Real-time visualization of processive myosin 5a-mediated vesicle movement in living astrocytes.
Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.
Stachelek
S J
SJ
Department of Cellular and Molecular Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655, USA.
Tuft
R A
RA
Lifschitz
L M
LM
Leonard
D M
DM
Farwell
A P
AP
Leonard
J L
JL
D000199:Actins / Q000502:physiology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism* / Q000502:physiology; D001483:Base Sequence; D002465:Cell Movement / Q000502:physiology*; D017931:DNA Primers; D009218:Myosins / Q000378:metabolism* / Q000502:physiology; D051381:Rats
D000199:Actins; D017931:DNA Primers; D009218:Myosins