Research

We are interested in understanding how cells achieve directional migration. This is an important biological problem in immune cell function, wound healing, cancer metastasis and axonal guidance. The neuronal growth cone is the highly motile structure at the tip of neuronal processes. It employs cell surface receptors to detect surrounding guidance cue information which is transduced via signal transduction cascades to the underlying actin and microtubule cytoskeleton. Our goal is to understand the underlying cellular and molecular mechanisms. Using advanced live cell imaging (Fluorescent Speckle Microscopy (FSM), FRET, DIC), biophysical, cell biological and molecular techniques and the extremely large growth cones from Aplysia californica, we are working on the following problems/questions.

Aplysia growth cone in DIC optics. Diameter: 50 um.

F-actin (red) and microtubule (green) labeling of the same growth cone.

Microtubule dynamics during adhesion-mediated growth

Using microtubule and actin FSM we have quantified cytoskeletal dynamics during directional growth cone responses triggered by the cell adhesion protein apCAM, focally presented with a microbead. We found that early microtubules explore the apCAM adhesion site due to partial uncoupling of microtubules from retrogradely moving actin filaments (Lee and Suter, 2008). These early microtubules may have a role in Src activation that strengthens the apCAM-actin coupling (Suter et al., 2004).  Strong apCAM-actin coupling in turn attenuates peripheral actin flow, which is followed by a concerted reorientation of actin and microtubule filaments towards the adhesion site.

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Movie 3 from Lee and Suter, 2008
DIC/microtubule FSM time-lapse movie showing early exploratory microtubules (colored in blue) extending towards apCAM-bead adhesion site. Time interval: 10 seconds; playback time: 50x real time. Scale bar: 5 um.

Dynamics and functions of Src tyrosine kinases in growth cones

Src tyrosine kinases have important signaling functions in growth cones; however, the dynamics of their distribution and the details of how they affect the cytoskeleton are not well understood. We have recently cloned two novel members of Src family kinases in Aplysia, termed Src1 and Src2, and investigated their localization and trafficking in growth cones. We found that microtubules play an important role in controlling the steady state distribution as well as activation state of Src in the growth cone periphery (Wu et al., 2008).

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Movie 2 from Wu et al, 2008
Time lapse movie showing Src2-EGFP localizing to the plasma membrane in growth cones. We also identified Src2-EGFP-positive endocytic vesicles moving in linear fashion (arrowhead). Time interval: 10 seconds; playback time: 30x real time. Scale bar: 10 µm.

Dynamics and functions of the Src substrate cortactin in growth cones

Cortactin is a Src substrate protein that regulates actin dynamics as well as plasma membrane actin interaction. Little is known about cortactin dynamics and functions in growth cones. We have cloned Aplysia cortactin and demonstrated that it localizes both the filopodial actin bundles as well as to apCAM adhesion sites (Decourt et al., 2009).

F-actin (red) and cortactin (green) labeling of an Aplysia growth cone.

Role of reactive oxygen species in regulating actin dynamics and growth cone motility

Reactive oxygen species (ROS) are now recognized to have physiological signaling role besides causing oxidative damage. In fibroblasts and endothelial cells there is increasing evidence for ROS regulating actin-dependent cell motility. We have recently shown that upon lowering growth cone ROS levels in general, or by inhibiting specific sources such as NADPH oxidases and lipoxygenases, F-actin organization and dynamics are severely impaired (Munnamalai and Suter, 2009). NADPH oxidase inhibition caused reduced actin assembly and retrograde flow in the peripheral domain, lipoxygenase inhibition resulted in increased contractility in the transition zone. These results suggest that localized ROS sources in the growth cone can regulate the growth cone actin cytoskeleton and related motility.

 

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NADPH oxidase inhibition by 1 µM phenyl arsine oxide (PAO) reduced F-actin (red) content and caused microtubule (green) extension. Scale bar: 10 µm.  
Movie 7 from Munnamalai and Suter, 2008
F-actin dynamics movie of growth cone in control condition and in the presence N-tert-butyl-a-phenylnitrone (PBN, an ROS scavenger). PBN caused initial leading edge and filopodial protrusion, slowing of F-actin flow and disassembly of F-actin structures. Time interval: 10 seconds; playback time: 100x real time. Scale bar:  10 um.

Topography and nanomechanics of growth cones

In collaboration with the laboratory of Dr. Gil Lee, University College, Dublin, Ireland, we have studied the surface topography and mechanical properties of neuronal growth cones using atomic force microscopy (Grzywa et al., 2006; Xiong et al., 2009). These studies measured the dimensions of distinct growth cones regions with nanometer resolution and demonstrated that there is a strong correlation between the elastic modulus and F-actin content and organization.

AFM imaging of filopodia and lamellipodia in live growth cone.