A One-Week Laboratory Practice: Introducing the Students to the Study of Plant Biochemistry and Signal Transduction

This report describes a one-week laboratory practice for students. An approach to study a Calcium dependent protein kinase (CDPK) involved in signal transduction processes in potato plants, is undertaken. A number of basic biochemical techniques including the partial purification of a protein kinase, protein kinase activity assays, protein determination, SDS-PAGE analysis of phosphorylated proteins and Western blot assays are described.Fil: Ulloa, Rita Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Raices, Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Tellez, Maria Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Nuclear pore complex-mediated modulation of TCR signaling is required for naïve CD4+ T cell homeostasis.

Nuclear pore complexes (NPCs) are channels connecting the nucleus with the cytoplasm. We report that loss of the tissue-specific NPC component Nup210 causes a severe deficit of naïve CD4+ T cells. Nup210-deficient CD4+ T lymphocytes develop normally but fail to survive in the periphery. The decreased survival results from both an impaired ability to transmit tonic T cell receptor (TCR) signals and increased levels of Fas, which sensitize Nup210-/- naïve CD4+ T cells to Fas-mediated cell death. Mechanistically, Nup210 regulates these processes by modulating the expression of Cav2 (encoding Caveolin-2) and Jun at the nuclear periphery. Whereas the TCR-dependent and CD4+ T cell-specific upregulation of Cav2 is critical for proximal TCR signaling, cJun expression is required for STAT3-dependent repression of Fas. Our results uncover an unexpected role for Nup210 as a cell-intrinsic regulator of TCR signaling and T cell homeostasis and expose NPCs as key players in the adaptive immune system

Uncoupling of Longevity and Telomere Length in C. elegans

The nematode Caenorhabditis elegans, after completing its developmental stages and a brief reproductive period, spends the remainder of its adult life as an organism consisting exclusively of post-mitotic cells. Here we show that telomere length varies considerably in clonal populations of wild-type worms, and that these length differences are conserved over at least ten generations, suggesting a length regulation mechanism in cis. This observation is strengthened by the finding that the bulk telomere length in different worm strains varies considerably. Despite the close correlation of telomere length and clonal cellular senescence in mammalian cells, nematodes with long telomeres were neither long lived, nor did worm populations with comparably short telomeres exhibit a shorter life span. Conversely, long-lived daf-2 and short-lived daf-16 mutant animals can have either long or short telomeres. Telomere length of post-mitotic cells did not change during the aging process, and the response of animals to stress was found independent of telomere length. Collectively, our data indicate that telomere length and life span can be uncoupled in a post-mitotic setting, suggesting separate pathways for replication-dependent and -independent aging

Organismal propagation in the absence of a functional telomerase pathway in Caenorhabditis elegans

Mutant worm strains that proliferate indefinitely without telomerase provide the first multicellular model for studying recombination-mediated telomere maintenance (ALT), a mechanism active in telomerase-deficient human tumours

C. elegans Telomeres Contain G-Strand and C-Strand Overhangs that Are Bound by Distinct Proteins

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in , chromosome termini possess 3′ G-strand overhangs as well as 5′ C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that telomere homeostasis relies on a novel mechanism that involves 5′ and 3′ single-stranded termini

Telomere Length Is Independent from Organismal Life Span and Thermo Tolerance

<div><p>(A) CF512 temperature-sensitive sterile worms were grown at 25 °C, and life span was monitored. Samples were taken at indicated times.</p><p>(B) DNA from CF512 temperature-sensitive sterile worms was extracted at the indicated time points, and telomere length was monitored in mixed and synchronous populations (for details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010030#s3" target="_blank">Materials and Methods</a>). Telomere length is indicated in kb. The asterisk points out a variable band-like signal resulting from gel drying.</p><p>(C) Seven different worm strains and <i>daf-2</i> and <i>daf-16</i> RNAi suppressed worms (indicated in different colors) with genetically determined telomere length (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010030#pgen-0010030-g001" target="_blank">Figure 1</a>D) were subjected to heat treatment as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010030#pgen-0010030-b31" target="_blank">31</a>]. Survival was assessed at the indicated time points.</p><p>(D) Two worm strains with long telomeres (CC2, TR403) and N2 worms with short telomeres were subjected to <i>daf-16</i> suppression by RNAi. Life spans were determined as described.</p></div

Organismal Life Span Is Independent of Telomere Length

<div><p>(A) Life span of N2 worms under control (green), <i>daf-2</i> (blue), and <i>daf-16</i> (red) RNAi suppression conditions. Worms were grown on bacteria expressing RNAi constructs targeting the indicated genes, and life span was assessed as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010030#pgen-0010030-b21" target="_blank">21</a>].</p><p>(B) Telomere length of nine individual control N2 clones was assessed as described. Samples were collected at the beginning (B) and end (E) of the experiment, and fragment size is indicated in kb.</p><p>(C) Telomere length of nine individual <i>daf-2</i> (RNAi) N2 clones was assessed as described in (B).</p><p>(D) Telomere length of nine individual <i>daf-16</i> (RNAi) N2 clones was assessed as described in (B).</p></div

Clonal Variation and Conservation of Telomere Length in <i>C. elegans</i>

<div><p>(A) AluI/MboI digests of genomic DNA from six Bristol N2 clones was separated by gel electrophoresis and hybridized with (TTAGGC)₄ or (GCCTAA)₄ probes as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0010030#pgen-0010030-b28" target="_blank">28</a>]. Fragment size is indicated in kb, and internal repeats are indicated by an asterisk.</p><p>(B) DNA from six individual N2 clones was incubated with Bal31 for the indicated time, and then processed as in (A).</p><p>(C) Six individual N2 clones were propagated for ten generations, and telomere length was determined as described.</p><p>(D) Seven different <i>C. elegans</i> strains were examined for their telomere length as described in (A).</p></div