The Dutch beggars

Thesis (B.L.)--University of Illinois, 1882.Ms.Bound with 8 other humanities theses. IU-

Localisation of Tartan protein in the eye.

<p>(A–A') The Trn antibody recognised endogenous levels of Trn in the 3^rd instar eye disc. The signal is absent in clones of <i>trn^28.4</i> null cells (marked by loss of GFP, green), confirming antibody specificity. (B–B') The Trn antibody signal was also lost in <i>caps^Del1trn^28.4</i> double null clones, marked by loss of GFP (green). (C–C') Z-section along the A-P axis of a wild type disc stained with anti-Trn. Trn is expressed only in the anterior half of the furrow and mostly in the apical surface of photoreceptors, as well as in some basolateral membranes. (D–D') Apical planar views of the corresponding discs in (C–C'). Trn is expressed in the furrow and in subset of photoreceptors after the furrow. The dashed line indicates the position of the sagittal section in C and C'. (E) Enlarged view of the disc in D. The inset shows an enlarged view of the marked ommatidium with the positions of each photoreceptor labelled. Trn expression is located in the expected positions of R1, 6, 7.</p

<i> caps trn</i> double null clones.

<p>(A) <i>caps^Del1 trn^28.4</i> double null clones in the 3^rd instar eye disc. Mutant tissue is marked by lack of GFP (green). Anti-Elav marks all photoreceptors. Anti-Armadillo (Arm) marks the apical surface of cells and is accumulated at a high level in the apically constricted cells of the furrow. At the border between wild type and <i>caps^Del1 trn^28.4</i> tissue, there is a reduction in Arm accumulation in the cells of the furrow and the apical surface of cells is expanded (yellow arrow A'). (A”) A Z-section along the furrow (between the two red arrows in A'). At the clone boundaries, cells are taller in the apical-basal direction, producing ‘bumps’ in the furrow (two yellow arrows). (B) A <i>caps^Del1trn^28.4</i> clone (marked by lack of GFP, green) where ommatidia near the clone boundaries are mis-positioned (circled in white). (C) <i>caps^Del1trn^28.4</i> clones in pupal retinae (marked by lack of GFP, green). Elav is used to mark the photoreceptors and Cut is used to mark cone cells (four per ommatidium) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001827#pone.0001827-Blochlinger1" target="_blank">[27]</a>. At mutant-wild type borders neighbouring ommatidia sometimes fuse with each other (red arrows in C'). Correct cone cell numbers are also sometimes disrupted (red arrow in C”). (D) <i>caps^pB1</i> clones in pupal retinae (marked by lack of GFP, green). No defects in mutant tissue or clone boundaries can be seen. (E) <i>trn^28.4</i> clones in pupal retinae (marked by lack of GFP, green). Again, the retinae are phenotypically wild type. (F) <i>caps^Del1trn^28.4</i> clones in the wing (marked by lack of GFP, green) do not visibly affect the DV boundary (between the two red arrows), as marked by anti-Senseless (Sens). Clones do not cross the DV boundary (white arrows).</p

<i> caps</i> and <i>trn</i> expression in the eye.

<p>The arrow in each panel marks the morphogenetic furrow (MF) and anterior is to the left in all images, unless otherwise stated. (A) <i>caps-lacZ</i> expression in 3^rd instar eye disc. Staining with anti-β−gal revealed <i>caps-lacZ</i> expression in the furrow and in subsets of cells after the furrow. (A'–A”) Co-staining with anti-Elav (a photoreceptor marker) and anti-Senseless (R8 specific marker) identified the cells eventually expressing high levels of <i>caps-lacZ</i> as photoreceptor R8. (B) <i>trn-lacZ</i> expression in 3^rd instar eye disc. Staining with anti-β−gal revealed <i>trn-lacZ</i> expression in the furrow and in a different subset of cells from <i>caps-lacZ</i> after the furrow. (B'–B”) Co-staining with anti-BarH1 (R1 and R6 specific marker) and anti-Prospero (R7 and cone cell marker) identified R1, 6 and 7 as the photoreceptors expressing high levels of <i>trn-lacZ</i>.</p

Generation of <i>caps</i> null and <i>caps trn</i> double null mutants.

<p>(A) Generating <i>caps^pB1</i> null. Two <i>piggyBac</i> element insertion lines were used for FLP-FRT based recombination to delete the entire coding sequence of <i>caps</i>, which is contained in exon 5. <i>pBacRB^e03402</i> is inserted upstream of exon 4 and <i>pBacRB^e03153</i> is inserted downstream of exon 5. Upon heatshock, recombination occurs between these two <i>piggyBac</i> elements, deleting the intervening region, regenerating a complete <i>piggyBac</i> element from half of each of the original <i>piggyBacs</i>. The deletion sites were confirmed by using genomic primers (marked) outside the <i>piggyBac</i> elements to amplify across the newly formed element (about 6 kb). Sequencing outward from either end of the PCR fragment identified the precise deletion sites. Flanking sequence of the new deletion is shown. (B) Generating <i>caps^Del1trn^28.4</i> double null. <i>caps¹⁶⁹⁶⁴</i> has a GS element inserted downstream of the <i>caps</i> gene. <i>trn^28.4</i> is a null allele of tartan generated by P-element excision. <i>caps¹⁶⁹⁶⁴</i> was used to induce male recombination with the <i>trn^28.4</i> chromosome. This allowed the simultaneous deletion of the <i>caps</i> gene and recombination onto the existing <i>trn^28.4</i> null chromosome. The GS element remains intact after the recombination allowing its precise new position, and any deletions, to be verified by inverse PCR and sequencing. The entire <i>caps</i> gene was deleted, but no other gene (apart from one tRNA gene) was affected. Flanking sequence of the new deletion is shown.</p

<i> caps</i> and <i>trn</i> single null clones.

<p>(A–B) <i>caps^pB1</i> clones in the 3^rd instar eye disc. Mutant tissue is marked by lack of GFP (green). Anti-Senseless (Sens), is used to identify R8 cells (A') and anti-Prospero (Prosp) is used to identify R7 and cone cells (A”). Anti-Elav marks all photoreceptor cells (B') and anti-Armadillo (Arm) marks the adherens junctions of cells, thereby outlining all cells (B”). No defects could be detected in <i>caps^pB1</i> mutant tissue. (C–D) <i>trn^28.4</i> clones in the 3^rd instar eye disc. Mutant tissue is marked by lack of GFP (green). As with <i>caps^pB1</i> clones, no defects were observed.</p

Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.</p

Cleavage of the pseudoprotease iRhom2 by the signal peptidase complex reveals an ER-to-nucleus signaling pathway

iRhoms are pseudoprotease members of the rhomboid-like superfamily and are cardinal regulators of inflammatory and growth factor signaling; they function primarily by recognizing transmembrane domains of their clients. Here, we report a mechanistically distinct nuclear function of iRhoms, showing that both human and mouse iRhom2 are non-canonical substrates of signal peptidase complex (SPC), the protease that removes signal peptides from secreted proteins. Cleavage of iRhom2 generates an N-terminal fragment that enters the nucleus and modifies the transcriptome, in part by binding C-terminal binding proteins (CtBPs). The biological significance of nuclear iRhom2 is indicated by elevated levels in skin biopsies of patients with psoriasis, tylosis with oesophageal cancer (TOC), and non-epidermolytic palmoplantar keratoderma (NEPPK); increased iRhom2 cleavage in a keratinocyte model of psoriasis; and nuclear iRhom2 promoting proliferation of keratinocytes. Overall, this work identifies an unexpected SPC-dependent ER-to-nucleus signaling pathway and demonstrates that iRhoms can mediate nuclear signaling.</p