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Overlapping functions of the two talin homologues in Dictyostelium.

ABSTRACT: Talin is a cytoskeletal protein involved in constructing and regulating focal adhesions in animal cells. The cellular slime mold Dictyostelium discoideum has two talin homologues, talA and talB, and earlier studies have characterized the single knockout mutants. talA(-) cells show reduced adhesion to the substrates and slightly impaired cytokinesis leading to a high proportion of multinucleated cells in the vegetative stage, while the development is normal. In contrast, talB(-) cells are characterized by reduced motility in the developmental stage, and they are arrested at the tight-mound stage. Here, we created and analyzed a double mutant with a disruption of both talA and talB. Defects in adhesion to the substrates, cytokinesis, and development were more severe in cells with a disruption of both talA and talB. The talA(-) talB(-) cells failed to attach to the substrates in the vegetative stage, exhibited a higher proportion of multinucleated cells than talA(-) cells, and showed more-reduced motility during the development and an earlier developmental arrest than talB(-) cells at the loose-mound stage. Moreover, overexpression of either talA or talB compensated for the loss of the other talin, respectively. The analysis of talA(-) talB(-) cells also revealed that talin was required for the formation of paxillin-rich adhesion sites and that there was another adhesion mechanism which is independent of talin in the developmental stage. This is the first study demonstrating overlapping functions of two talin homologues, and our data further indicate the importance of talin.

SUBMITTER: Tsujioka M

PROVIDER: S-EPMC2394970 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.

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Overlapping functions of the two talin homologues in Dictyostelium.

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