Sebastien Phan

Transcellular degradation of axonal mitochondria.

Significance Mitochondria are organelles that perform many essential functions, including providing the energy to cells. Cells remove damaged mitochondria through a process called mitophagy. Mitophagy is considered a subset of a process called autophagy, by which damaged organelles are enwrapped and delivered to lysosomes for degradation. Implicit in the categorization of mitophagy as a subset of autophagy, which means “self-eating,” is the assumption that a cell degrades its own mitochondria. However, we show here that in a location called the optic nerve head, large numbers of mitochondria are shed from neurons to be degraded by the lysosomes of adjoining glial cells. This finding calls into question the assumption that a cell necessarily degrades its own organelles. It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

A close-up view inside the nucleus The nuclei of human cells contain 2 meters of genomic DNA. How does it all fit? Compaction starts with the DNA wrapping around histone octamers to form nucleosomes, but it is unclear how these further compress into mitotic chromosomes. Ou et al. describe a DNA-labeling method that allows them to visualize chromatin organization in human cells (see the Perspective by Larson and Misteli). They show that chromatin forms flexible chains with diameters between 5 and 24 nm. In mitotic chromosomes, chains bend back on themselves to pack at high density, whereas during interphase, the chromatin chains are more extended. Science, this issue p. eaag0025; see also p. 354 A new technique reveals that chromatin is a disordered 5- to 24-nanometer chain that is packed at different concentration densities according to the cell cycle. INTRODUCTION In human cells, 2 m of DNA are compacted in the nucleus through assembly with histones and other proteins into chromatin structures, megabase three-dimensional (3D) domains, and chromosomes that determine the activity and inheritance of our genomes. The long-standing textbook model is that primary 11-nm DNA–core nucleosome polymers assemble into 30-nm fibers that further fold into 120-nm chromonema, 300- to 700-nm chromatids, and, ultimately, mitotic chromosomes. Further extrapolating from this model, silent heterochromatin is generally depicted as 30- and 120-nm fibers. The hierarchical folding model is based on the in vitro structures formed by purified DNA and nucleosomes and on chromatin fibers observed in permeabilized cells from which other components had been extracted. Unfortunately, there has been no method that enables DNA and chromatin ultrastructure to be visualized and reconstructed unambiguously through large 3D volumes of intact cells. Thus, a remaining question is, what are the local and global 3D chromatin structures in the nucleus that determine the compaction and function of the human genome in interphase cells and mitotic chromosomes? RATIONALE To visualize and reconstruct chromatin ultrastructure and 3D organization across multiple scales in the nucleus, we developed ChromEMT, which combines electron microscopy tomography (EMT) with a labeling method (ChromEM) that selectivity enhances the contrast of DNA. This technique exploits a fluorescent dye that binds to DNA, and upon excitation, catalyzes the deposition of diaminobenzidine polymers on the surface, enabling chromatin to be visualized with OsO4 in EM. Advances in multitilt EMT allow us to reveal the chromatin ultrastructure and 3D packing of DNA in both human interphase cells and mitotic chromosomes. RESULTS ChromEMT enables the ultrastructure of individual chromatin chains, heterochromatin domains, and mitotic chromosomes to be resolved in serial slices and their 3D organization to be visualized as a continuum through large nuclear volumes in situ. ChromEMT stains and detects 30-nm fibers in nuclei purified from hypotonically lysed chicken erythrocytes and treated with MgCl2. However, we do not observe higher-order fibers in human interphase and mitotic cells in situ. Instead, we show that DNA and nucleosomes assemble into disordered chains that have diameters between 5 and 24 nm, with different particle arrangements, densities, and structural conformations. Chromatin has a more extended curvilinear structure in interphase nuclei and collapses into compact loops and interacting arrays in mitotic chromosome scaffolds. To analyze chromatin packing, we create 3D grid maps of chromatin volume concentrations (CVCs) in situ. We find that interphase nuclei have subvolumes with CVCs ranging from 12 to 52% and distinct spatial distribution patterns, whereas mitotic chromosome subvolumes have CVCs >40%. CONCLUSION We conclude that chromatin is a flexible and disordered 5- to 24-nm-diameter granular chain that is packed together at different concentration densities in interphase nuclei and mitotic chromosomes. The overall primary structure of chromatin polymers does not change in mitotic chromosomes, which helps to explain the rapid dynamics of chromatin condensation and how epigenetic interactions and structures can be inherited through cell division. In contrast to rigid fibers that have longer fixed persistence lengths, disordered 5- to 24-nm-diameter chromatin chains are flexible and can bend at various lengths to achieve different levels of compaction and high packing densities. The diversity of chromatin structures is exciting and provides a structural basis for how different combinations of DNA sequences, interactions, linker lengths, histone variants, and modifications can be integrated to fine-tune the function of genomic DNA in the nucleus to specify cell fate. Our data also suggest that the assembly of 3D domains in the nucleus with different chromatin concentrations, rather than higher-order folding, determines the global accessibility and activity of DNA. ChromEMT reveals the in situ chromatin ultrastructure, 3D packing, and organization of DNA. EMT sample volume-DNA-nucleosome chains are black. Chromatin is a structurally disordered 5- to 24-nm granular chain that is packed together at different 3D CVC densities in human interphase nuclei and mitotic chromosomes (red, high density; yellow, medium density; blue, low density). The chromatin structure of DNA determines genome compaction and activity in the nucleus. On the basis of in vitro structures and electron microscopy (EM) studies, the hierarchical model is that 11-nanometer DNA-nucleosome polymers fold into 30- and subsequently into 120- and 300- to 700-nanometer fibers and mitotic chromosomes. To visualize chromatin in situ, we identified a fluorescent dye that stains DNA with an osmiophilic polymer and selectively enhances its contrast in EM. Using ChromEMT (ChromEM tomography), we reveal the ultrastructure and three-dimensional (3D) organization of individual chromatin polymers, megabase domains, and mitotic chromosomes. We show that chromatin is a disordered 5- to 24-nanometer-diameter curvilinear chain that is packed together at different 3D concentration distributions in interphase and mitosis. Chromatin chains have many different particle arrangements and bend at various lengths to achieve structural compaction and high packing densities.

A split horseradish peroxidase for the detection of intercellular protein–protein interactions and sensitive visualization of synapses

Intercellular protein–protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection, and synaptic transmission, but they are challenging to visualize because existing techniques have insufficient sensitivity and/or specificity. Here we report a split horseradish peroxidase (sHRP) as a sensitive and specific tool for the detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable to studying mechanisms of communication between a variety of cell types.

A Rab5 endosomal pathway mediates Parkin-dependent mitochondrial clearance

Damaged mitochondria pose a lethal threat to cells that necessitates their prompt removal. The currently recognized mechanism for disposal of mitochondria is autophagy, where damaged organelles are marked for disposal via ubiquitylation by Parkin. Here we report a novel pathway for mitochondrial elimination, in which these organelles undergo Parkin-dependent sequestration into Rab5-positive early endosomes via the ESCRT machinery. Following maturation, these endosomes deliver mitochondria to lysosomes for degradation. Although this endosomal pathway is activated by stressors that also activate mitochondrial autophagy, endosomal-mediated mitochondrial clearance is initiated before autophagy. The autophagy protein Beclin1 regulates activation of Rab5 and endosomal-mediated degradation of mitochondria, suggesting cross-talk between these two pathways. Abrogation of Rab5 function and the endosomal pathway results in the accumulation of stressed mitochondria and increases susceptibility to cell death in embryonic fibroblasts and cardiac myocytes. These data reveal a new mechanism for mitochondrial quality control mediated by Rab5 and early endosomes.

Three-Dimensional Reconstruction of Serial Mouse Brain Sections: Solution for Flattening High-Resolution Large-Scale Mosaics

Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy (Chow et al., 2006; Price et al., 2006). With this technology, researchers can collect and analyze high-resolution digitized volumes of whole brain sections down to 0.2 μm. However, until recently, scientists lacked the tools to easily handle these large high-resolution datasets. Furthermore, artifacts resulting from specimen preparation limited volume reconstruction using this technique to only a single tissue section. In this paper, we carefully describe the steps we used to digitally reconstruct a series of consecutive mouse brain sections labeled with three stains, a stain for blood vessels (DiI), a nuclear stain (TO-PRO-3), and a myelin stain (FluoroMyelin). These stains label important neuroanatomical landmarks that are used for stacking the serial sections during reconstruction. In addition, we show that the use of two software applications, ir-Tweak and Mogrifier, in conjunction with a volume flattening procedure enable scientists to adeptly work with digitized volumes despite tears and thickness variations within tissue sections. These applications make processing large-scale brain mosaics more efficient. When used in combination with new database resources, these brain maps should allow researchers to extend the lifetime of their specimens indefinitely by preserving them in digital form, making them available for future analyses as our knowledge in the field of neuroscience continues to expand.

TxBR montage reconstruction for large field electron tomography

Electron tomography (ET) has been proven an essential technique for imaging the structure of cells beyond the range of the light microscope down to the molecular level. Large-field high-resolution views of biological specimens span more than four orders of magnitude in spatial scale, and, as a consequence, are rather difficult to generate directly. Various techniques have been developed towards generating those views, from increasing the sensor array size to implementing serial sectioning and montaging. Datasets and reconstructions obtained by the latter techniques generate multiple three-dimensional (3D) reconstructions, that need to be combined together to provide all the multiscale information. In this work, we show how to implement montages within TxBR, a tomographic reconstruction software package. This work involves some new application of mathematical concepts related to volume preserving transformations and issues of gauge ambiguity, which are essential problems arising from the nature of the observation in an electron microscope. The purpose of TxBR is to handle those issues as generally as possible in order to correct for most distortions in the 3D reconstructions and allow for a seamless recombination of ET montages.

Assembly of Excitatory Synapses in the Absence of Glutamatergic Neurotransmission

Synaptic excitation mediates a broad spectrum of structural changes in neural circuits across the brain. Here, we examine the morphologies, wiring, and architectures of single synapses of projection neurons in the murine hippocampus that developed in virtually complete absence of vesicular glutamate release. While these neurons had smaller dendritic trees and/or formed fewer contacts in specific hippocampal subfields, their stereotyped connectivity was largely preserved. Furthermore, loss of release did not disrupt the morphogenesis of presynaptic terminals and dendritic spines, suggesting that glutamatergic neurotransmission is unnecessary for synapse assembly and maintenance. These results underscore the instructive role of intrinsic mechanisms in synapse formation.

Tomography of Large Format Electron Microscope Tilt Series: Image Alignment and Volume Reconstruction

Image alignment is a critical step in obtaining high quality reconstructions. Bundle adjustment, based on a general projective model, combined with calculation of the envelope of backprojected tangents to a surface of revolution around the rotation axis establish the parameters of the projection maps, rotation angles and axis of rotation. Subsequent regression techniques may be used to calculate higher order polynomial corrections to the projection maps, and can compensate for curvilinear trajectories through the object, sample warping, and optical aberration. Backprojection from properly filtered images along the nominal electron trajectories yields high quality 3D reconstructions. We report here on recent extensions to the electron microscope tomography code, TxBR. In addition we discuss the basis for this code in the theory of Fourier integral operators.

Electron tomography and multiscale biology

Electron tomography (ET) is an emerging technology for the three dimensional imaging of cellular ultrastructure. In combination with other techniques, it can provide three dimensional reconstructions of protein assemblies, correlate 3D structures with functional investigations at the light microscope level and provide structural information which extends the findings of genomics and molecular biology. Realistic physical details are essential for the task of modeling over many spatial scales. While the electron microscope resolution can be as low as a fraction of a nm, a typical 3D reconstruction may just cover 1/1015 of the volume of an optical microscope reconstruction. In order to bridge the gap between those two approaches, the available spatial range of an ET reconstruction has been expanded by various techniques. Large sensor arrays and wide-field camera assemblies have increased the field dimensions by a factor of ten over the past decade, and new techniques for serial tomography and montaging make possible the assembly of many three-dimensional reconstructions. The number of tomographic volumes necessary to incorporate an average cell down to the protein assembly level is of the order 104, and given the imaging and algorithm requirements, the computational problem lays well in the exascale range. Tomographic reconstruction can be made parallel to a very high degree, and their associated algorithms can be mapped to the simplified processors comprising, for example, a graphics processor unit. Programming this on a GPU board yields a large speedup, but we expect that many more orders of magnitude improvement in computational capabilities will still be required in the coming decade. Exascale computing will raise a new set of problems, associated with component energy requirements (cost per operation and costs of data transfer) and heat dissipation issues. As energy per operation is driven down, reliability decreases, which in turn raises difficult problems in validation of computer models (is the algorithmic approach faithful to physical reality), and verification of codes (is the computation reliably correct and replicable). Leaving aside the hardware issues, many of these problems will require new mathematical and algorithmic approaches, including, potentially, a re-evaluation of the Turing model of computation.

3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography

Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding.