Super-resolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact C. elegans tissue

AbstractWhen cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC), and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the three-dimensional organization of components within the axes, which consist of cohesin complexes and additional meiosis-specific proteins. Here we investigate the molecular organization of meiotic chromosome axes in C. elegans through STORM and PALM superresolution imaging of intact germline tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using three-dimensional averaging, we determined the 3D-organization of all known components within synapsed chromosome axes to a precision of 2-5 nanometers. We find that meiosis-specific HORMA-domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Cohesin complexes containing COH-3 or -4 kleisins form a central core in the central plane of the axes, whereas complexes containing REC-8 kleisin protrude above and below the plane defined by the SC. This splayed organization may help to explain the role of the chromosome axes in promoting inter-homolog repair of meiotic double strand breaks by inhibiting inter-sister repair.

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Superresolution microscopy reveals the three-dimensional organization of meiotic chromosome axes in intact Caenorhabditis elegans tissue

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1702312114 ◽

2017 ◽

Vol 114 (24) ◽

pp. E4734-E4743 ◽

Cited By ~ 40

Author(s):

Simone Köhler ◽

Michal Wojcik ◽

Ke Xu ◽

Abby F. Dernburg

Keyword(s):

Caenorhabditis Elegans ◽

Fluorescent Protein ◽

Germ Line ◽

Meiotic Chromosome ◽

Three Dimensional ◽

Three Dimensions ◽

Molecular Organization ◽

Superresolution Microscopy ◽

Specific Proteins ◽

Optical Reconstruction

When cells enter meiosis, their chromosomes reorganize as linear arrays of chromatin loops anchored to a central axis. Meiotic chromosome axes form a platform for the assembly of the synaptonemal complex (SC) and play central roles in other meiotic processes, including homologous pairing, recombination, and chromosome segregation. However, little is known about the 3D organization of components within the axes, which include cohesin complexes and additional meiosis-specific proteins. Here, we investigate the molecular organization of meiotic chromosome axes in Caenorhabditis elegans through STORM (stochastic optical reconstruction microscopy) and PALM (photo-activated localization microscopy) superresolution imaging of intact germ-line tissue. By tagging one axis protein (HIM-3) with a photoconvertible fluorescent protein, we established a spatial reference for other components, which were localized using antibodies against epitope tags inserted by CRISPR/Cas9 genome editing. Using 3D averaging, we determined the position of all known components within synapsed chromosome axes to high spatial precision in three dimensions. We find that meiosis-specific HORMA domain proteins span a gap between cohesin complexes and the central region of the SC, consistent with their essential roles in SC assembly. Our data further suggest that the two different meiotic cohesin complexes are distinctly arranged within the axes: Although cohesin complexes containing the kleisin REC-8 protrude above and below the plane defined by the SC, complexes containing COH-3 or -4 kleisins form a central core, which may physically separate sister chromatids. This organization may help to explain the role of the chromosome axes in promoting interhomolog repair of meiotic double-strand breaks by inhibiting intersister repair.

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POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2019071118 ◽

2021 ◽

Vol 118 (11) ◽

pp. e2019071118

Author(s):

Ayana Sugizaki ◽

Keisuke Sato ◽

Kazuyoshi Chiba ◽

Kenta Saito ◽

Masahiko Kawagishi ◽

...

Keyword(s):

Actin Filaments ◽

Molecular Orientation ◽

Fluorescent Protein ◽

Three Dimensional ◽

Super Resolution ◽

Living Cells ◽

Test Case ◽

Polarization Microscopy ◽

Universal Method ◽

Early Embryos

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

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Targeted Gene Knockout Reveals a Role in Meiotic Recombination for ZHP-3, a Zip3-Related Protein in Caenorhabditis elegans

Molecular and Cellular Biology ◽

10.1128/mcb.24.18.7998-8006.2004 ◽

2004 ◽

Vol 24 (18) ◽

pp. 7998-8006 ◽

Cited By ~ 69

Author(s):

Verena Jantsch ◽

Pawel Pasierbek ◽

Michael M. Mueller ◽

Dieter Schweizer ◽

Michael Jantsch ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Meiotic Recombination ◽

Fluorescent Protein ◽

Gene Knockout ◽

Chiasma Formation ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

C Elegans ◽

Reciprocal Recombination

ABSTRACT The meiotically expressed Zip3 protein is found conserved from Saccharomyces cerevisiae to humans. In baker's yeast, Zip3p has been implicated in synaptonemal complex (SC) formation, while little is known about the protein's function in multicellular organisms. We report here the successful targeted gene disruption of zhp-3 (K02B12.8), the ZIP3 homolog in the nematode Caenorhabditis elegans. Homozygous zhp-3 knockout worms show normal homologue pairing and SC formation. Also, the timing of appearance and the nuclear localization of the recombination protein Rad-51 seem normal in these animals, suggesting proper initiation of meiotic recombination by DNA double-strand breaks. However, the occurrence of univalents during diplotene indicates that C. elegans ZHP-3 protein is essential for reciprocal recombination between homologous chromosomes and thus chiasma formation. In the absence of ZHP-3, reciprocal recombination is abolished and double-strand breaks seem to be repaired via alternative pathways, leading to achiasmatic chromosomes and the occurrence of univalents during meiosis I. Green fluorescent protein-tagged C. elegans ZHP-3 forms lines between synapsed chromosomes and requires the SC for its proper localization.

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C. elegans DSB-3 Reveals Conservation and Divergence among Protein Complexes Promoting Meiotic Double-Strand Breaks

10.1101/2021.05.14.444243 ◽

2021 ◽

Author(s):

Albert W Hinman ◽

Hsin-Yi Yeh ◽

Baptiste Roelens ◽

Kei Yamaya ◽

Alexander Woglar ◽

...

Keyword(s):

Meiotic Recombination ◽

Protein Complexes ◽

Meiotic Chromosome ◽

Structured Illumination ◽

Dna Breaks ◽

Structured Illumination Microscopy ◽

Strand Breaks ◽

C Elegans ◽

Double Strand Dna Breaks ◽

Essential Components

Meiotic recombination plays dual roles in the evolution and stable inheritance of genomes: recombination promotes genetic diversity by reassorting variants, and it establishes temporary connections between pairs of homologous chromosomes that ensure for their future segregation. Meiotic recombination is initiated by generation of double-strand DNA breaks (DSBs) by the conserved topoisomerase-like protein Spo11. Despite strong conservation of Spo11 across eukaryotic kingdoms, auxiliary complexes that interact with Spo11 complexes to promote DSB formation are poorly conserved. Here, we identify DSB-3 as a DSB-promoting protein in the nematode Caenorhabditis elegans. Mutants lacking DSB-3 are proficient for homolog pairing and synapsis but fail to form meiotic crossovers. Lack of crossovers in dsb-3 mutants reflects a requirement for DSB-3 in meiotic DSB formation. DSB-3 concentrates in meiotic nuclei with timing similar to DSB-1 and DSB-2 (predicted homologs of yeast/mammalian Rec114/REC114), and DSB-1, DSB-2, and DSB-3 are interdependent for this localization. Bioinformatics analysis and interactions among the DSB proteins support the identity of DSB-3 as a homolog of MEI4 in conserved DSB-promoting complexes. This identification is reinforced by colocalization of pairwise combinations of DSB-1, DSB-2, and DSB-3 foci in structured illumination microscopy images of spread nuclei. However, unlike yeast Rec114, DSB-1 can interact directly with SPO-11, and in contrast to mouse REC114 and MEI4, DSB-1, DSB-2 and DSB-3 are not concentrated predominantly at meiotic chromosome axes. We speculate that variations in the meiotic program that have co-evolved with distinct reproductive strategies in diverse organisms may contribute to and/or enable diversification of essential components of the meiotic machinery.

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High Resolution View on the Regulation of Recombinase Accumulation in Mammalian Meiosis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.672191 ◽

2021 ◽

Vol 9 ◽

Author(s):

Aditya N. Mhaskar ◽

Lieke Koornneef ◽

Alex N. Zelensky ◽

Adriaan B. Houtsmuller ◽

Willy M. Baarends

Keyword(s):

Molecular Mechanisms ◽

Single Cells ◽

Somatic Cells ◽

Super Resolution ◽

Repair Process ◽

Molecular Organization ◽

Dna Double Strand Breaks ◽

Subsequent Formation ◽

Strand Breaks ◽

Meiotic Dsbs

A distinguishing feature of meiotic DNA double-strand breaks (DSBs), compared to DSBs in somatic cells, is the fact that they are induced in a programmed and specifically orchestrated manner, which includes chromatin remodeling prior to DSB induction. In addition, the meiotic homologous recombination (HR) repair process that follows, is different from HR repair of accidental DSBs in somatic cells. For instance, meiotic HR involves preferred use of the homolog instead of the sister chromatid as a repair template and subsequent formation of crossovers and non-crossovers in a tightly regulated manner. An important outcome of this distinct repair pathway is the pairing of homologous chromosomes. Central to the initial steps in homology recognition during meiotic HR is the cooperation between the strand exchange proteins (recombinases) RAD51 and its meiosis-specific paralog DMC1. Despite our understanding of their enzymatic activity, details on the regulation of their assembly and subsequent molecular organization at meiotic DSBs in mammals have remained largely enigmatic. In this review, we summarize recent mouse data on recombinase regulation via meiosis-specific factors. Also, we reflect on bulk “omics” studies of initial meiotic DSB processing, compare these with studies using super-resolution microscopy in single cells, at single DSB sites, and explore the implications of these findings for our understanding of the molecular mechanisms underlying meiotic HR regulation.

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Single-molecule spectroscopy and imaging over the decades

Faraday Discussions ◽

10.1039/c5fd00149h ◽

2015 ◽

Vol 184 ◽

pp. 9-36 ◽

Cited By ~ 40

Author(s):

W. E. Moerner ◽

Yoav Shechtman ◽

Quan Wang

Keyword(s):

Single Molecule ◽

Optical Switching ◽

Fluorescent Protein ◽

Single Molecules ◽

Three Dimensional ◽

Super Resolution ◽

Laser Frequency ◽

High Resolution Spectroscopy ◽

Focal Volume ◽

Ensemble Averaging

As of 2015, it has been 26 years since the first optical detection and spectroscopy of single molecules in condensed matter. This area of science has expanded far beyond the early low temperature studies in crystals to include single molecules in cells, polymers, and in solution. The early steps relied upon high-resolution spectroscopy of inhomogeneously broadened optical absorption profiles of molecular impurities in solids at low temperatures. Spectral fine structure arising directly from the position-dependent fluctuations of the number of molecules in resonance led to the attainment of the single-molecule limit in 1989 using frequency-modulation laser spectroscopy. In the early 1990s, a variety of fascinating physical effects were observed for individual molecules, including imaging of the light from single molecules as well as observations of spectral diffusion, optical switching and the ability to select different single molecules in the same focal volume simply by tuning the pumping laser frequency. In the room temperature regime, researchers showed that bursts of light from single molecules could be detected in solution, leading to imaging and microscopy by a variety of methods. Studies of single copies of the green fluorescent protein also uncovered surprises, especially the blinking and photoinduced recovery of emitters, which stimulated further development of photoswitchable fluorescent protein labels. All of these early steps provided important fundamentals underpinning the development of super-resolution microscopy based on single-molecule localization and active control of emitting concentration. Current thrust areas include extensions to three-dimensional imaging with high precision, orientational analysis of single molecules, and direct measurements of photodynamics and transport properties for single molecules trapped in solution by suppression of Brownian motion. Without question, a huge variety of studies of single molecules performed by many talented scientists all over the world have extended our knowledge of the nanoscale and many microscopic mechanisms previously hidden by ensemble averaging.

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Cell biology of the future: Nanometer-scale cellular cartography

The Journal of Cell Biology ◽

10.1083/jcb.201508021 ◽

2015 ◽

Vol 211 (2) ◽

pp. 211-214 ◽

Cited By ~ 2

Author(s):

Justin W. Taraska

Keyword(s):

Electron Microscopy ◽

Cell Biology ◽

Cellular Structure ◽

Three Dimensional ◽

Super Resolution ◽

Molecular Organization ◽

Nanometer Scale ◽

Cellular Regulation ◽

New Developments ◽

Quantitative Image

Understanding cellular structure is key to understanding cellular regulation. New developments in super-resolution fluorescence imaging, electron microscopy, and quantitative image analysis methods are now providing some of the first three-dimensional dynamic maps of biomolecules at the nanometer scale. These new maps—comprehensive nanometer-scale cellular cartographies—will reveal how the molecular organization of cells influences their diverse and changeable activities.

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Sensory cilia act as a specialized venue for regulated EV biogenesis and signaling

10.1101/2021.02.04.429799 ◽

2021 ◽

Cited By ~ 1

Author(s):

Juan Wang ◽

Inna A. Nikonorova ◽

Malan Silva ◽

Jonathon D. Walsh ◽

Peter Tilton ◽

...

Keyword(s):

Fluorescent Protein ◽

Coiled Coil ◽

Super Resolution ◽

Intraflagellar Transport ◽

Sensory Stimulation ◽

Intercellular Signaling ◽

C Elegans ◽

Sensory Cilia ◽

Ciliated Neurons

AbstractExtracellular vesicles play major roles in intercellular signaling, yet fundamental aspects of their biology remain poorly understood. Ciliary EV shedding is evolutionary conserved. Here we use super resolution, real time imaging of fluorescent-protein tagged EV cargo combined with in vivo bioassays to study signaling EVs in C. elegans. We find that neuronal sensory cilia shed the TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites - the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. Blocking ciliary tip shedding results in excessive EV shedding from the base. Finally, we demonstrate that C. elegans male ciliated neurons modulate EV cargo composition in response to sensory stimulation by hermaphrodite mating partners. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

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POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos

10.1101/2020.06.20.116178 ◽

2020 ◽

Author(s):

Ayana Sugizaki ◽

Keisuke Sato ◽

Kazuyoshi Chiba ◽

Kenta Saito ◽

Masahiko Kawagishi ◽

...

Keyword(s):

Actin Filaments ◽

Molecular Orientation ◽

Fluorescent Protein ◽

Three Dimensional ◽

Super Resolution ◽

Living Cells ◽

Test Case ◽

Polarization Microscopy ◽

Universal Method ◽

Early Embryos

AbstractBiomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring orientation of fluorescently labeled molecules within living cells in real-time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but due to difficulties in the rational three-dimensional design of protein connection, universal method for constrained tagging with fluorophore was not available. Here we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner and can target arbitrary biomolecules by combining with phage-display screening. As an initial test case of POLArIS, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens including whole bodies of developing embryos and animals, and also expressed in a cell-type/tissue specific manner.

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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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