Real-time analysis of clathrin-mediated endocytosis during cell migration

Abstract

. Real-time analysis of clathrin-mediated endocytosis during cell migration. J. Cell Sci. 116, 847-855.In both the online and print versions of this paper, in Materials and Methods, the construct pEGFP-dynamin2 was incorrectly identified as a human isoform. It is a rat isoform. AUTHOR CORRECTION IntroductionThe internalization of integral membrane proteins from the cell surface through receptor-mediated endocytosis occurs via clathrin-coated pits (Schmid, 1997;Takei and Haucke, 2001). Examples of molecules that undergo clathrin-mediated endocytosis include nutrients such as iron, via transferrin uptake (Hopkins et al., 1985;Conrad et al., 1999), growth factors and cytokines, through receptors such as the epidermalgrowth-factor receptor (Lamaze and Schmid, 1995;Carter and Sorkin, 1998), and cellular adhesion molecules such as integrins (Raub and Kuentzel, 1989). Additionally, numerous pathogenic organisms and molecules have been observed to gain entry into cells by exploiting the endocytosis machinery (Doxsey et al., 1987;Sieczkarski and Whittaker, 2002). Endocytosis has also been implicated in cell migration (Bretscher, 1996;Palecek et al., 1996;Sheetz et al., 1999;Bajno et al., 2000;Kamiguchi and Lemmon, 2000). In particular, it has been proposed that an increased rate of endocytosis at the trailing edge would contribute to the polarized cycling of either bulk membrane or cellular adhesion molecules, such as integrins, to the leading edge (Bretscher, 1996;Palecek et al., 1996;Sheetz et al., 1999).Great strides have recently been made in the analysis of the different factors involved in the production of clathrin-coated vesicles (Takei and Haucke, 2001). The presence and interaction of such components as the AP-2 adapter complex (Benmerah et al., 1998;Kamiguchi et al., 1998) and the accessory proteins AP180 (Takei and Haucke, 2001), Eps15 (Benmerah et al., 1998;Benmerah et al., 1999) and Hip1R (Engqvist-Goldstein et al., 2001) are beginning to be well characterized. Many of the proteins that are involved in clathrin-mediated endocytosis have been identified and cloned, their crystal structures have been determined (ter Haar et al., 1998;Collins et al., 2002), and detailed models for the production and internalization of clathrin-coated vesicles have been suggested (Schmid, 1997;Takei and Haucke, 2001;Kirchhausen, 2002). However, numerous questions regarding the events and interactions relevant to endocytosis remain unanswered. For example, it is not clear whether activated receptors recruit AP-2 which results in the polymerization of clathrin coat components, or if clathrin-coated pits are preformed and recruit activated receptors. Additionally, the precise molecular function(s) of the GTPase dynamin in endocytosis remains to be resolved (Takei et al., 1995;Cao et al., 1998;McNiven et al., 2000;Ochoa et al., 2000;Schmid and Sorkin, 2002;Tsuboi et al., 2002). Although previous studies have focused on the neuron-specific dynamin1 isoform (Tsuboi et al., 2002), there is also a functional role in endocytosis for the...

On the move: endocytic trafficking in cell migration

“…The finding that CCPs per se are not specifically enriched at the leading edge is in line with the broad distribution of AP-2 observed in migrating ECV304 cells [49] and the non-polarized uptake of the bona fide CME cargo transferrin in migrating fibroblasts [50]. When looking more specifically at CCP dynamics in different cell regions, the number of disappearing CCPs per area was reported to be increased in the front region [48]. However, as this number was not normalized to the total, this difference might originate from the likewise increased total number of clathrin puncta.…”

“…When dividing migrating cells into a lagging, middle and leading region, Rappoport et al observed an enrichment of clathrin, dynamin2 and transferrin in the middle and front region versus the rear of the cell. However, there was no difference between middle and front region arguing for a rather weak polarization of the endocytic machinery in the direction of cell migration [48]. The finding that CCPs per se are not specifically enriched at the leading edge is in line with the broad distribution of AP-2 observed in migrating ECV304 cells [49] and the non-polarized uptake of the bona fide CME cargo transferrin in migrating fibroblasts [50].…”

“…However, there are also clathrin-independent internalization routes like caveolinmediated endocytosis or the clathrin-independent carrier (CLIC) pathway [47]. The first attempts to study the localization and dynamics of CME during cell migration were made more than a decade ago by combining overexpression of fluorescently tagged endocytic proteins with dual-color total internal reflection fluorescence (TIRF) microscopy of MDCK cells in a scratched monolayer [48]. When dividing migrating cells into a lagging, middle and leading region, Rappoport et al observed an enrichment of clathrin, dynamin2 and transferrin in the middle and front region versus the rear of the cell.…”

Quand les microtubules rencontrent les puits recouverts de clathrine et permettent aux cellules de tenir le cap

“…Dans la plupart des cellules, les puits recouverts de clathrine sont distribués de façon homogène et aléatoire à la membrane plasmique. Cependant, il n'en va pas de même au cours de la migration cellulaire, et nous avons confirmé des observations plus anciennes [7] montrant que les puits recouverts de clathrine s'accumulent préférentiellement à l'avant des cellules cancéreuses en migration ( Figure 2A). Cette distribution asymétrique des puits recouverts de clathrine est en soi remarquable et doit être mise en parallèle avec d'autres observations ayant éta-bli que les microtubules acétylés sont essentiellement dirigés vers l'avant des cellules en migration [8,9].…”

“…Nous avons donc mis au point un système L'excès de centrosomes contribue à une réduction de la taille du cerveau Les mutations identifiées chez des patients atteints de MCPH ont été incriminées dans la diminution du nombre de centrosomes fonctionnels dans la population des cellules souches neuronales (CSN) [5,6]. Notre équipe a récemment développé et caractérisé une lignée de souris chez laquelle l'amplification centrosomale est restreinte à la population des CSN [7]. Nous avons choisi de perturber la machinerie de duplication …”