Letter
Nature 453, 241-245 (8 May 2008) | doi:10.1038/nature06857; Received 9 January 2008; Accepted 19 February 2008; Published online 2 April 2008
Imaging of Rab5 activity identifies essential regulators for phagosome maturation
Masahiro Kitano1,3, Michio Nakaya2,4, Takeshi Nakamura1, Shigekazu Nagata2,4 & Michiyuki Matsuda1
- Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, and,
- Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
- Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
- Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Correspondence to: Takeshi Nakamura1 Correspondence and requests for materials should be addressed to T.N. (Email: tnakamr@path1.med.kyoto-u.ac.jp).
Efficient phagocytosis of apoptotic cells is crucial for tissue homeostasis and the immune response1, 2. Rab5 is known as a key regulator of the early endocytic pathway3 and we have recently shown that Rab5 is also implicated in apoptotic cell engulfment4; however, the precise spatio-temporal dynamics of Rab5 activity remain unknown. Here, using a newly developed fluorescence resonance energy transfer biosensor, we describe a change in Rab5 activity during the engulfment of apoptotic thymocytes. Rab5 activity on phagosome membranes began to increase on disassembly of the actin coat encapsulating phagosomes. Rab5 activation was either continuous or repetitive for up to 10 min, but it ended before the collapse of engulfed apoptotic cells. Expression of a dominant-negative mutant of Rab5 delayed this collapse of apoptotic thymocytes, showing a role for Rab5 in phagosome maturation. Disruption of microtubules with nocodazole inhibited Rab5 activation on the phagosome membrane without perturbing the engulfment of apoptotic cells. Furthermore, we found that Gapex-5 is the guanine nucleotide exchange factor essential for Rab5 activation during the engulfment of apoptotic cells. Gapex-5 was bound to a microtubule-tip-associating protein, EB1, whose depletion inhibited Rab5 activation during phagocytosis. We therefore propose a mechanistic model in which the recruitment of Gapex-5 to phagosomes through the microtubule network induces the transient Rab5 activation.