Vincent Galy

Postfertilization Autophagy of Sperm Organelles Prevents Paternal Mitochondrial DNA Transmission

Maternal inheritance of mitochondrial DNA results from autophagy-dependent clearance of paternal mitochondria. In sexual reproduction of most animals, the spermatozoon provides DNA and centrioles, together with some cytoplasm and organelles, to the oocyte that is being fertilized. Paternal mitochondria and their genomes are generally eliminated in the embryo by an unknown degradation mechanism. We show that, upon fertilization, a Caenorhabditis elegans spermatozoon triggers the recruitment of autophagosomes within minutes and subsequent paternal mitochondria degradation. Whereas the nematode-specific sperm membranous organelles are ubiquitinated before autophagosome formation, the mitochondria are not. The degradation of both paternal structures and mitochondrial DNA requires an LC3-dependent autophagy. Analysis of fertilized mouse embryos shows the localization of autophagy markers, which suggests that this autophagy event is evolutionarily conserved to prevent both the transmission of paternal mitochondrial DNA to the offspring and the establishment of heteroplasmy.

MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly

The metazoan nuclear envelope (NE) breaks down and re‐forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re‐forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL‐28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL‐28 interacts with the Nup107–160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107–160 complex to chromatin. We suggest that MEL‐28 acts as a seeding point for NPC assembly.

Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly

Barrier‐to‐autointegration factor (BAF) is an essential, highly conserved, metazoan protein. BAF interacts with LEM (LAP2, emerin, MAN1) domain‐carrying proteins of the inner nuclear membrane. We analyzed the in vivo function of BAF in Caenorhabditis elegans embryos using both RNA interference and a temperature‐sensitive baf‐1 gene mutation and found that BAF is directly involved in nuclear envelope (NE) formation. NE defects were observed independent of and before the chromatin organization phenotype previously reported in BAF‐depleted worms and flies. We identified vaccinia‐related kinase (VRK) as a regulator of BAF phosphorylation and localization. VRK localizes both to the NE and chromatin in a cell‐cycle‐dependent manner. Depletion of VRK results in several mitotic defects, including impaired NE formation and BAF delocalization. We propose that phosphorylation of BAF by VRK plays an essential regulatory role in the association of BAF with chromatin and nuclear membrane proteins during NE formation.

MEL-28, a Novel Nuclear-Envelope and Kinetochore Protein Essential for Zygotic Nuclear-Envelope Assembly in C. elegans

The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.

EhPAK, a member of the p21-activated kinase family, is involved in the control of Entamoeba histolytica migration and phagocytosis.

Entamoeba histolytica migration is essential for the development of amoebiasis, a human disease characterised by invasion and destruction of tissues. Amoebic motility requires both polarisation of the cell and formation of a predominant pseudopod. As p21-activated kinases PAKs are known to regulate eukaryotic cell motility and morphology, we investigated the role of PAK in E. histolytica. We showed that the C-terminal domain of EhPAK comprised a constitutive kinase activity in vitro and that overproduction of this fragment, in E. histolytica, caused a significant reduction in amoeboid migration, as measured by dynamic image analysis, indicating an involvement of EhPAK in this process. A dramatic loss of polarity, as indicated by the increased number of membrane extensions all around E. histolytica, was also observed, suggesting that the N-terminal domain of EhPAK was necessary for maintenance of cell polarity. To support this view, we showed that despite the absence of the consensus motif to bind to Rac and Cdc42, the N-terminal domain of EhPAK bound to Rac1, suggesting that the N-terminal region was a regulatory domain. In addition, we also found an increased rate of human red blood cell phagocytosis, suggesting for the first time an active role for a PAK protein in this process. Taking together, the results suggest strongly that EhPAK is a key regulatory element in polarity, motility and phagocytosis of E. histolytica.

A role for gp210 in mitotic nuclear-envelope breakdown

The cytoplasmic and nuclear compartments of animal cells mix during mitosis on disassembly of the nuclear envelope (NE). NE breakdown (NEBD) involves the dispersion of the nuclear membranes and associated proteins, including nuclear pore complexes (NPCs) and the nuclear lamina. Among the approximately 30 NPC components known, few contain transmembrane domains. gp210 is a single-pass transmembrane glycoprotein of metazoan NPCs. We show that both RNAi-mediated depletion and mutation of Caenorhabditis elegans gp210 affect NEBD in early embryonic cells, preventing lamin depolymerization and leading to the formation of twinned nuclei after mitosis owing to physical interference with normal chromosome alignment and segregation. When added to in vitro assembled nuclei, antibodies specific for the C-terminal cytoplasmic tail of gp210 completely blocked NEBD. This treatment inhibited mitotic hyper-phosphorylation of gp210. Phosphorylation of gp210 is proposed to be mediated by cyclin-B–cdc2 and we show that depletion of cyclin B from C. elegans embryos also leads to a nuclear-twinning phenotype. In summary, we show that gp210 is important for efficient NPC disassembly and NEBD and suggest that phosphorylation of gp210 is an early event in NEBD that is required for lamin disassembly and other aspects of NEBD.

CLLD8/KMT1F Is a Lysine Methyltransferase That Is Important for Chromosome Segregation

Proteins bearing a SET domain have been shown to methylate lysine residues in histones and contribute to chromatin architecture. Methylation of histone H3 at lysine 9 (H3K9) has emerged as an important player in the formation of heterochromatin, chromatin condensation, and transcriptional repression. Here, we have characterized a previously undescribed member of the histone H3K9 methyltransferase family named CLLD8 (or SETDB2 or KMT1F). This protein contributes to the trimethylation of both interspersed repetitive elements and centromere-associated repeats and participates in the recruitment of heterochromatin protein 1 to centromeres. Consistently, depletion in CLLD8/KMT1F coincides with a loss of CENP proteins and delayed mitosis, suggesting that this protein participates in chromosome condensation and segregation. Altogether, our results provide evidence that CLLD8/KMT1F is recruited to heterochromatin regions and contributes in vivo to the deposition of trimethyl marks in concert with SUV39H1/KMT1A.

A quantitative method for measuring phototoxicity of a live cell imaging microscope.

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.

Allophagy: a macroautophagic process degrading spermatozoid-inherited organelles.

In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.In most animals, during oocyte fertilization the spermatozoon provides DNA and centrioles together with some cytoplasm and organelles, but paternal mitochondria are generally eliminated in the embryo. Using the model animal C. elegans we have shown that paternal organelle degradation is dependent on the formation of autophagosomes a few minutes after fertilization. This macroautophagic process is preceded by an active ubiquitination of some spermatozoon-inherited organelles. Analysis of fertilized mouse embryos suggests that this autophagy event is evolutionarily conserved.

A generic classification-based method for segmentation of nuclei in 3D images of early embryos

BackgroundStudying how individual cells spatially and temporally organize within the embryo is a fundamental issue in modern developmental biology to better understand the first stages of embryogenesis. In order to perform high-throughput analyses in three-dimensional microscopic images, it is essential to be able to automatically segment, classify and track cell nuclei. Many 3D/4D segmentation and tracking algorithms have been reported in the literature. Most of them are specific to particular models or acquisition systems and often require the fine tuning of parameters.ResultsWe present a new automatic algorithm to segment and simultaneously classify cell nuclei in 3D/4D images. Segmentation relies on training samples that are interactively provided by the user and on an iterative thresholding process. This algorithm can correctly segment nuclei even when they are touching, and remains effective under temporal and spatial intensity variations. The segmentation is coupled to a classification of nuclei according to cell cycle phases, allowing biologists to quantify the effect of genetic perturbations and drug treatments. Robust 3D geometrical shape descriptors are used as training features for classification. Segmentation and classification results of three complete datasets are presented. In our working dataset of the Caenorhabditis elegans embryo, only 21 nuclei out of 3,585 were not detected, the overall F-score for segmentation reached 0.99, and more than 95% of the nuclei were classified in the correct cell cycle phase. No merging of nuclei was found.ConclusionWe developed a novel generic algorithm for segmentation and classification in 3D images. The method, referred to as Adaptive Generic Iterative Thresholding Algorithm (AGITA), is freely available as an ImageJ plug-in.