Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans

Kemphues, Kenneth J.; Wolf, Nurit; Wood, William B.; Hirsh, David

doi:10.1016/0012-1606(86)90180-6

Cited by 78 publications

(68 citation statements)

References 24 publications

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“…The embryos were then collected and fixed in methanol/acetone using the freeze-cracking protocol (25). For whole mounted immunostaining, mixed-stage population of larvae and adults were collected and washed in PBS and Ruvkun Fixation Buffer before being fixed and permeabilized using 1% paraformaldehyde in Ruvkun Fixation Buffer for 30 min and two freeze-thaw cycles in dry/ethanol bath.…”

Section: Detection Of the Native Ce-cpz-1 Protein In C Elegans By Immentioning

confidence: 99%

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Hashmi¹,

Zhang²,

Oksov

et al. 2004

Journal of Biological Chemistry

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We have analyzed the expression and function of Cecpz-1, a Caenorhabditis elegans cathepsin Z-like cysteine protease gene, during development of the worm. The cpz-1 gene is expressed in various hypodermal cells of all developmental stages and is specifically expressed in the gonads and the pharynx of adult worms. Disruption of cpz-1 function by RNA interference or cpz-1(ok497) deletion mutant suggests that cpz-1 has a role in the molting pathways. The presence of the native CPZ-1 protein in the hypodermis/cuticle of larval and adult stages and along the length of the pharynx of adult worms, as well as the cyclic expression of the transcript during larval development, supports such function. We hypothesize that the CPZ-1 enzyme functions directly as a proteolytic enzyme degrading cuticular proteins before ecdysis and/or indirectly by processing other proteins such as proenzymes and/or other proteins that have an essential role during molting. Notably, an impressive level of the CPZ-1 native protein is present in both the new and the old cuticles during larval molting, in particular in the regions that are degraded prior to shedding and ecdysis. The similar localization of the related Onchocerca volvulus cathepsin Z protein suggests that the function of CPZ-1 during molting might be conserved in other nematodes. Based on the cpz-1 RNA interference and cpz-1 (ok497) deletion mutant phenotypes, it appears that cpz-1 have additional roles during morphogenesis. Deletion of cpz-1 coding sequence or inhibition of cpz-1 function by RNA interference also caused morphological defects in the head or tail region of larvae, improperly developed gonad in adult worms and embryonic lethality. The CPZ-1 native protein in these affected regions may have a role in the cuticular and the basement membrane extracellular matrix assembly process. The present findings have defined a critical role for cathepsin Z in nematode biology.Based on the identification and characterization of a cysteine protease in human brain, a new subfamily of cysteine proteases of the papain family, the cathepsin Z-like enzyme, was recently classified (1). The human brain cathepsin Z was, however, found to be widely expressed in many tissues, suggesting that this enzyme is possibly involved in the normal intracellular protein degradation that takes place in all cell types. Notably, the enzyme was also found in many cancer cell lines and in primary tumors from different sources, which also suggested a role for this enzyme in tumor progression (1). The human cathepsin Z contains distinctive features that separate it from other human cysteine proteases (2). Cathepsin Z is characterized by an unusual and unique 3-amino acid insertion (HIP) in the highly conserved region between the glutamine of the putative oxyanion hole and the active site cysteine, which might confer special properties to the enzyme (3). It functional diversity is generated by the addition of the HIP residues including His 23 in the mature enzyme that provides an anchor for the C terminus of a s...

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Section: Detection Of the Native Ce-cpz-1 Protein In C Elegans By Immentioning

confidence: 99%

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Hashmi¹,

Zhang²,

Oksov

et al. 2004

Journal of Biological Chemistry

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“…The embryos were then collected and fixed in methanol/acetone using the freeze-cracking protocol (27). For whole mounted immunostaining, mixed-stage population of larvae and adults were collected and washed in PBS and Ruvkun Fixation buffer before being fixed and permeabilized using 1% paraformaldehyde in Ruvkun Fixation buffer for 30 min and two freezethaw cycles in a dry ice/ethanol bath (28).…”

Section: Stage-specific Transcript Of Ce-cpl-1 Using Rt-pcr-c Elegansmentioning

confidence: 99%

Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans

Hashmi¹,

Britton²,

Liu³

et al. 2002

Journal of Biological Chemistry

111

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Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/ degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.Cysteine proteases of the papain superfamily have long been recognized for their role in intracellular and extracellular protein degradation in a range of cellular processes (1). Within the papain family, the cathepsins can be subdivided into more than 10 subfamilies on the basis of their primary sequence and enzymatic activity (2). The family includes cathepsin B, C, L, and Z, all of which contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, such as substrate specificity and pH stability. Cathepsin B-like cysteine protease genes occur as a large multigene family in a wide range of parasitic and free-living nematodes. Several cathepsin B genes were reported to be expressed in Caenorhabditis elegans, some of which were restricted to the intestines of larval and adult transgenic worms (3-5). Interestingly, the structurally similar Hemonchus contortus (3-5) and Schistosoma mansoni (6) cathepsin B homologues were also expressed in the gut and were suggested to be potentially involved in feeding (5, 7), such as nutrient digestion. Heterologous transformation of C. elegans with an H. contortus cathepsin B gene promoter has demonstrated also conservation of the mechanisms controlling its spatial expression in free-living and parasitic nematodes (8), and therefore both enzymes were hypothesized to be not only structurally similar, but also...

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“…In most species, including Caenorhabditis elegans, Arabidopsis, Drosophila, and yeasts, a reduced level of the respective DdCP224 homolog leads to mitotic spindle defects and, during interphase, to shorter and/or reduced numbers of microtubules (Kemphues et al, 1986;Whittington et al, 2001). In HeLa cells, depletion of the DdCP224 homolog colonic and hepatic tumor overexpressed gene (ch-TOG) by RNA interference caused a fivefold increase in mitotic index, concomitant with failure of proper metaphase plate organization (Gergely et al, 2003).…”

Section: Ddcp224 Is Required For Microtubule Growthmentioning

confidence: 99%

Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

Gräf

Euteneuer

et al. 2003

MBoC

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The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over-and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis. INTRODUCTIONIn dividing cells, control of centrosome number is essential for the fidelity of mitosis and maintenance of euploidy. Supernumerary centrosomes are a hallmark of tumor cells, although it is still a matter of discussion whether their appearance is a cause or a consequence of carcinogenesis (Lingle et al., 2002;Nigg, 2002). However, it is widely accepted that supernumerary centrosomes contribute to the formation of multipolar spindles and thus to defective chromosome segregation. Although most of the daughter cells resulting from such mitotic chaos will die, some of them will acquire the potential for neoplastic growth in spite of supernumerary centrosomes and aneuploidy, as they regain the ability to form a bipolar spindle. Brinkley (2001) suggested two mechanisms how this can be achieved: first, "deamplification" of supernumerary centrosomes by elimination or inactivation, and second, fusion or "coalescence" of supernumerary centrosomes resulting in one or two compound microtubule-organizing centers (MTOCs).Herein, we have studied the fate of supernumerary centrosomes in a simple model system. In Dictyostelium amoebae, one molecular component involved in the formation of supernumerary centrosomes is DdCP224, a member of the XMAP215 family of microtubule-associated proteins (Gräf et al., 2000). In most species, these proteins are long, thin, monomeric molecules with a size of ϳ 230 kDa (Cassimeris et al., 2001). Their ubiquitous occurrence, even in plants, suggests indispensible functions (Ohkura et al., 2001). XMAP215, for instance, is a promoter of microtubule elongation due to a suppression of catastrophe events induced by the Kin I family kinesin ...

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Two loci required for cytoplasmic organization in early embryos of Caenorhabditis elegans

Cited by 78 publications

References 24 publications

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

The Caenorhabditis elegans Cathepsin Z-like Cysteine Protease, Ce-CPZ-1, Has a Multifunctional Role during the Worms' Development

Cathepsin L Is Essential for Embryogenesis and Development ofCaenorhabditis elegans

Regulated Expression of the Centrosomal Protein DdCP224 Affects Microtubule Dynamics and Reveals Mechanisms for the Control of Supernumerary Centrosome Number

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