Erika Hartwieg

Induction of apoptosis in fibroblasts by IL-1β-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3

The mammalian interleukin-1 beta-converting enzyme (ICE) has sequence similarity to the C. elegans cell death gene ced-3. We show here that overexpression of the murine ICE (mICE) gene or of the C. elegans ced-3 gene causes Rat-1 cells to undergo programmed cell death. Point mutations in a region homologous between mICE and CED-3 eliminate the ability of mICE and ced-3 to cause cell death. The cell death caused by mICE can be suppressed by overexpression of the crmA gene, a specific inhibitor of ICE, as well as by bcl-2, a mammalian oncogene that can act to prevent programmed cell death. Our results suggest that ICE may function during mammalian development to cause programmed cell death.

Multipotent neural cell lines can engraft and participate in development of mouse cerebellum

Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.

Odorant-selective genes and neurons mediate olfaction in C. elegans

Olfaction is a versatile and sensitive mechanism for detecting volatile odorants. We show that the nematode C. elegans detects many volatile chemicals, which can be attractants, repellents, or attractants at low concentrations and repellents at high concentrations. Through laser ablation, we have identified chemosensory neurons that detect volatile odorants. Chemotaxis to volatile odorants requires different sensory neurons from chemotaxis to water-soluble attractants, indicating that C. elegans might have senses that correspond to smell and taste, respectively. Single neurons have complex sensory properties, since six distinguishable volatile odorants are sensed by only two types of sensory neurons. Chemotaxis to subsets of volatile odorants is disrupted by mutations in the odr genes, which might be involved in odorant sensation or signal transduction.

CED-1 Is a Transmembrane Receptor that Mediates Cell Corpse Engulfment in C. elegans

We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.

Induction of apoptosis by Drosophila reaper, hid and grim through inhibition of IAP function

Induction of apoptosis in Drosophila requires the activity of three closely linked genes, reaper, hid and grim. Here we show that the proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti‐apoptotic activity of the Drosophila IAP1 (diap1) protein. In a genetic modifier screen, both loss‐of‐function and gain‐of‐function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain‐of‐function mutations in diap1 strongly suppressed reaper‐, hid‐ and grim‐induced apoptosis. Sequence analysis of these alleles revealed that they were caused by single amino acid changes in the baculovirus IAP repeat domains of diap1, a domain implicated in binding REAPER, HID and GRIM. Significantly, the corresponding mutant DIAP1 proteins displayed greatly reduced binding of REAPER, HID and GRIM, indicating that REAPER, HID and GRIM kill by forming a complex with DIAP1. These data provide strong in vivo evidence for a previously published model of cell death regulation in Drosophila.

The Caenorhabditis elegans Gene unc-25 Encodes Glutamic Acid Decarboxylase and Is Required for Synaptic Transmission But Not Synaptic Development

The neurotransmitter GABA has been proposed to play a role during nervous system development. We show that theCaenorhabditis elegans gene unc-25encodes glutamic acid decarboxylase (GAD), the GABA biosynthetic enzyme. unc-25 is expressed specifically in GABAergic neurons. Null mutations in unc-25 eliminate the UNC-25 protein or alter amino acids conserved in all known GADs, result in a complete lack of GABA, and cause defects in all GABA-mediated behaviors. In unc-25 mutants the GABAergic neurons have normal axonal trajectories and synaptic connectivity, and the size and shape of synaptic vesicles are normal. The number of synaptic vesicles at GABAergic neuromuscular junctions is slightly increased. Cholinergic ventral nerve cord neurons, which innervate the same muscles as GABAergic ventral cord neurons, have normal morphology, connectivity, and synaptic vesicles. We conclude that GAD activity and GABA are not necessary for the development or maintenance of neuromuscular junctions in C. elegans.

The C. elegans PH Domain Protein CED-12 Regulates Cytoskeletal Reorganization via a Rho/Rac GTPase Signaling Pathway

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 Crkll, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.

The Caenorhabditis elegans mucolipin-like gene cup-5 is essential for viability and regulates lysosomes in multiple cell types

The misregulation of programmed cell death, or apoptosis, contributes to the pathogenesis of many diseases. We used Nomarski microscopy to screen for mutants containing refractile cell corpses in a C. elegans strain in which all programmed cell death is blocked and such corpses are absent. We isolated a mutant strain that accumulates refractile bodies resembling irregular cell corpses. We rescued this mutant phenotype with the C. elegans mucolipidosis type IV (ML-IV) homolog, the recently identified cup-5 (coelomocyte-uptake defective) gene. ML-IV is a human autosomal recessive lysosomal storage disease characterized by psychomotor retardation and ophthalmological abnormalities. Our null mutations in cup-5 cause maternal-effect lethality. In addition, cup-5 mutants contain excess lysosomes in many and possibly all cell types and contain lamellar structures similar to those observed in ML-IV cell lines. The human ML-IV gene is capable of rescuing both the maternal-effect lethality and the lysosome-accumulation abnormality of cup-5 mutants. cup-5 mutants seem to contain excess apoptotic cells as detected by staining with terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. We suggest that the increased apoptosis seen in cup-5 mutants is a secondary consequence of the lysosomal defect, and that abnormalities in apoptosis may be associated with human lysosomal storage disorders.

Extracellular Proteins Organize the Mechanosensory Channel Complex in C. elegans Touch Receptor Neurons

Specialized extracellular matrix (ECM) is associated with virtually every mechanosensory system studied. C. elegans touch receptor neurons have specialized ECM and attach to the surrounding epidermis. The mec-1 gene encodes an ECM protein with multiple EGF and Kunitz domains. MEC-1 is needed for the accumulation of the collagen MEC-5 and other ECM components, attachment, and, separately, for touch sensitivity. MEC-1 and MEC-5 bind to touch processes uniformly and in puncta. These puncta colocalize with and localize the mechanosensory channel complex in the touch neurons. In turn, the production of the MEC-1 and MEC-5 puncta appears to rely on interactions with the neighboring epidermal tissue. These and other observations lead us to propose that extracellular, but not cytoskeletal, tethering of the degenerin channel is needed for mechanosensory transduction. Additionally, our experiments demonstrate an important role of the ECM in organizing the placement of the channel complex.

Axons degenerate in the absence of mitochondria in C. elegans

Many neurodegenerative disorders are associated with mitochondrial defects [1-3]. Mitochondria can play an active role in degeneration by releasing reactive oxygen species and apoptotic factors [4-7]. Alternatively, mitochondria can protect axons from stress and insults, for example by buffering calcium [8]. Recent studies manipulating mitochondria lend support to both of these models [9-13]. Here, we identify a C. elegans mutant, ric-7, in which mitochondria are unable to exit the neuron cell bodies, similar to the kinesin-1/unc-116 mutant. When axons lacking mitochondria are cut with a laser, they rapidly degenerate. Some neurons even spontaneously degenerate in ric-7 mutants. Degeneration can be suppressed by forcing mitochondria into the axons of the mutants. The protective effect of mitochondria is also observed in the wild-type: a majority of axon fragments containing a mitochondrion survive axotomy, whereas those lacking mitochondria degenerate. Thus, mitochondria are not required for axon degeneration and serve a protective role in C. elegans axons.