PLS3 function

PLS3 is an actin binding protein with its main function in actin filament cross-linking. Generally, plastins are located in focal adhesions, ruffling membranes, lamellipodia, filopodia, or in specialized surface structures with highly ordered microfilament bundles such as microvilli and stereocilia. Sometimes they co-localize with stress fibers (Bretscher and Weber, 1980, Delanote et al., 2005). PLS3 function in actin bundling and the consequences of overexpression have nicely been demonstrated in vitro in fibroblast–like CV-1 and epithelial LLC-PK1 cells (Arpin et al., 1994). In CV-1 cells, PLS3 overexpression resulted in a rounding-up of cells, concomitantly with a significant reorganization of the actin cytoskeleton. In this regard, actin filaments became organized into polygonal networks in the cortical region of the cells. In LLC-PK1 epithelial cells, PLS3 overexpression led to a high increase in length and density of brush border microvilli, particularly at the periphery of the cells. Furthermore, PLS3 overexpression resulted in a significant diminution of focal contacts, presumably due to extensive remodeling of the actin cytoskeleton, implying a possible involvement of PLS3 in cell movement processes. Another study focused on the mechanism underlying PLS3 induced cytoskeletal rearrangements (Giganti et al., 2005). Here, beads were coated with the VCA domain of Wiskott/Aldrich-syndrome protein (WASP) to recruit the

actin-branching and polymerizing complex Arp2/3. This alone resulted in actin polymerization at the surface of the beads and movements of such when placed in cell-free extracts. Importantly, it was shown that PLS3 increased the velocity of VCA beads movement, stabilized actin comets and concomitantly displaced cofilin, an actin-depolymerizing protein. Since a mutated form of the ABD1 domain unable in cross-linking was sufficient to induce the above described effects, Giganti et al. propose a novel mechanism of action for PLS3 in which this protein might contribute to Arp2/3 mediated actin assembly independently of cross-link formation. In other words: PLS3 on the one hand stabilizes actin filaments through cross-linking and additionally triggers Arp2/3-mediated actin polymerization. Since the Arp2/3 complex nucleates a branched actin network found, e.g., in the lamellipodium at the leading edge of migrating cells, this fact highlights the significance of PLS3 function for protrusive membrane-outgrowth and more generally, cell motility. A role of PLS3 in cell motility was further confirmed in studies by Serio et al., who found that T-plastin is important for actin tail formation in the pathogenic bacteria Rickettsia parkeri (R. parkeri) and for its actin-based motility in mammalian cells (Serio et al., 2010). In line with this, PLS3 was shown to play an important role in invasion of the bacteria Shigella flexneri and Salmonella typhimurium (Prevost et al., 1992, Adam et al., 1995, Zhou et al., 1999a). In both cases, PLS3 causes actin cytoskeleton rearrangements in the host cells that lead to the formation of actin protrusions engulfing the bacteria and finally resulting in phagocytosis and uptake of the pathogen.

PLS3 was furthermore implicated to play a role in DNA repair and cell cycle control and consequentially also in cancer. By differential mRNA display, it has been demonstrated that PLS3 is 12-fold more abundant in cisplatin-resistant cell lines from bladder, prostatic, head and neck cancer (Hisano et al., 1996). In this regard, cisplatin is an anti-cancer drug intercalating with DNA and interfering with DNA-repair. Accordingly, the downregulation of PLS3 was found to be associated with higher cisplatin sensitivity. Additionally, the increased sensitivity of cancer cells to DNA interfering drugs as caused by plastin3 downregulation, has also been shown for the Topoisomerase II-blocking agent VP-16 (Ikeda et al., 2005). In line with these findings, higher PLS3 levels were also detected in UV-radiation resistant cell lines (Higuchi et al., 1998). Also in Sézary syndrom, the most common form of cutaneous T-cell lymphoma, the presence of sézary cells was correlated with high levels of PLS3 in peripheral blood mononuclear cells (PBMCs) (Capriotti et al., 2008, Tang et al., 2010). Vice versa, PLS3 levels dropped upon chemotherapeutic treatment and completely disappeared after bone marrow transplantation in a patient. In another study, higher levels of PLS3 were correlated with G2-phase arrest upon X-radiation in Chinese hamster ovary (CHO) cells (Sasaki et al., 2002). However, when PLS3 was downregulated via antisense knockdown, radiation-induced G2 arrest was short and decreased in transfected cells. The authors have

therefore speculated that downregulation of PLS3 may be involved in cancer development through G2/M cell-cycle control in mammalian cells. These results highlight low levels of PLS3 as possibly causative for cancer development, different from the before mentioned findings, where high PLS3 levels were associated with cancerous state. Possibly, species- or cell type specific differences might explain these opposite observations. In this line, it is also unclear whether up- or downregulation of PLS3 in the various cancer forms is causative for, or is a result from cancer development. Next to PLS3, the L-plastin isoform is an even more prevalent cancer marker, since it is expressed in a high percentage of tumor-derived cells, but never in normal diploid cells of solid tissues (Leavitt and Kakunaga, 1980, Leavitt, 1994). Apart from cancer, PLS3 is also involved in the human autoimmune disease systemic lupus erythematosus (SLE). In an animal model for this disease as well as in human patients, antibodies against L- and T-plastin were found in serum (De Mendonca Neto et al., 1992, Mine et al., 1998, Shinomiya et al., 2003, Delanote et al., 2005). However, it has to be determined in how far these antibodies contribute to the loss of cell function or other aspects of SLE.

Due to interaction with ataxin-2 (ATX2) in yeast and also murine brain, PLS3 has additionally been implicated in the regulation of RNA metabolism and translational pathway. In this context, ATX2 is known to interact with cytoplasmic poly(A)-binding protein (PABP) that functions in translation initiation and mRNA decay regulation and forms part of stress granules (Ralser et al., 2005a). The finding of an interaction of PLS3 and ATX2 has been further underlined by the observation of a stabilizing effect of ATX2 on PLS3 when overexpressed (Ralser et al., 2005b).