ACT-5 is an essential Caenorhabditis elegans actin required for intestinal microvilli formation.

Investigation of Caenorhabditis elegans act-5 gene function revealed that intestinal microvillus formation requires a specific actin isoform. ACT-5 is the most diverged of the five C. elegans actins, sharing only 93% identity with the other four. Green fluorescent protein reporter and immunofluorescence analysis indicated that act-5 gene expression is limited to microvillus-containing cells within the intestine and excretory systems and that ACT-5 is apically localized within intestinal cells. Animals heterozygous for a dominant act-5 mutation looked clear and thin and grew slowly. Animals homozygous for either the dominant act-5 mutation, or a recessive loss of function mutant, exhibited normal morphology and intestinal cell polarity, but died during the first larval stage. Ultrastructural analysis revealed a complete loss of intestinal microvilli in homozygous act-5 mutants. Forced expression of ACT-1 under the control of the act-5 promoter did not rescue the lethality of the act-5 mutant. Together with immuno-electron microscopy experiments that indicated ACT-5 is enriched within microvilli themselves, these results suggest a microvillus-specific function for act-5, and further, they raise the possibility that specific actins may be specialized for building microvilli and related structures.

Genome-wide responses to mitochondrial dysfunction.

Mitochondrial dysfunction can lead to diverse cellular and organismal responses. We used DNA microarrays to characterize the transcriptional responses to different mitochondrial perturbations in Saccharomyces cerevisiae. We examined respiratory-deficient petite cells and respiratory-competent wild-type cells treated with the inhibitors of oxidative phosphorylation antimycin, carbonyl cyanide m-chlorophenylhydrazone, or oligomycin. We show that respiratory deficiency, but not inhibition of mitochondrial ATP synthesis per se, induces a suite of genes associated with both peroxisomal activities and metabolite-restoration (anaplerotic) pathways that would mitigate the loss of a complete tricarboxylic acid cycle. The array data suggested, and direct microscopic observation of cells expressing a derivative of green fluorescent protein with a peroxisomal matrix-targeting signal confirmed, that respiratory deficiency dramatically induces peroxisome biogenesis. Transcript profiling of cells harboring null alleles of RTG1, RTG2, or RTG3, genes known to control signaling from mitochondria to the nucleus, suggests that there are multiple pathways of cross-talk between these organelles in yeast.

Asymmetric segregation of PIE-1 in C. elegans is mediated by two complementary mechanisms that act through separate PIE-1 protein domains.

The CCCH finger protein PIE-1 is a regulator of germ cell fate that segregates with the germ lineage in early embryos. At each asymmetric division, PIE-1 is inherited preferentially by the germline daughter and is excluded from the somatic daughter. We show that this asymmetry is regulated at the protein level by two complementary mechanisms. The first acts before cell division to enrich PIE-1 in the cytoplasm destined for the germline daughter. The second acts after cell division to eliminate any PIE-1 left in the somatic daughter. The latter mechanism depends on PIE-1's first CCCH finger (ZF1), which targets PIE-1 for degradation in somatic blastomeres. ZF1s in two other germline proteins, POS-1 and MEX-1, are also degraded in somatic blastomeres, suggesting that localized degradation also acts on these proteins to exclude them from somatic lineages.

Regulated nuclear translocation of the Mig1 glucose repressor.

Glucose represses the transcription of many genes in bakers yeast (Saccharomyces cerevisiae). Mig1 is a Cys2-His2 zinc finger protein that mediates glucose repression of several genes by binding to their promoters and recruiting the general repression complex Ssn6-Tup1. We have found that the subcellular localization of Mig1 is regulated by glucose. Mig1 is imported into the nucleus within minutes after the addition of glucose and is just as rapidly transported back to the cytoplasm when glucose is removed. This regulated nuclear localization requires components of the glucose repression signal transduction pathway. An internal region of the protein separate from the DNA binding and repression domains is necessary and sufficient for glucose-regulated nuclear import and export. Changes in the phosphorylation status of Mig1 are coincident with the changes in its localization, suggesting a possible regulatory role for phosphorylation. Our results suggest that a glucose-regulated nuclear import and/or export mechanism controls the activity of Mig1.

Movement of cortical actin patches in yeast.

In yeast, actin forms patches associated with the plasma membrane. Patch distribution correlates with polarized growth during the cell cycle and in response to external stimuli. Using green fluorescent protein fused to capping protein to image actin patches in living cells, we find that patches move rapidly and over long distances. Even patches in clusters, such as at the incipient bud site, show movement. Patches move independently of one another and generally over small distances in a local area, but they can also move larger distances, including through the mother-bud neck. Changes in patch polarization occur quickly through the cell cycle. These observations provide important new parameters for a molecular analysis of the regulation and function of actin.

Transient localized accumulation of actin in Caenorhabditis elegans blastomeres with oriented asymmetric divisions.

During Caenorhabditis elegans embryogenesis, specific cells in the P1 lineage rotate their duplicated centrosome pair onto the anterior-posterior axis; this rotation is correlated with and necessary for a differential inheritance of cytoplasmic determinants in the daughter cells. Centrosome pair rotation is sensitive to inhibitors of actin and microtubule polymerization and may require microtubule attachment to a specific cortical site. We show that actin and the barbed-end binding protein, capping protein, transiently accumulate at this cortical site, possibly by assembly onto persistent remnants of previous cell divisions. Based on these observations, we propose a model for the molecular basis of centrosome rotation that is consistent with the dependence of rotation on actin filaments and microtubules.

The alpha and beta subunits of nematode actin capping protein function in yeast.

We cloned and analyzed two genes, cap-1 and cap-2, which encode the alpha and beta subunits of Caenorhabditis elegans capping protein (CP). The nematode CP subunits are 55% (cap-1) and 66% (cap-2) identical to the chicken CP subunits and 32% (cap-1) and 48% (cap-2) identical to the yeast CP subunits. Purified nematode CP made by expression of both subunits in yeast is functionally similar to chicken skeletal muscle CP in two different actin polymerization assays. The abnormal cell morphology and disorganized actin cytoskeleton of yeast CP null mutants are restored to wild-type by expression of the nematode CP subunits. Expression of the nematode CP alpha or beta subunit is sufficient to restore viability to yeast cap1 sac6 or cap2 sac6 double mutants, respectively. Therefore, despite evolution of the nematode actin cytoskeleton to a state far more complex than that of yeast, one important component can function in both organisms.

Localization of CapZ during myofibrillogenesis in cultured chicken muscle.

Actin filaments undergo dramatic changes in their organization during myofibrillogenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to "cap" each actin filament of the sacromere. Double immunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, alpha-actinin, titin, and myosin during myofibrillogenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric alpha-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric alpha-actinin in young myotubes was not coincident; in older myotubes, both CapZ and alpha-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes.

The chloroplast genome of an exsymbiotic Chlorella-like green alga.

Chloroplast DNA was isolated and cloned from Chlorella, strain N1a, exsymbiotic with Paramecium bursaria. BamHI, SalI, KpnI and Xho I restriction fragments of the DNA were assembled into a circular map. The genome consists of approximately 120 kbp of DNA, has a G/C content of 38%, and contains only a single copy of the rRNA cistron. The rRNA cistron is small, 5000-8000 bp, and the 16S and 23S gene are separated by less than 2000 bp.

The mitochondrial genome of an exsymbiotic Chlorella-like green alga.

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F0-F1 ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.

cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle.

Three cDNAs encoding the beta chain of Cap Z have been isolated from lambda gt11 chicken libraries screened with antibodies. The coding region, which is identical among the cDNAs, contains 831 basepairs encoding a protein with 277 amino acid residues and Mr = 31,352. The predicted protein sequence contains eight regions that match perfectly the NH2-terminal sequence of eight peptides isolated from muscle Cap Z beta. The amino acid compositions of the protein predicted from the cDNA sequence and Cap Z beta are similar. Comparison of the cDNA sequence and the predicted protein sequence with those of other actin-binding proteins and all sequences in the GenBank and NBRF Protein databases shows no remarkable similarities. Two of the three cDNAs contain the complete coding region. Both coding regions begin with a consensus translation start site; however, their 5'-untranslated regions are different. Northern analysis of whole chicken embryos and adult chicken tissues shows two mRNA species. The embryo and non-muscle tissues have transcripts of 1.35 and 2.00 kilobases, and the muscle tissues have transcripts of 2.15 and 1.45 kilobases. Southern analysis of chicken genomic DNA shows that there are probably two related genes, one more similar to the cDNA than the other.