A coat of filamentous actin prevents clustering of late-endosomal vacuoles in vivo.
The endocytic pathway depends on the actin cytoskeleton. Actin contributes to internalization at the plasma membrane and to subsequent trafficking steps like propulsion through the cytoplasm, fusion of phagosomes with early endosomes, and transport from early to late endosomes. In vitro studies with mammalian endosomes and yeast vacuoles implicate actin in membrane fusion. Here, we investigate the function of the actin coat that surrounds late endosomes in Dictyostelium. Latrunculin treatment leads to aggregation of these endosomes into grape-like clusters and completely blocks progression of endocytic marker. In addition, the cells round up and stop moving. Because this drug treatment perturbs all actin assemblies in the cell simultaneously, we used a novel targeting approach to specifically study the function of the cytoskeleton in one subcellular location. To this end, we constructed a hybrid protein targeting cofilin, an actin depolymerizing protein, to late endosomes. As a consequence, the endosomal compartments lost their actin coats and aggregated, but these cells remained morphologically normal, and the kinetics of endocytic marker trafficking were unaltered. Therefore, the actin coat prevents the clustering of endosomes, which could be one safeguard mechanism precluding their docking and fusion.
Drengk
Anja
A
Abteilung Zellbiologie and Center for Interdisciplinary Nanostructure Science and Technology (CINSaT), Universität Kassel, Heinrich Plett Str. 40, 34132, Kassel, Germany.
Fritsch
Jürgen
J
Schmauch
Christian
C
Rühling
Harald
H
Maniak
Markus
M
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004023:Dictyostelium / Q000166:cytology* / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D015800:Protozoan Proteins; D013050:Spectrometry, Fluorescence
D051339:Actin Depolymerizing Factors; D000199:Actins; D003598:Cytoskeletal Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C105803:vac A protein, Dictyostelium discoideum; D049452:Green Fluorescent Proteins