Fndc-1 contributes to paternal mitochondria elimination in C. elegans.

Paternal mitochondria are eliminated following fertilization by selective autophagy, but the mechanisms that restrict this process to sperm-derived organelles are not well understood. FUNDC1 (FUN14 domain containing 1) is a mammalian mitophagy receptor expressed on the mitochondrial outer membrane that contributes to mitochondrial quality control following hypoxic stress. Like FUNDC1, the C. elegans ortholog FNDC-1 is widely expressed in somatic tissues and mediates hypoxic mitophagy. Here, we report that FNDC-1 is strongly expressed in sperm but not oocytes and contributes to paternal mitochondria elimination. Paternal mitochondrial DNA is normally undetectable in wildtype larva, but can be detected in the cross-progeny of fndc-1 mutant males. Moreover, loss of fndc-1 retards the rate of paternal mitochondria degradation, but not that of membranous organelles, a nematode specific membrane compartment whose fusion is required for sperm motility. This is the first example of a ubiquitin-independent mitophagy receptor playing a role in the selective degradation of sperm mitochondria.

Autophagosomal Sperm Organelle Clearance and mtDNA Inheritance in C. elegans.

The nematode C. elegans represents a powerful experimental system with key properties and advantages to study the mechanisms underlying mitochondrial DNA maternal inheritance and paternal components sorting. First, the transmission is uniparental and maternal as in many animal species; second, at fertilization sperm cells contain both mitochondria and mtDNA; and third, the worm allows powerful genetics and cell biology approaches to characterize the mechanisms underlying the uniparental and maternal transmission of mtDNA. Fertilization of C. elegans oocyte occurs inside the transparent body when the mature oocyte resumes meiosis I and passes through the spermatheca. One amoeboid sperm cell fuses with the oocyte and delivers its whole content. Among the structures entering the embryo, the sperm mitochondria and a fraction of the nematode-specific membranous organelles are rapidly degraded, whereas others like centrioles and sperm genomic DNA are transmitted. In this chapter, we will review the knowledge acquired on sperm inherited organelles clearance during the recent years using C. elegans.

Sperm-inherited organelle clearance in C. elegans relies on LC3-dependent autophagosome targeting to the pericentrosomal area.

Macroautophagic degradation of sperm-inherited organelles prevents paternal mitochondrial DNA transmission in C. elegans. The recruitment of autophagy markers around sperm mitochondria has also been observed in mouse and fly embryos but their role in degradation is debated. Both worm Atg8 ubiquitin-like proteins, LGG-1/GABARAP and LGG-2/LC3, are recruited around sperm organelles after fertilization. Whereas LGG-1 depletion affects autophagosome function, stabilizes the substrates and is lethal, we demonstrate that LGG-2 is dispensable for autophagosome formation but participates in their microtubule-dependent transport toward the pericentrosomal area prior to acidification. In the absence of LGG-2, autophagosomes and their substrates remain clustered at the cell cortex, away from the centrosomes and their associated lysosomes. Thus, the clearance of sperm organelles is delayed and their segregation between blastomeres prevented. This allowed us to reveal a role of the RAB-5/RAB-7 GTPases in autophagosome formation. In conclusion, the major contribution of LGG-2 in sperm-inherited organelle clearance resides in its capacity to mediate the retrograde transport of autophagosomes rather than their fusion with acidic compartments: a potential key function of LC3 in controlling the fate of sperm mitochondria in other species.

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

BACKGROUND: Studying 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. RESULTS: We 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. CONCLUSION: We 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.

An antibody free approach to probe the presence of poly-ubiquitin chains on C. elegans sperm derived organelles after fertilization.

Upon C. elegans 's oocyte fertilization, the sperm brings mitochondria and membranous organelles (MOs) which are rapidly eliminated by autophagy. Their poly-ubiquitylation is suspected to be a signal for their recognition and degradation but mitochondria poly-ubiquitylation remains debated. Using fluorescent Tandem-repeated Ubiquitin-Binding Entities (TUBEs) we confirmed the presence of K48- and K63-ubiquitin chains on MOs contrasting with the absence of signal on sperm mitochondria. This new and sensitive approach confirmed the poly-ubiquitylation of the MOs while providing additional arguments for the absence of substantial poly-ubiquitylation of sperm-derived mitochondria, suggesting that K63- and K48-poly-ubiquitylation are unlikely acting as a common targeting signal for their degradation.

Fast and easy method to culture and obtain large populations of male nematodes.

Caenorhabditis elegans is a model system widely used in fundamental research. Even though, nematodes are easy to maintain in the laboratory, obtaining large populations of worms require a lot of work and is time consuming. Furthermore, because C. elegans are mainly hermaphrodite it is even more complicated to obtain large amounts of males which make high-throughput experiments using C. elegans males very challenging. In order to overcome these limitations, we developed affordable and rapid methods to: (1) grow large synchronous worm populations (2) easily obtain large amounts of males We developed a culture method on plates to grow big synchronized worm populations with the standard incubators used on all worm labs. We also established an easy filtration method allowing to obtain large male populations in an hour. After filtering, the worm population contains more than 90% of adult males and no adult hermaphrodites since all the contaminants are larva and embryos. The culture and the filtering methods we developed are easy to implement and require a very limited investment in equipment and consumables beside the standard one present in worm labs. In addition, this filtering method could be applied to nematode's species similar in size to C. elegans.

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.

Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast.

Recent experiments have shown that gene repression can be correlated with relocation of genes to heterochromatin-rich silent domains. Here, we investigate whether nuclear architecture and spatial positioning can contribute directly to the transcriptional activity of a genetic locus in Saccharomyces cerevisiae. By disassembling telomeric silent domains without altering the chromatin-mediated silencing machinery, we show that the transcriptional activity of silencer--reporter constructs depends on intranuclear position. This demonstrates that telomeric silent domains are actively involved in transcriptional silencing. Employing fluorescent in situ hybridization (FISH) in combination with genetic assays, we demonstrate that telomeres control the establishment of transcriptional states by reversible partitioning with the perinuclear silencing domains. Anchoring telomeres interferes with their ability to assume an active state, whereas disassembly of silencing domains prevents telomeres from assuming a repressed state. Our data support a model in which domains of enriched transcriptional regulators allow genes to determine transcriptional states by spatial positioning.

Mitophagy of polarized sperm-derived mitochondria after fertilization.

Loss of membrane potential of sperm mitochondria has been regarded as the first step preceding mitophagy degradation after their entry into the C. elegans oocyte at fertilization. This is in line with the classical view of mitophagy of defective or abnormal mitochondria and could serve as a recognition signal for their specific and quick autophagy degradation. Here, using TMRE (tetramethylrhodamine ethyl ester) and live imaging we show that this is not the case. Instead, sperm inherited mitochondria show a stable labeling with TMRE before and at the time of autophagosomes formation. Interestingly, this labeling remains in late-stage-embryos of autophagy-defective-mutants suggesting that the loss of membrane potential occurs upon the entry of the mitochondria into the autophagy pathway. These stabilized and still polarized sperm mitochondria remain distinct but associated with the maternal-derived mitochondrial network suggesting a mechanism that prevents their fusion and represents an efficient additional protective system against fertilization-induced heteroplasmy.

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.

Caenorhabditis elegans nucleoporins Nup93 and Nup205 determine the limit of nuclear pore complex size exclusion in vivo.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate communication between the nucleus and the cytoplasm. To obtain insight into the structure and function of NPCs of multicellular organisms, we have initiated an extensive analysis of Caenorhabditis elegans nucleoporins. Of 20 assigned C. elegans nucleoporin genes, 17 were found to be essential for embryonic development either alone or in combination. In several cases, depletion of nucleoporins by RNAi caused severe defects in nuclear appearance. More specifically, the C. elegans homologs of vertebrate Nup93 and Nup205 were each found to be required for normal NPC distribution in the nuclear envelope in vivo. Depletion of Nup93 or Nup205 caused a failure in nuclear exclusion of nonnuclear macromolecules of approximately 70 kDa without preventing active nuclear protein import or the assembly of the nuclear envelope. The defects in NPC exclusion were accompanied by abnormal chromatin condensation and early embryonic arrest. Thus, the contribution to NPC structure of Nup93 and Nup205 is essential for establishment of normal NPC function and for cell viability.

Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos.

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin alpha and beta, are essential for both spindle assembly and nuclear formation in early embryos.

Modern tools to study nuclear pore complexes and nucleocytoplasmic transport in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is characterized by many features that make it highly attractive to study nuclear pore complexes (NPCs) and nucleocytoplasmic transport. NPC composition and structure are highly conserved in nematodes and being amenable to a variety of genetic manipulations, key aspects of nuclear envelope dynamics can be observed in great details during breakdown, reassembly, and interphase. In this chapter, we provide an overview of some of the most relevant modern techniques that allow researchers unfamiliar with C. elegans to embark on studies of nucleoporins in an intact organism through its development from zygote to aging adult. We focus on methods relevant to generate loss-of-function phenotypes and their analysis by advanced microscopy. Extensive references to available reagents, such as mutants, transgenic strains, and antibodies are equally useful to scientists with or without prior C. elegans or nucleoporin experience.

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.

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.

Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission.

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.

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.

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.