Daniel Rube et al. (2001) International C. elegans Meeting "Parallels between Mitochondrial Division and Endocytosis"

Ashley Wright, Daniel Rube, Alexander van der Bliek, Ayako Hasegawa

[International C. elegans Meeting, 2001]

Recently it has become clear that mitochondria are filamentous structures that continuously divide and fuse with one another throughout the life of a cell. We have been studying mitochondrial division in the C. elegans body wall muscle cells using organelle specific GFP markers. This cell type is ideally suited to look at subtle changes in mitochondrial morphology as the mitochondria are planar and thus easy to focus on using fluorescence microscopy, are sufficiently large in size, and are distributed evenly throughout the muscle cell. We first studied DRP-1, a homologue to the endocytosis protein, dynamin (DYN-1). By expressing both drp-1 ::GFP along with each of several organelle markers under the body wall muscle specific promoter, myo-3 , we found that DRP-1 was localized to the mitochondria. We then found that expression of a dominant negative form of DRP-1 blocks division of the mitochondrial outer membrane but division of the inner membrane still occurs. Furthermore, overexpressing drp-1 brings about more mitochondrial division. We concluded that DRP-1 acts to bring about division of the mitochondrial outer membrane. Additional confirmation was given by time-lapse observations of DRP-1 at sites where mitochondrial fission. Dynamin proteins act in both mitochondrial division and endocytosis. The prominent role of the same type of protein in these processes suggests that they may share a common evolutionary origin. It is not difficult to imagine that the first endosymbiotic bacteria used dynamin of the host cell during entry at the plasma membrane and used it again during the division of its outer membrane. Dynamin may not have only been the only protein used in this process as several additional proteins act along with dynamin in the endocytic process. To investigate whether these or similar proteins also play a role in mitochondrial division, we have been expressing antisense and snapback constructs of them to see if we can replicate the drp-1 loss-of-function phenotype observed. In preliminary results we have seen aberrant mitochondria in synaptojanin antisense but not endophilin antisense. However, we have seen abnormal mitochondria with antisense of an endophilin-related protein. The phenotypes seen suggest a mild division defect but we are seeking a stronger division defect through the use of double mutants and snapback constructs as an alternative to antisense. We are then planning to determine at what stage these proteins act in relation to DRP-1.

Daniel A Rube et al. (2003) International Worm Meeting "The role of ERP-1 in mitochondrial division and apoptosis."

Shilpa S Gandre, Daniel A Rube, Alexander M van der Bliek

[International Worm Meeting, 2003]

Mitochondria form a dynamic tubular network that undergoes both fission and fusion. Mitochondrial division is necessary to distribute mitochondria to progenitor cells. A role for mitochondrial division in apoptosis has been shown recently in mammalian systems. Our laboratory has studied proteins believed to be involved in mitochondrial division that are homologs of endocytosis proteins. The most well-studied is DRP-1, a homolog of dynamin that is required for mitochondrial division. More recently, we have been examining ERP-1, an endophilin homolog, as well as amphiphysin and synaptojanin. Here we provide evidence that endophilin A, a neuronal protein that induces membrane curvature at the necks of budding endocytic vesicles, also has a mitochondrial counterpart, which we call ERP-1 in C. elegans. ERP-1 is homologous to endophilin B in mammals. We find that loss of ERP-1 function in C. elegans muscle cells inhibits mitochondrial division, causing the mitochondria to form a contiguous network. We also find that ERP-1 loss of function does not affect endocytosis or secretion. In contrast, overexpression of ERP-1 causes mitochondria to fragment. Furthermore, a functional ERP-1::YFP fusion protein colocalizes with CFP::DRP-1 in spots on mitochondria. We conclude that ERP-1 cooperates with DRP-1 during mitochondrial division. It was shown recently that DRP-1 is required for apoptosis and cytochrome C release in mammalian cells. It was also recently shown that mammalian endophilin B interacts with mammalian Bax, a proapoptotic protein. The significance of the latter interaction is not known, though we believe it may enhance the rate of mitochondrial division during apoptosis. Since there is no obvious Bax homolog in C. elegans, we are looking for other ERP-1 interacting proteins.

Daniel A Rube et al. (2002) West Coast Worm Meeting "The endophilin-related protein ERP-1 regulates mitochondrial division"

Daniel A Rube, Ashley P Wright, Alexander M van der Bliek

[West Coast Worm Meeting, 2002]

Mitochondria form a dynamic tubular network that undergoes both fission and fusion. Mitochondrial division is necessary in order to distribute mitochondria to progenitor cells and also plays a role in apoptosis. We think that there are two different mitochondrial division mechanisms, one for the inner membrane and one from the outer membrane. Our hypothesis is that the inner membrane division mechanism is more closely related to that of bacterial division while the outer membrane division mechanism is similar to that of endocytosis. The scope of this investigation is to explore the function of variants of endocytosis proteins in mitochondrial division. The role of dynamin in endocytic vesicle formation is mirrored by the role of dynamin-related protein DRP-1 in mitochondrial division. We now provide evidence that endophilin, a protein that induces membrane curvature at the necks of budding endocytic vesicles, also has a mitochondrial counterpart, which we call ERP-1 in C. elegans. We find that loss of ERP-1 function in C. elegans muscle cells selectively inhibits mitochondrial division, causing the mitochondria to be nearly contiguously networked. In contrast, overexpression of ERP-1 causes mitochondria to fragment. Time lapse photography shows that an ERP-1::YFP fusion protein colocalizes with CFP::DRP-1 in spots on mitochondria. We conclude that ERP-1 cooperates with DRP-1 during mitochondrial division. The discovery that two very different endocytic proteins have relatives required for division of the mitochondrial outer membrane suggests that the machineries for these two processes share a common evolutionary origin.

(2024) Development "The people behind the papers - Nadia Manzi and Daniel Dickinson."

[Development, 2024]

The mitotic kinase Aurora A has been shown to regulate the anterior-posterior polarity in developing Caenorhabditis elegans embryos. In a new study, Daniel Dickinson and colleagues find that Aurora A has temporally distinct roles in coordinating the localization of Partitioning defective (PAR) proteins to establish cell polarity during development. To find out more about the story behind the paper, we caught up with first author Nadia Manzi and corresponding author Daniel Dickinson, Assistant Professor at the University of Texas at Austin.

(2019) Development "The people behind the papers - Daniel Osorio, Elaine Chan, Joana Saramago and Ana Carvalho."

[Development, 2019]

Animal cytokinesis is driven by an actomyosin ring that assembles at the cell equator and constricts to physically separate the two daughters. Although myosin is known to be essential for cytokinesis in multiple systems, whether this requirement reflects its motor or actin crosslinking activities has recently been a matter of contention. A new paper in Development now addresses this problem using the first divisions of the <i>Caenorhabditis elegans</i> embryo as a model. We caught up with the paper's three first authors Daniel Osorio, Elaine Chan and Joana Saramago, and their supervisor Ana Carvalho, Principal Investigator at the University of Porto's i3S consortium, to find out more about the story.

Vo, An A. et al. (2021) MicroPubl Biol

Ward, Jordan D., Ragle, James Matthew, Levenson, Max T., Vo, An A.

[MicroPubl Biol, 2021]

The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Zappaterra MD et al. (2000) West Coast Worm Meeting "Loss of a dynamin related protein MGM-1 causes excessive mitochondrial fragmentation"

van der Bliek A, Zappaterra MD

[West Coast Worm Meeting, 2000]

Our lab studies the functions of dynamin and dynamin related proteins. These proteins form a small family of large GTP-binding proteins. We have recently shown that the C. elegans dynamin related protein DRP-1, is involved in the scission of the mitochondrial outer membrane (1). Since little is known about mitochondrial division and mitochondrial morphology in animal cells, we are investigating the mechanisms that regulate these processes. MGM-1 is another member of the dynamin family present in animal cells. As of now we know that this protein is present in yeast, C. elegans, and humans. Unlike other members of the dynamin family, MGM-1 has an N-terminal mitochondrial leader sequence. MGM-1 is also more closely related to bacterial dynamin-like proteins than it is to dynamin or DRP-1, suggesting that this protein has followed a different evolutionary path. We speculate that MGM-1 was introduced into eukaryotic cells by the progenitors of mitochondria. Expression studies using -galactosidase under the control of the mgm-1 promoter show high levels of expression in intestines, body wall muscles, and neurons. These high levels might reflect high metabolic rates in those tissues, because the MGM-1 expression pattern is similar to that of another dynamin-related protein, DRP-1, which is also important for mitochondrial maintenance. Mgm-1 RNAi drastically slows the growth rate and approximately doubles the life span of C. elegans. We show that excessive fragmentation of mitochondria is induced by RNAi and mgm-1 antisense cDNA. We present evidence that MGM-1 is localized to the mitochondrial matrix where it might regulate the morphology of the mitochondrial inner membrane. 1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular Cell 4: 815-826. 1999.

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223

Astrid Kleinert et al. (2006) European Worm Meeting "Analysis of Insulin-Like Peptides in C. elegans by Mass Spectrometry"

Astrid Kleinert, Daniel Hess, Joy Alcedo, Jan Hofsteenge

[European Worm Meeting, 2006]

Astrid Kleinert, Daniel Hess, Jan Hofsteenge and Joy Alcedo. Genetic analyses in C. elegans have implicated an endocrine signaling pathway, the insulin/IGF-1 pathway, in regulating lifespan. Mutations in the gene daf-2, which encodes an insulin/IGF-1 receptor homologue, can increase worm lifespan by more than 100% (1). In addition, the insulin and IGF-1 pathways have been shown to influence fly and mouse lifespan (2-5).. Although downstream components of the DAF-2 pathway have been studied in detail, far less is known about the putative DAF-2 ligands predicted to be encoded by 38 insulin-like genes in the worm. It remains unknown which of the predicted insulin-like peptides are present in wild type or in longevity mutants. In order to determine this, we have initiated a project to analyze the polypeptide subfractions of the C. elegans proteome by mass spectrometry. Worm lysates will be prepared from mixed stage, wild-type worms, excluding membrane-bound proteins. Acid-ethanol extraction and gel filtration will be tested as methods to obtain a subfraction enriched in small polypeptides. Peptide identification will be done by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data analyses will be carried out using a MASCOT search database, which contains a subset of the UNIPROT database and a database generated of relevant C. elegans polypeptides. Finally, using the SILAC method for quantification (6), we plan to compare differences in the levels of polypeptides, e.g., insulin-like peptides, between wild-type worms and longevity mutants. . References: (1) Kenyon et al., 1993. Nature 366: 461-4. (2) Clancy et al., 2001. Science 292: 104-6. (3) Tatar et al., 2001. Science 292: 107-110. (4) Holzenberger et al., 2003. Nature 421: 182-7. (5) Blher et al., 2003. Science 299:572-4. (6) Krijgsveld et al., 2003. Nature Biotech. 21: 927-931.