Peroxisome elongation and constriction but not fission can occur independently of dynamin-like protein 1.
The mammalian dynamin-like protein DLP1 belongs to the dynamin family of large GTPases, which have been implicated in tubulation and fission events of cellular membranes. We have previously shown that the expression of a dominant-negative DLP1 mutant deficient in GTP hydrolysis (K38A) inhibited peroxisomal division in mammalian cells. In this study, we conducted RNA interference experiments to 'knock down' the expression of DLP1 in COS-7 cells stably expressing a GFP construct bearing the C-terminal peroxisomal targeting signal 1. The peroxisomes in DLP1-silenced cells were highly elongated with a segmented morphology. Ultrastructural and quantitative studies confirmed that the tubular peroxisomes induced by DLP1-silencing retained the ability to constrict their membranes but were not able to divide into spherical organelles. Co-transfection of DLP1 siRNA with Pex11pbeta, a peroxisomal membrane protein involved in peroxisome proliferation, induced further elongation and network formation of the peroxisomal compartment. Time-lapse microscopy of living cells silenced for DLP1 revealed that the elongated peroxisomes moved in a microtubule-dependent manner and emanated tubular projections. DLP1-silencing in COS-7 cells also resulted in a pronounced elongation of mitochondria, and in more dispersed, elongated Golgi structures, whereas morphological changes of the rER, lysosomes and the cytoskeleton were not detected. These observations clearly demonstrate that DLP1 acts on multiple membranous organelles. They further indicate that peroxisomal elongation, constriction and fission require distinct sets of proteins, and that the dynamin-like protein DLP1 functions primarily in the latter process.
Department of Cell Biology and Cell Pathology, Robert Koch Strasse 6, University of Marburg, Marburg, 35037, Germany.
Annett
A
Koch
Gabriele
G
Schneider
Georg H
GH
Lüers
Michael
M
Schrader
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D034281:Dynamins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020868:Gene Silencing; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D020675:Peroxisomes / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C490452:Pex11b protein, mouse; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases; C486391:Dnm1l protein, mouse; D034281:Dynamins