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    • We also found that CCDC can associate with stabilize and

    2018-10-23

    We also found that CCDC170 can associate with, stabilize, and bundle, perinuclear MTs (Figs. 2 and 4), possibly creating a perinuclear “track” for Golgi organization (Fig. 3 and Fig. S7). We also showed both by imaging and Western blotting that overexpression of CCDC170 leads to increases in the level of ac-α-tubulin, a marker for stable MTs. Expression of CCDC170 stabilized MTs even in the presence of the MT depolymerizing agent nocodazole (Figs. 4 and 5). In addition, we observed MT-bundling/stabilization after expression of CCDC170 fragments 1-649 and 1-591, as well as the clinically relevant 355–715 fragment (Fig. 2 and Fig. S6). Lastly, we identified the MAP4 MT-stabilizing protein (Holmfeldt et al., 2009) as a potential functional binding partner of CCDC170 (Fig. 7). Although our results clearly show that CCDC170 can stabilize MTs, the experimental approaches described here do not distinguish between roles in dynamic growth of MTs versus stabilization of existing MTs. As we found that CCDC170 can associate with both the Golgi and MTs, we propose that its normal function is to link the Golgi to, and stabilize, perinuclear MTs. As such, the function of CCDC170 seems to be similar to another CCDC protein that associates with Golgi, CCDC165/MTCL1 (Sato et al., 2014). Overexpression analysis of CCDC170 was informative in terms of its function, as it revealed a role for CCDC170 in organizing the Golgi and MTs. Also, these results suggest that changes in the level, or other perturbations, of CCDC170 could affect Golgi organization. Our findings suggest that the regulation of the Golgi-MT network by CCDC proteins might be impacted in disease. Regarding a breast cancer mechanism, the Golgi is now understood to control cell polarity and directional migration through Golgi-derived MTs that extend throughout the buy ANA 12 (discussed below). As CCDC170 functions as a structural and organizational protein, we assessed how breast cancer-associated CCDC170 truncations might impact Golgi and MT associations, and we also performed functional mapping of CCDC170 Golgi and MT binding domains (Fig. 2 and Fig. S6). We found that clinically relevant truncations of CCDC170 (1–48, 1–405, 355–715, 593–715) lost Golgi localization. Combined with our mapping analyses, it was evident that the extreme C-terminal region of CCDC170 was necessary, but not sufficient, for Golgi localization. For example the 1–649 and 1–591 truncations resulted in loss of Golgi localization, with retention of MT association. However, the 593–715 fragment was not sufficient for Golgi association and showed whole cell localization, as did the 1–405 fragment. These findings indicate that positioning of CCDC170 at the Golgi requires multiple protein anchors, while MT-association was more modular and mapped between positions 405 and 591. Taken together, our findings implicate a clear model for how CCDC170 alterations might play a role in the breast cancer cell phenotype. As CCDC170 contributes to Golgi organization through MT-binding and stabilization, we hypothesized that breast cancer-associated changes in CCDC170 could affect Golgi organization. Disorganization of the Golgi could, in turn, affect the positioning of Golgi-derived MTs that control cell polarity and migration, as follows. The Golgi has recently been identified as a MT-organizing center (MTOC) and mediates the nucleation of a subset of non-centrosomal MTs (Zhu and Kaverina, 2013). Unlike dynamic centrosomal MTs, Golgi-derived microtubules are stable and acetylated (Skoufias et al., 1990; Thyberg and Moskalewski, 1993). As mentioned, these stable microtubules function in the positioning of the Golgi, but more importantly, Golgi-derived MTs have been found to extend into the cytoplasm and be essential for maintaining cell polarity. Furthermore, in migrating cells, the Golgi is normally positioned at the side of the nucleus facing the leading edge, and buy ANA 12 motile cells lacking Golgi-derived MTs are defective for polarized migration (Miller et al., 2009; Rios, 2014). Overall, very little is known about how dysfunction of Golgi-derived MTs can lead to cellular defects or disease. We tested whether CCDC170 levels could affect polarized cell migration and found that overexpression could slow directional migration, while knockout increased wound healing (Fig. 6). Furthermore, in cells overexpressing CCDC170, there was an uncoupling between Golgi positioning and directional migration (Fig. 6f). Thus, regarding the mechanisms by which CCDC170 may contribute to breast cancer, we hypothesize, and provide evidence that, CCDC170 regulates cell polarity and motility and that CCDC170 MT-stabilizing activity may be critical for these processes (Fig. 7d). In addition to a role of CCDC170 in Golgi organization (with coordinated impact on Golgi-associated MT organization), it is possible that CDCC170 perturbations may affect cell mobility or invasion by influencing the exocytic pathway (Eaton and Martin-Belmonte, 2014). We are currently investigating this possibility.