Dynamics of clathrin-mediated endocytosis and its requirement for organelle biogenesis in Dictyostelium

Abstract

SummaryThe protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. In single-cell eukaryotes, such as Saccharomyces cerevisiae, the gene encoding clathrin is not an essential gene, raising the question of whether clathrin conveys specific advantages for multicellularity. Furthermore, in contrast to mammalian cells, endocytosis in S. cerevisiae is not dependent on either clathrin or adaptor protein 2 (AP2), an endocytic adaptor molecule. In this study, we investigated the requirement for components of clathrin-mediated endocytosis (CME) in another unicellular organism, the amoeba Dictyostelium. We identified a heterotetrameric AP2 complex in Dictyostelium that is similar to that which is found in higher eukaryotes. By simultaneously imaging fluorescently tagged clathrin and AP2, we found that, similar to higher eukaryotes, these proteins colocalized to membrane puncta that move into the cell together. In addition, the contractile vacuole marker protein, dajumin-green fluorescent protein (GFP), is trafficked via the cell membrane and internalized by CME in a clathrin-dependent, AP2-independent mechanism. This pathway is distinct from other endocytic mechanisms in Dictyostelium. Our finding that CME is required for the internalization of contractile vacuole proteins from the cell membrane explains the contractile vacuole biogenesis defect in Dictyostelium cells lacking clathrin. Our results also suggest that the machinery for CME and its role in organelle maintenance appeared early during eukaryotic evolution. We hypothesize that dependence of endocytosis on specific components of the CME pathway evolved later, as demonstrated by internalization independent of AP2 function.

Resolving the homology—function relationship through comparative genomics of membrane-trafficking machinery and parasite cell biology

“…D. discoideum AP-2 localizes to distinct puncta near the cell surface which co-localize with clathrin; both AP-2 and clathrin also partially localize to the contractile vacuole network [67]. Similarly, the single beta subunit involved in both AP-1 and AP-2 complexes in D. discoideum localizes to the plasma membrane and also to intracellular structures [68].…”

“…Similarly, the single beta subunit involved in both AP-1 and AP-2 complexes in D. discoideum localizes to the plasma membrane and also to intracellular structures [68]. Consistent with a role in endocytosis, AP-2 interacts with an Eps15-related protein [67], but also with the SNARE VAMP7 [69], which is known to associate with the contractile vacuole [70,71]. Oddly, knockout of AP-2 subunits does not affect the internalization of the contractile vacuole marker dajumin [67], or the localization of p25 or p80 endosomal markers [72].…”

“…Consistent with a role in endocytosis, AP-2 interacts with an Eps15-related protein [67], but also with the SNARE VAMP7 [69], which is known to associate with the contractile vacuole [70,71]. Oddly, knockout of AP-2 subunits does not affect the internalization of the contractile vacuole marker dajumin [67], or the localization of p25 or p80 endosomal markers [72]. Comparatively, knockout of the lone AP-1/2β subunit results in pleiomorphic defects, including impaired osmotic stress response [68], likely due to its function in both complexes.…”

Imaging and Modeling the Dynamics of Clathrin-Mediated Endocytosis

“…Genome editing, however, might be the method of choice to study EAPs that play a more direct regulatory and/or structural role, without perturbing their normal dynamic behavior. Given a robust fiduciary of the lifetime of a CCP, extremely low signals derived from endogenously Kaksonen et al 2006, Taylor et al 2011, and Macro et al 2012 tagged EAPs can be detected with high sensitivity using a master/slave approach, in which the fiduciary signal defines time and location of the signal readout in the low SNR slave channel (Aguet et al 2013). …”

“…2D). As in yeast, actin is required for CME (Brady et al 2010), whereas AP2 is dispensable (Macro et al 2012). However, in contrast to yeast but similar to higher eukaryotic cells, micropinocytosis requires clathrin (O'Halloran and Anderson 1992;Ruscetti et al 1994) and only recently, a bona fide cargo that requires clathrin for internalization has been identified (Macro et al 2012).…”