Dynamin (Dyn) is a GTPase involved in the endocytosis of various kinds of cells [5, 6]. Three Dyn family proteins, Dyn1–3, have been identified to date; whereas Dyn1 and Dyn3 are expressed primarily in neurons and testis, respectively, Dyn2 is expressed in diverse cell types [7]. Dyn mediates the scission of newly formed vesicles from the membrane of one intracellular compartment and typically promotes their targeting to, and fusion with, another compartment. It has been implicated in several well-known endocytic activities including clathrin- and caveolin -mediated endocytosis, where they mediate the scission of vesicular compartments from the unit membranes [6].

In this chapter, we demonstrate the involvement of Dyn2 in the formation of DVs in urinary bladder umbrella cells. Precise methods and results have been reported in the previous paper [1]. Dyn2 was immunostained in the cytoplasm of UCs. By immunoelectron microscopy for Dyn2 detection, its specific aggregation was detected around DV membranes (inset in Fig. 27.1d). As Dyn2 is known to control organization of actin microfilaments in relation to the endocytosis of culture cells [6, 8], the quick-freezing and deep-etching (QF-DE) method was used to examine ultrastructure of UCs (Fig. 27.1d). Paraformaldehyde-fixed mouse urinary bladder was permeabilized with detergent, and some were immunostained for Dyn2. The bladder was quickly frozen and deep-etched in freeze-dry machine as shown in Fig. 27.1c, and replica membranes were made, allowing us to observe cytoskeletons with transmission electron microscope . At the E-surface of freeze-fractured membranes (ES in Fig. 27.1d), the DVs have characteristic hexagonal pattern of globular intramembranous particles organized on the outer leaflet [9]. Distinct and often branched microfilaments, which were demonstrated by decoration of granular immunoproducts on the DV membranes with immunostaining with the Dyn2-specific antibody , connected to the cytoplasmic sides of each DV membrane (arrows in Fig. 27.1d) and intermediate filaments (arrowheads in Fig. 27.1d).

Dynasore , a cell-permeable Dyn-GTPase inhibitor [10, 11], was used for the treatment of mouse bladders in vivo. Under anesthesia, the bladders were exposed by opening the abdominal cavities of the mice, and urine in the bladders was sucked out with a syringe. After 5 min, the dynasore solution was gradually injected into the bladder lumen and incubated for 15 min. The dynasore treatment drastically reduced the number of DVs in the UCs and induced the formation of large membrane invaginations in the apical cell surface (Fig. 27.1b). This finding indicates that the Dyn2 probably moves along the membranes and/or interacts with proteins, such as microfilament -related proteins, during GTPase inhibition.