Microtubule-mediated Src tyrosine kinase trafficking in neuronal growth cones.
Src family tyrosine kinases are important signaling enzymes in the neuronal growth cone, and they have been implicated in axon guidance; however, the detailed localization, trafficking, and cellular functions of Src kinases in live growth cones are unclear. Here, we cloned two novel Aplysia Src kinases, termed Src1 and Src2, and we show their association with both the plasma membrane and the microtubule cytoskeleton in the growth cone by live cell imaging, immunocytochemistry, and cell fractionation. Activated Src2 is enriched in filopodia tips. Interestingly, Src2-enhanced green fluorescent protein-positive endocytic vesicles and tubulovesicular structures undergo microtubule-mediated movements that are bidirectional in the central domain and mainly retrograde in the peripheral domain. To further test the role of microtubules in Src trafficking in the growth cone, microtubules were depleted with either nocodazole or vinblastine treatment, resulting in an increase in Src2 plasma membrane levels in all growth cone domains. Our data suggest that microtubules regulate the steady-state level of active Src at the plasma membrane by mediating retrograde recycling of endocytosed Src. Expression of constitutively active Src2 results in longer filopodia that protrude from smaller growth cones, implicating Src2 in controlling the size of filopodia and lamellipodia.
Wu
Bingbing
B
Department of Biological Sciences, Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, USA.
Decourt
Boris
B
Zabidi
Muhammad A
MA
Wuethrich
Levi T
LT
Kim
William H
WH
Zhou
Zhigang
Z
MacIsaac
Keira
K
Suter
Daniel M
DM
D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000166:cytology / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D004705:Endocytosis; D049452:Green Fluorescent Proteins / Q000378:metabolism; D020439:Growth Cones / Q000201:enzymology*; D008870:Microtubules / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D021381:Protein Transport; D011554:Pseudopodia / Q000201:enzymology; D022142:Secretory Vesicles / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D019061:src-Family Kinases