Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

During meiosis, homologous chromosomes associate to form the synaptonemal complex (SC), a structure essential for fertility. Information about the epigenetic features of chromatin within this structure at the level of superresolution microscopy is largely lacking. We combined single-molecule localization microscopy (SMLM) with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be nonrandomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin [trimethylation of histone H3 at lysine (Lys) 27 (H3K27me3)] are found at 500-nm intervals along the SC, whereas one of the ends of the SC displays a large and dense cluster of centromeric histone mark [trimethylation of histone H3 at Lys 9 (H3K9me3)]. Chromatin associated with active transcription [trimethylation of histone H3 at Lys 4 (H3K4me3)] is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 with an average spread larger than their periodicity. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher order genome organization is yet to be elucidated.

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Super-resolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes

10.1101/023432 ◽

2015 ◽

Author(s):

Kirti Prakash ◽

David Fournier ◽

Stefan Redl ◽

Gerrit Best ◽

Máté Borsos ◽

...

Keyword(s):

Single Molecule ◽

Histone H3 ◽

Super Resolution ◽

Periodic Patterns ◽

Meiotic Chromosomes ◽

H3k4me3 Mark ◽

Loop Pattern ◽

Active Transcription ◽

Dense Cluster ◽

Small Clusters

During meiosis, homologous chromosomes associate to form a unique structure called synaptonemal complex (SC) whose disruption leads to infertility. Information about the epigenetic features of chromatin within this structure at the level of super-resolution microscopy is largely lacking. We combined single molecule localization microscopy with quantitative analytical methods to describe the epigenetic landscape of meiotic chromosomes at the pachytene stage in mouse oocytes. DNA is found to be non-randomly distributed along the length of the SC in condensed clusters. Periodic clusters of repressive chromatin (trimethylation of histone H3 at lysine 27, H3K27me3) are found at 500 nm intervals along the SC, while one of the ends of SC displays a large and dense cluster of centromeric histone mark (trimethylation of histone H3 at lysine 9, H3K9me3). Chromatin associated with active transcription (trimethylation of histone H3 at lysine 4, H3K4me3) is arranged in a radial hair-like loop pattern emerging laterally from the SC. These loops seem to be punctuated with small clusters of H3K4me3 mark with an average spread larger than their spacing. Our findings indicate that the nanoscale structure of the pachytene chromosomes is constrained by periodic patterns of chromatin marks, whose function in recombination and higher-order genome organisation is yet to be elucidated.

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Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0661 ◽

2019 ◽

Vol 30 (12) ◽

pp. 1369-1376 ◽

Cited By ~ 9

Author(s):

Tim N. Baldering ◽

Marina S. Dietz ◽

Karl Gatterdam ◽

Christos Karathanasis ◽

Ralph Wieneke ◽

...

Keyword(s):

Membrane Proteins ◽

Single Molecule ◽

Fusion Proteins ◽

Fluorescent Proteins ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Molecular Information ◽

Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

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High-numerical-aperture cryogenic light microscopy for increased precision of superresolution reconstructions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618206114 ◽

2017 ◽

Vol 114 (15) ◽

pp. 3832-3836 ◽

Cited By ~ 15

Author(s):

Marc Nahmani ◽

Conor Lanahan ◽

David DeRosier ◽

Gina G. Turrigiano

Keyword(s):

Single Molecule ◽

Room Temperature ◽

Fluorescent Protein ◽

Numerical Aperture ◽

Fluorescent Imaging ◽

Objective Lens ◽

High Numerical Aperture ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Photoactivated Localization Microscopy

Superresolution microscopy has fundamentally altered our ability to resolve subcellular proteins, but improving on these techniques to study dense structures composed of single-molecule-sized elements has been a challenge. One possible approach to enhance superresolution precision is to use cryogenic fluorescent imaging, reported to reduce fluorescent protein bleaching rates, thereby increasing the precision of superresolution imaging. Here, we describe an approach to cryogenic photoactivated localization microscopy (cPALM) that permits the use of a room-temperature high-numerical-aperture objective lens to image frozen samples in their native state. We find that cPALM increases photon yields and show that this approach can be used to enhance the effective resolution of two photoactivatable/switchable fluorophore-labeled structures in the same frozen sample. This higher resolution, two-color extension of the cPALM technique will expand the accessibility of this approach to a range of laboratories interested in more precise reconstructions of complex subcellular targets.

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Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804725115 ◽

2018 ◽

Vol 115 (51) ◽

pp. 12991-12996 ◽

Cited By ~ 11

Author(s):

Pablo A. Gómez-García ◽

Erik T. Garbacik ◽

Jason J. Otterstrom ◽

Maria F. Garcia-Parajo ◽

Melike Lakadamyali

Keyword(s):

Single Molecule ◽

Cross Talk ◽

Learning Algorithm ◽

Imaging Modality ◽

Fluorescence Emission ◽

Acquisition Time ◽

Color Channel ◽

Superresolution Microscopy ◽

Superresolution Imaging ◽

Frequency Multiplexing

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore’s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as “frequency multiplexed DNA-PAINT,” or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.

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Novel fibrillar structure in the inversin compartment of primary cilia revealed by 3D single-molecule superresolution microscopy

Molecular Biology of the Cell ◽

10.1091/mbc.e19-09-0499 ◽

2020 ◽

Vol 31 (7) ◽

pp. 619-639 ◽

Cited By ~ 2

Author(s):

Henrietta W. Bennett ◽

Anna-Karin Gustavsson ◽

Camille A. Bayas ◽

Petar N. Petrov ◽

Nancie Mooney ◽

...

Keyword(s):

Molecular Structure ◽

Cell Lines ◽

Single Molecule ◽

Primary Cilia ◽

Three Dimensional ◽

Fibrillar Structure ◽

Systematic Analysis ◽

Superresolution Microscopy ◽

Superresolution Imaging

Using three-dimensional single-molecule superresolution imaging and systematic analysis of knockout cell lines, we have determined the molecular structure and composition of the inversin compartment in primary cilia. INVS establishes fibrillar structures that recruit ANKS6-NEK8 complexes to sequester NPHP3 in this unique periaxonemal compartment.

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Laser-free super-resolution microscopy

10.1101/121061 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kirti Prakash

Keyword(s):

Single Molecule ◽

Super Resolution ◽

Superresolution Microscopy ◽

Correlative Imaging ◽

Deep Blue ◽

Meiotic Chromosomes ◽

Enhanced Resolution ◽

Blue Excitation ◽

Localisation Microscopy ◽

Super Resolution Microscopy

We report that single-molecule superresolution microscopy can be achieved with a conventional epifluorescence microscope setup and a Mercury arc lamp. The configuration termed as laser-free super-resolution microscopy (LFSM), is an extension of single molecule localisation microscopy (SMLM) techniques and allows single molecules to be switched on and off (a phenomenon termed as “blinking”), detected and localised. The use of a short burst of deep blue excitation (350-380 nm) can be further used to reactivate the blinking, once the blinking process has slowed or stopped. A resolution of 90 nm is achieved on test specimens (mouse and amphibian meiotic chromosomes). Finally, we demonstrate that STED and LFSM can be performed on the same biological sample using a simple imaging medium. It is hoped that this type of correlative imaging will provide a basis for a further enhanced resolution.

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Extraction of accurate cytoskeletal actin velocity distributions from noisy measurements

10.1101/2020.08.13.247304 ◽

2020 ◽

Author(s):

Cayla M. Miller ◽

Elgin Korkmazhan ◽

Alexander R. Dunn

Keyword(s):

Single Molecule ◽

Actin Filaments ◽

Dynamical Behavior ◽

Jump Process ◽

Pairwise Distance ◽

Actin Dynamics ◽

Cell Cortex ◽

Superresolution Microscopy ◽

Distance Distributions ◽

Primary Fibroblasts

Dynamic remodeling of the actin cytoskeleton allows cells to migrate, change shape, and exert mechanical forces on their surroundings. How the complex dynamical behavior of the cytoskeleton arises from the interactions of its molecular components remains incompletely understood. Tracking the movement of individual actin filaments in living cells can in principle provide a powerful means of addressing this question. However, single-molecule fluorescence imaging measurements that could provide this information are limited by low signal-to-noise ratios, with the result that the localization errors for individual fluorophore fiducials attached to filamentous (F)-actin are comparable to the distances traveled by actin filaments between measurements. In this study we tracked the movement F-actin labeled with single-molecule densities of the fluorogenic label SiR-actin in primary fibroblasts and endothelial cells. We then used a Bayesian statistical approach to estimate true, underlying actin filament velocity distributions from the tracks of individual actin-associated fluorophores along with quantified localization uncertainties. This analysis approach is broadly applicable to inferring statistical pairwise distance distributions arising from noisy point localization measurements such as occur in superresolution microscopy. We found that F-actin velocity distributions were better described by a statistical jump process, in which filaments exist in mechanical equilibria punctuated by abrupt, jump-like movements, than by models incorporating combinations of diffusive motion and drift. A model with exponentially distributed time- and length-scales for filament jumps recapitulated F-actin velocity distributions measured for the cell cortex, integrin-based adhesions, and actin stress fibers, indicating that a common physical model can potentially describe F-actin dynamics in a variety of cellular contexts.

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Two classes of active transcription sites and their roles in developmental regulation

10.1101/2020.06.23.167338 ◽

2020 ◽

Author(s):

Sarah Robinson-Thiewes ◽

John McCloskey ◽

Judith Kimble

Keyword(s):

Single Molecule ◽

Developmental Regulation ◽

Full Length ◽

Genes Encoding ◽

Transcription Sites ◽

Core Components ◽

Active Transcription ◽

Multiple Levels ◽

Nascent Transcripts ◽

Transcriptional Output

AbstractGenes encoding powerful developmental regulators are exquisitely controlled, often at multiple levels. Here, we use single molecule FISH (smFISH) to investigate nuclear active transcription sites (ATS) and cytoplasmic mRNAs of three key regulatory genes along the C. elegans germline developmental axis. The genes encode ERK/MAP kinase and core components of the Notch-dependent transcription complex. Using differentially-labeled probes spanning either a long first intron or downstream exons, we identify two ATS classes that differ in transcriptional progression: iATS harbor partial nascent transcripts while cATS harbor full-length nascent transcripts. Remarkably, the frequencies of iATS and cATS are patterned along the germline axis in a gene-, stage- and sex-specific manner. Moreover, regions with more frequent iATS make fewer full-length nascent transcripts and mRNAs, whereas those with more frequent cATS produce more of them. We propose that the regulated balance of these two ATS classes has a major impact on transcriptional output during development.

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H3K9me2 protects lifespan against the transgenerational burden of germline transcription in C. elegans

10.1101/782136 ◽

2019 ◽

Cited By ~ 1

Author(s):

Teresa W. Lee ◽

Heidi S. David ◽

Amanda K. Engstrom ◽

Brandon S. Carpenter ◽

David J. Katz

Keyword(s):

Caenorhabditis Elegans ◽

Histone H3 ◽

Complex Trait ◽

Wild Type ◽

C Elegans ◽

Germline Transcription ◽

Active Transcription

ABSTRACTDuring active transcription, the COMPASS complex methylates histone H3 at lysine 4 (H3K4me). In Caenorhabditis elegans, mutations in COMPASS subunits, including WDR-5, extend lifespan and enable the inheritance of increased lifespan in wild-type descendants. Here we show that the increased lifespan of wdr-5 mutants is itself a transgenerational trait that manifests after eighteen generations and correlates with changes in the heterochromatin factor H3K9me2. Additionally, we find that wdr-5 mutant longevity and its inheritance requires the H3K9me2 methyltransferase MET-2 and can be recapitulated by a mutation in the putative H3K9me2 demethylase JHDM-1. These data suggest that lifespan is constrained by reduced H3K9me2 due to transcription-coupled H3K4me. wdr-5 mutants alleviate this burden, extending lifespan and enabling the inheritance of increased lifespan. Thus, H3K9me2 functions in the epigenetic establishment and inheritance of a complex trait. Based on this model, we propose that lifespan is limited by the germline in part because germline transcription reduces heterochromatin.

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Single-Molecule Imaging and Labelling with DNA-Based Superresolution Microscopy

Biophysical Journal ◽

10.1016/j.bpj.2019.11.920 ◽

2020 ◽

Vol 118 (3) ◽

pp. 146a

Author(s):

Mingjie Dai ◽

Ninning Liu ◽

Sinem K. Saka ◽

Peng Yin

Keyword(s):

Single Molecule ◽

Single Molecule Imaging ◽

Superresolution Microscopy

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