A Window into the Dynamics of Rab5 Activity

Science Signaling  13 May 2008:
Vol. 1, Issue 19, pp. ec176
DOI: 10.1126/stke.119ec176

The small guanosine triphosphatase (GTPase) Rab5, known for its role in early endocytosis, has also been implicated in the engulfment of apoptotic cells, a process important in limiting the inflammatory response. Kitano et al. used Raichu-Rab5, a fluorescence resonance energy transfer (FRET) probe in which FRET reflects an increase in the active GTP-bound form of Rab, or Raichu-Rab5/PM (a form targeted to the plasma membrane) to monitor Rab5 activation in Swiss3T3 cells stably expressing integrin αvβ3 or in a macrophage cell line. Live cell imaging showed that the duration of Rab5 activation varied among phagosomes and that, in a substantial number, Rab5 was reactivated after termination of the initial period of activation. Simultaneous imaging of fluorescently labeled actin revealed that Rab5 activation and recruitment to phagosomes occurred during disassembly of the actin network around the phagosome. Rab5 inactivation took place before the breakdown of the apoptotic cell, a process that was delayed (but not blocked) by a Rab5 dominant-negative mutant. Pharmacological manipulations indicated that microtubule integrity was required for Rab5 activation and that its repetitive activation depended on their dynamic instability. Depletion of the Rab5 guanine nucleotide exchange factor (GEF) Gapex-5 with short interfering RNA (siRNA) inhibited Rab5 activation during phagocytosis, whereas depletion of other Rab5 GEFs (Rabex-5 and Rin) did not. EB1, a protein that associates with the tips of microtubules, bound to Gapex-5 in pull-down assays, and EB1 depletion with siRNA decreased Rab5 activation. The authors develop a model in which actin disassembly during closure of the phagosome enables extension of microtubules to the area, allowing the delivery of Gapex-5 to the phagosome and thereby Rab5 activation.

M. Kitano, M. Nakaya, T. Nakamura, S. Nagata, M. Matsuda, Imaging of Rab5 activity identifies essential regulators for phagosome maturation. Nature 453, 241-245 (2008). [PubMed]