Identification and characterization of aplysia Src tyrosine kinases in neuronal growth cones

Bingbing Wu, Purdue University

Abstract

Src family tyrosine kinases are important signaling molecules that are involved in cell proliferation, differentiation, adhesion, migration and endocytosis. There is accumulating evidence that Src tyrosine kinases are also involved in axon guidance. However, the detailed localization, trafficking, and function of Src kinases in growth cones are poorly understood. I used live cell fluorescent microscopy, immunocytochemistry, and cell fractionation analysis to study the Src tyrosine kinases in Aplysia neuronal growth cones. I was able to clone two novel Aplysia Src kinases, termed Aplysia Src1 and Src2. Both Src1 and Src2 have been found to be partially associated with plasma membrane and microtubule cytoskeleton by live cell imaging, immunocytochemistry, and cell fractionation. Active Src2 kinases are enriched at the tips of filopodia. I have observed Src2-enhanced green fluorescent protein-positive puncta and tubulovesicular structure undergoing bidirectional movement in the central domain and mainly retrograde movement in the peripheral domain of the growth cones. By applying endocytic markers in the growth cones, I was able to determine that the majority of the Src2-EGFP positive structures are endocytic vesicles. To further test the role of microtubules in Src2 distribution in the growth cone, microtubules were depleted from the growth cone with either nocodazole or vinblastine treatment, abolishing Src2-EGFP positive endocytic vesicle movements and resulting an increase of Src2 signal 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. Furthermore, constitutively active Src2 is localized at the plasma membrane and filopodia tips but not associated with endocytic vesicles. However, general growth cone endocytosis is not affected by overexpression of constitutively active Src2. Taken together these results indicate Src2 activity affects growth cone morphology and is negatively regulates its own endocytosis.

Degree

Ph.D.

Advisors

Suter, Purdue University.

Subject Area

Neurosciences|Cellular biology

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