Actin-based motility of isolated axoplasmic organelles.
We previously showed that axoplasmic organelles from the squid giant axon move toward the barbed ends of actin filaments and that KI-washed organelles separated from soluble proteins by sucrose density fractionation retain a 235-kDa putative myosin. Here, we examine the myosin-like activities of KI-washed organelles after sucrose density fractionation to address the question whether the myosin on these organelles is functional. By electron microscopy KI-washed organelles bound to actin filaments in the absence of ATP but not in its presence. Analysis of organelle-dependent ATPase activity over time and with varying amounts of organelles revealed a basal activity of 350 (range: 315-384) nmoles Pi/mg/min and an actin-activated activity of 774 (range: 560-988) nmoles/mg/min, a higher specific activity than for the other fractions. By video microscopy washed organelles moved in only one direction on actin filaments with a net velocity of 1.11 +/- .03 microns/s and an instantaneous velocity of 1.63 +/- 0.29 microns/s. By immunogold electronmicroscopy, 7% of KI-washed organelles were decorated with an anti-myosin antibody as compared to 0.5% with non-immune serum. Thus, some axoplasmic organelles have a tightly associated myosin-like activity.
Bearer
E L
EL
Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912, USA.
DeGiorgis
J A
JA
Medeiros
N A
NA
Reese
T S
TS
D000199:Actins / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D001370:Axonal Transport / Q000187:drug effects* / Q000502:physiology; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D049832:Decapodiformes; D004789:Enzyme Activation / Q000187:drug effects; D006737:Horseshoe Crabs; D007150:Immunohistochemistry; D009218:Myosins / Q000032:analysis / Q000276:immunology; D015388:Organelles / Q000201:enzymology / Q000502:physiology*; D011193:Potassium Iodide
D000199:Actins; D011193:Potassium Iodide; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins