Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells.
Fluorescence recovery after photobleaching measurements with high spatial resolution are performed to elucidate the impact of the actin cytoskeleton on translational mobility of green fluorescent protein (GFP) in aqueous domains of Dictyostelium discoideum amoebae. In vegetative Dictyostelium cells, GFP molecules experience a 3.6-fold reduction of their translational mobility relative to dilute aqueous solutions. In disrupting the actin filamentous network using latrunculin-A, the intact actin cytoskeletal network is shown to contribute an effective viscosity of 1.36 cP, which accounts for 53% of the restrained molecular diffusion of GFP. The remaining 47% of hindered protein motions is ascribed to other mechanical barriers and the viscosity of the cell liquid. A direct correlation between the density of the actin network and its limiting action on protein diffusion is furthermore established from measurements under different osmotic conditions. In highly locomotive polarized cells, the obstructing effect of the actin filamentous network is seen to decline to 0.46 cP in the non-cortical regions of the cell. Our results indicate that the meshwork of actin filaments constitutes the primary mechanical barrier for protein diffusion and that any noticeable reorganization of the network is accompanied by altered intracellular protein mobility.
Potma
E O
EO
Ultrafast Laser and Spectroscopy Laboratory, Materials Science Centre, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
de Boeij
W P
WP
Bosgraaf
L
L
Roelofs
J
J
van Haastert
P J
PJ
Wiersma
D A
DA
D000199:Actins / Q000494:pharmacology / Q000502:physiology* / Q000648:ultrastructure; D000818:Animals; D016764:Cell Polarity / Q000502:physiology*; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D004023:Dictyostelium / Q000378:metabolism*; D004058:Diffusion / Q000187:drug effects; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D007700:Kinetics; D008164:Luminescent Proteins / Q000187:drug effects / Q000378:metabolism*; D009997:Osmotic Pressure; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D014177:Translations
D000199:Actins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins