Cytoskeletal dynamics in neuronal growth cones during adhesion-mediated guidance

Aih Cheun Lee, Purdue University

Abstract

During nervous system development, growing axons need to be directed to an appropriate target area in order to make functional connections. Axonal growth is lead by growth cones, which sense guidance signals in the environment, and transduce them into directional movement. Microtubules (MTs) undergo rearrangement during growth cone steering events; however, it is unclear whether MTs extend to adhesion sites because of changes in plus-end polymerization and/or translocation dynamics, because of changes in actin-microtubule interactions, or because they follow the reorganization of the actin cytoskeleton. In the current study, I used fluorescent speckle microscopy (FSM) to directly quantify MT and actin dynamics in Aplysia growth cones steering towards beads coated with the cell adhesion molecule apCAM. I conclude that adhesion molecules guide neuronal growth cones and underlying MT rearrangements by differentially regulating MT-actin coupling and actin movements according to growth cone region and not by controlling plus-end polymerization rates. MTs exploring the adhesion site in the early phases of cue exposure are less coupled to retrograde actin flow, allowing them to extend into the growth cone periphery. In the later phases of adhesion-mediated outgrowth, actin and MTs rearrange in concert in response to strong traction towards the adhesion site. Furthermore, I have studied the role of Src tyrosine kinase and tyrosine phosphorylation in the regulation of cytoskeletal dynamics and adhesion-mediated responses in the growth cone. Live cell imaging of growth cones expressing GFP-tagged Aplysia Src1 and Src2, and immunocytochemistry with Aplysia Src-specific antibodies revealed that adhesion formation by apCAM beads results in Src2 accumulation, but tension application is needed to activate Src2. Pharmacological studies with a Src inhibitor showed that Src activity plays a role in the regulation of actin dynamics and organization related to lamellipodial protrusion, filopodia stabilization, and retrograde actin flow, while not affecting microtubule dynamics in growth cones. Using a phosphotyrosine (PY) location reporter, I observed that PY signal is strongly enhanced in filopodial tips on the axis of outgrowth during the traction phase of growth cone steering. These studies provide insight on how microtubules are regulated in response to adhesive guidance cues, and point to intracellular proteins and signaling cascades that might change cytoskeletal behavior as a result of specific guidance cue signaling. To gain knowledge on how the growth cone cytoskeleton changes during guidance is important not only for understanding the wiring of the developing nervous system, but also for developing new therapeutic strategies to treat nervous system injuries and neurodegenerative diseases.

Degree

Ph.D.

Advisors

Suter, Purdue University.

Subject Area

Neurosciences|Cellular biology

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