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 main objective is to understand the underlying biochemical and biophysical mechanisms of axonal growth and guidance during nervous system development. We use advanced imaging (such as Fluorescent Speckle Microscopy (FSM), DIC, STORM, SEM), and biophysical, cell biological and molecular techniques to investigate the role of cytoskeletal and signaling proteins as well as of forces in axonal growth and guidance. We conduct our studies in two model systems: cultured neurons from Aplysia californica and developing zebrafish embryos.

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

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

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). We have also recently shown that Src2 and cortactin positively regulate the actin network density and protrusion of lamellipodia as well as the formation, stability and length of filopodia in growth cones (He et al., 2015). Our results indicate that Src2-dependent phosphorylation of cortactin is critical for several of these functions.

 
 

Dual color STORM images of cortactin and pSrc2 labeling in Aplysia growth cones. Co-localization of the two proteins is marked by arrowheads in (a) and shown enlarged in a single filopodium in (b). From He et al. (2015).

 

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. We have further demonstrated that a NOX-2 type NADPH oxidase complex exists in Aplysia growth cones. NOX-2 exhibits an interesting bidirectional relationship with the actin cytoskeleton (Munnamalai et al., 2014). We are currently investigating the role of ROS signaling in neuronal development including axonal growth and guidance also in a new model system, zebrafish. We have found a broad expression pattern of four different NADPH oxidases, nox1, nox2/cybb, nox5 and duox, in the developing zebrafish central nervous system (Weaver et al., 2016). Ongoing studies address the function of NADPH oxidase in zebrafish nervous system development.

 

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Aplysia bag cell growth cone stained for F-actin (red), gp91phox (green), and p40phox (blue).  Both gp91phox and p40phox exhibit partial co-localization with F-actin. From Munnamalai et al. 2014  
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.
   

In situ hybridization of nox2/cybb in developing zebrafish embryos shows broad expression of Nox2/Cybb in forebrain, eyes, and midbrain during the first 2 days of development (A-D whole mount; E-P cryo sections). From Weaver et al., 2016

   

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.

In collaboration with the lab of Dr. Arvind Raman, Mechanical Engineering, Purdue University, we have recently measured the retrograde traction force developed by Aplysia growth cones as they undergo adhesion-mediated growth cone advance. We have used two different approaches: force-calibrated microneedles and AFM. We have found that Aplysia growth cones can produce a wide range of retrograde traction forces (1-100 nN) as they advance in response to a local adhesion substrate. Our biophysical studies further revealed a linear relationship between substrate stiffness and force development suggesting a reinforcement mechanism. Ongoing studies address the nature of the seemingly constant substrate deformation observed during adhesion-mediated growth cone advance.

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DIC time-lapse movie showing an Aplysia growth cone responding to a force-calibrated needle coated with the cell adhesion molecule apCAM. After a latency period of 10 min the growth cone develops a traction force of 15 nN. Scale bar: 10 mm. Time compression: 75x. From Athamneh et al. 2015.

 

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.