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Dynein‐mediated Lysosome Trafficking in Autophagic Flux of Mouse Coronary Arterial Myocytes
Author(s) -
Xu Ming,
Li Xiaoxue,
Zhang Yang,
Li PinLan
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1092.6
Subject(s) - lysosome , microbiology and biotechnology , dynein , autophagy , colocalization , autophagosome , bafilomycin , biology , chemistry , microtubule , biochemistry , enzyme , apoptosis
Dynein is a motor protein in a variety of mammalian cells that regulates lysosome trafficking. The present study hypothesized that dynein‐mediated lysosome trafficking contributes to autophagic flux, an important autophagy maturation process in coronary arterial myocytes (CAMs) and that nicotinic acid adenine dinucleotide phosphate (NAADP)‐induced lysosome Ca 2+ release regulates dynein‐mediated lysosome trafficking and autophagic flux. Pretreatment of CAMs with EHNA (erythro‐9‐(2‐hydroxy‐3‐nonyl)adenine), an dynein inhibitor or overexpression of dynamitin that disrupts the dynein complex enhanced 7‐ketocholesterol (7‐Ket)‐induced expression of autophagic marker LC3B by 90.3 % and 82.5%, respectively. Similarly, EHNA and dynamitin cDNA increased the cellular level of p62, a selective substrate for autophagy, by 58.7 % and 52%, respectively. Confocal microscopy demonstrated that LC3B colocalization with Lamp‐1, a lysosome protein increased upon 7‐Ket and that their colocalization coefficient was reduced by 57.3 % and 54.1%, respectively with pretreatment of CAMs with EHNA or dynamitin cDNA. By flow cytometry, it was found that both EHNA and dynamitin cDNA increased 7‐Ket‐induced formation of autophagosomes (APs), but reduced the number of autophagolysosomes (lysosome‐autophagosome fused, APLs) in CAMs. In addition, 7‐Ket was found to increase lysosomal Ca 2+ release as detected by colocalization of Ca 2+ (by Fluo4) and lysosome staining (by Rho) in living CAMs, which was blocked by 92.7 % when these cells were treated by NAADP inhibitors, NED‐19 or PPADS. These results suggest that atherogenic stimulus, 7‐Ket can enhance autophagy in CAMs and this 7‐Ket‐induced autophagy is associated with dynein‐mediated lysosome trafficking (supported by NIH grants HL057244, HL091464 and HL075316).