SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1.

Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.

Artificially decreasing cortical tension generates aneuploidy in mouse oocytes.

Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.

Laser Ablation of Microtubule-Chromosome Attachment in Mouse Oocytes.

Laser ablation is a powerful tool to study forces within biological systems. This technique has been extensively used to study mitotic spindle formation and chromosome segregation. This chapter describes laser ablation of microtubule-chromosome attachments coupled to fluorescence live microscopy and quantitative analysis of individual chromosome movement after microtubule severing. This method allows to gain insight into the organization and dynamics of the meiotic spindle and chromosomes in metaphase I mouse oocytes.

Separation and Loss of Centrioles From Primordidal Germ Cells To Mature Oocytes In The Mouse.

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

Chromosome structural anomalies due to aberrant spindle forces exerted at gene editing sites in meiosis.

Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

Interactions between endosomal maturation and autophagy: analysis of ESCRT machinery during Caenorhabditis elegans development.

Endocytosis and autophagy are key vesicular pathways involved in degradation and recycling of cellular material. Both degradative pathways finally fuse with lysosome but are indeed interconnected at several levels. In particular, the fusion between endosomes and autophagosomes can generate intermediate vesicles named amphisomes. We analyzed the physiological and developmental roles of the ESCRT machinery in a model organism, the nematode Caenorhabditis elegans and showed that the blockage of the endosomal maturation triggers the induction of autophagic activity. This chapter describes several methods for studying endocytosis, autophagy, and their interconnection in C. elegans. A series of genetic, biochemical, and microscopy analyses has been used to study at the cellular and developmental levels, the cross talks between autophagy and endocytosis.

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant.

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.

The C. elegans LC3 acts downstream of GABARAP to degrade autophagosomes by interacting with the HOPS subunit VPS39.

The formation of the autophagic vesicles requires the recruitment of ubiquitin-like Atg8 proteins to the membrane of nascent autophagosomes. Seven Atg8 homologs are present in mammals, split into the LC3 and the GABARAP/GATE-16 families, whose respective functions are unknown. Using Caenorhabditis elegans, we investigated the functions of the GABARAP and the LC3 homologs, LGG-1 and LGG-2, in autophagosome biogenesis. Both LGG-1 and LGG-2 localize to the autophagosomes but display partially overlapping patterns. During allophagy, a developmentally stereotyped autophagic flux, LGG-1 acts upstream of LGG-2 to allow its localization to autophagosomes. LGG-2 controls the maturation of LGG-1-positive autophagosomes and facilitates the tethering with the lysosomes through a direct interaction with the VPS-39 HOPS complex subunit. Genetic analyses sustain a sequential implication of LGG-1, LGG-2, RAB-7, and HOPS complex to generate autolysosomes. The duplications of Atg8 in metazoans thus allowed the acquisition of specialized functions for autophagosome maturation.

Need an ESCRT for autophagosomal maturation?

Several reports in fly, nematode and mammalian cells have revealed that the inactivation of endosomal sorting complexes required for transport (ESCRT) blocks the endosomal maturation but also leads to the increased number of autophagosomal structures. In this review we compare these data and conclude that the way ESCRT mutations affect the relationships between autophagosomes and endosomes cannot be generalized but depends on the studied species. We propose that the effect of ESCRT mutations on autophagy is directly dependent of the level of interaction between autophagosomes and endosomes. In particular, the formation of amphisomes during autophagosomal maturation could be the key point to explain the differences observed between species. These observations highlight the importance of multiple model organisms to decipher the complexity of relationships between such dynamic vesicles.