Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells.
We have reconstituted fusion between phagosomes and lysosomes in streptolysin O-permeabilized J774-E macrophages. Fusion was assessed by measuring the delivery of avidin-conjugated horseradish peroxidase pre-internalized into lysosomes to phagosomes containing biotinylated beta-glucuronidase-conjugated paramagnetic beads (1-2 microm). Fusion was dependent on energy and exogenously supplied cytosol. Phagosome-lysosome fusion was greatly inhibited when microtubules were depolymerized by nocodazole treatment, suggesting that fusion occurs via microtubule-dependent transport. Furthermore, fusion was inhibited by GTPgammaS and Rab GDP dissociation inhibitor. These results suggest that rab proteins are involved in the regulation of fusion. Lastly, anti-NEM-sensitive factor (NSF) antibodies inhibited fusion, and addition of recombinant NSF wild type partially restored the fusogenic activity, indicating that NSF is required for fusion between phagosomes and lysosomes.
Funato
K
K
Animal and Cellular Systems Lab, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan.
Beron
W
W
Yang
C Z
CZ
Mukhopadhyay
A
A
Stahl
P D
PD
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000494:pharmacology; D002459:Cell Fusion; D002460:Cell Line; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D020730:Guanine Nucleotide Dissociation Inhibitors; D006160:Guanosine Triphosphate / Q000378:metabolism; D008247:Lysosomes / Q000502:physiology*; D051379:Mice; D052066:N-Ethylmaleimide-Sensitive Proteins; D010588:Phagosomes / Q000502:physiology*; D013301:Streptolysins / Q000494:pharmacology*; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; C086796:GDP dissociation inhibitor 1; D020730:Guanine Nucleotide Dissociation Inhibitors; D013301:Streptolysins; D033921:Vesicular Transport Proteins; C025964:streptolysin O; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D005033:Ethylmaleimide