Dynamin triple knockout cells reveal off target effects of commonly used dynamin inhibitors

Abstract

SummaryDynamin, which is encoded by three genes in mammals, is a GTPase implicated in endocytic membrane fission. Dynamin 1 and 3 are predominantly expressed in brain, whereas dynamin 2 is ubiquitously expressed. With the goal of assessing the impact of the lack of dynamin on cell physiology, we previously generated and characterized dynamin 1 and 2 double knockout (DKO) fibroblasts. These DKO cells were unexpectedly viable in spite of a severe impairment of clathrin-mediated endocytosis. As low-level expression of the dynamin 3 gene in these cells could not be excluded, we have now engineered dynamin 1, 2 and 3 triple KO (TKO) fibroblasts. These cells did not reveal any additional defects beyond what was previously observed in DKO fibroblasts. Surprisingly, although fluid-phase endocytosis and peripheral membrane ruffling were not impaired by the lack of all three dynamins, two structurally similar, widely used dynamin inhibitors, dynasore and Dyngo-4a, robustly inhibited these two processes both in wild-type and TKO cells. Dynamin TKO cells will be useful tools for the further exploration of dynamin-dependent processes and the development of more specific dynamin inhibitors.

Dynamin 2 regulates biphasic insulin secretion and plasma glucose homeostasis

“…A similar scenario has been reported at rodent nerve terminals lacking dynamin 1 (36,75), in which dynamin 3 partially supports vesicle recycling (38,39). It is unclear whether the dynamin-independent endocytosis at some nonsecretory cells (45,71) and nerve terminals (i.e., bulk endocytosis) (40,72,76) also occurs in dynamin 2 KO β cells. Future study using triple dynamin KO cells will shed new light on this question.…”

Endocytic proteins drive vesicle growth via instability in high membrane tension environment

“…8C has a maximum in-plane stress of 0.46 mN/m, almost an order of magnitude less than the rupture tension, thereby, making it stable. We note that vesicles with elongated tubular domains have also been observed in dynamin-mutant mammalian cells (58). Because actin burst in mammalian cells (under low resting tension) occurs just before scission, our work suggests that actin forces lead to elongation of the vesicles but are unable to dissociate vesicles from the plasma membrane due to inadequate scission stress.…”

Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes

“…This suggests that continued signaling after internalization represents a conserved mechanism shared by both lineages of lymphocytes. Our genetic approach has the advantage of avoiding potential off-target effects of pharmacological dynamin inhibition (22). We propose a model where TCR signaling from the plasma membrane represents "wave 1" of signaling, which is followed by TCR internalization and signaling from intracellular compartments (such as endosomes) that constitutes "wave 2" of signaling.…”