquitinated Proteins ◽

The Many ◽

Super Resolution Microscopy ◽

Insight Into

AbstractMacroautophagy is a conserved degradation pathway, which mediates cellular homeostasis by the delivery of harmful substances into lysosomes. This is achieved by the sequestration of these substances referred to as cargo within double membrane vesicles, the autophagosomes, which form de novo. Among the many cargoes that are targeted by autophagy are condensates containing p62 and ubiquitinated proteins. p62 recruits the FIP200 protein to initiate autophagosome formation at the condensates. How FIP200 in turn organizes the autophagy machinery is unclear. Here we show that FIP200 is dispensable for the recruitment of the upstream autophagy machinery to the condensates, but it is necessary for phosphatidylinositol 3-phosphate formation and WIPI2 recruitment. We further find that FIP200 is required for the activation of the ULK1 kinase. Surprisingly, ULK1 kinase activity is not strictly required for autophagosome formation at p62 condensates. Super-resolution microscopy of p62 condensates revealed that FIP200 surrounds the condensates where it spatially organizes ATG13 and ATG9A for productive autophagosome formation. Our data provide a mechanistic insight into how FIP200 orchestrates autophagosome initiation at the cargo.

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Stochastic association of neighboring replicons creates replication factories in budding yeast

The Journal of Cell Biology ◽

10.1083/jcb.201306143 ◽

2013 ◽

Vol 202 (7) ◽

pp. 1001-1012 ◽

Cited By ~ 37

Author(s):

Nazan Saner ◽

Jens Karschau ◽

Toyoaki Natsume ◽

Marek Gierliński ◽

Renata Retkute ◽

...

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Budding Yeast ◽

Super Resolution ◽

Replication Proteins ◽

Replication Factory ◽

Genome Wide ◽

Nuclear Structures ◽

Super Resolution Microscopy ◽

Insight Into

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

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Novel insight into the potential pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication mutations in patients with Pelizaeus–Merzbacher disease

10.21203/rs.3.rs-216270/v1 ◽

2021 ◽

Author(s):

Ruoyu Duan ◽

Liuju Li ◽

Huifang Yan ◽

Miao He ◽

Kai Gao ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Morphological Changes ◽

Super Resolution ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Potential Pathogenicity ◽

Pelizaeus Merzbacher Disease ◽

Super Resolution Microscopy ◽

First Time ◽

Insight Into

Abstract Among the hypomyelinating leukodystrophies, Pelizaeus–Merzbacher disease (PMD) is a representative disorder. The disease is caused by different types of PLP1 mutations, among which PLP1 duplication accounts for ~ 70% of the mutations. Previous studies have shown that PLP1 duplications lead to PLP1 retention in the endoplasmic reticulum (ER); in parallel, recent studies have demonstrated that PLP1 duplication can also lead to mitochondrial dysfunction. As such, the respective roles and interactions of the ER and mitochondria in the pathogenesis of PLP1 duplication are not clear. In both PLP1 patients’ and healthy fibroblasts, we measured mitochondrial respiration with a Seahorse XF Extracellular Analyzer and examined the interactions between the ER and mitochondria with super-resolution microscopy (spinning-disc pinhole-based structured illumination microscopy, SD-SIM). For the first time, we demonstrated that PLP1 duplication mutants had closer ER-mitochondrion interfaces mediated through structural and morphological changes in both the ER and mitochondria-associated membranes (MAMs). These changes in both the ER and mitochondria then led to mitochondrial dysfunction, as reported previously. This work highlights the roles of MAMs in bridging PLP1 expression in the ER and pathogenic dysfunction in mitochondria, providing novel insight into the pathogenicity of mitochondrial dysfunction resulting from PLP1 duplication. These findings suggest that interactions between the ER and mitochondria may underlie pathogenic mechanisms of hypomyelinating leukodystrophies diseases at the organelle level.

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Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽

10.32607/2075851-2017-9-4-42-51 ◽

2017 ◽

Vol 9 (4) ◽

pp. 42-51

Author(s):

S. S. Ryabichko ◽

◽

A. N. Ibragimov ◽

L. A. Lebedeva ◽

E. N. Kozlov ◽

...

Keyword(s):

Cell Nucleus ◽

Super Resolution ◽

Structure And Function ◽

And Function ◽

Super Resolution Microscopy

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Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

10.26434/chemrxiv.9209450.v1 ◽

2019 ◽

Author(s):

Jeffrey Chang ◽

Matthew Romei ◽

Steven Boxer

Keyword(s):

Rational Design ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Super Resolution ◽

Unit Cell Size ◽

Chlorine Substituent ◽

Gfp Chromophore ◽

The One ◽

Green Fluorescent ◽

Super Resolution Microscopy

Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

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