Feng-Song Wang

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Neurofilament subunits can undergo axonal transport without incorporation into Triton‐insoluble structures

We examined the form(s) in which NF subunits undergo axonal transport. Pulse-chase radiolabeling analyses with 35S-methioinine revealed that newly synthesized Triton-soluble NF subunits accumulated within axonal neurites elaborated by NB2a/d1 neuroblastoma prior to the accumulation of Triton-insoluble subunits. Gel chromatographic, immunological, ultrastructural, and autoradiographic analyses of Triton-soluble axonal fractions demonstrated that radiolabeled, Triton-soluble subunits were associated with NFs. Triton-soluble, radiolabeled axonal NF subunits were also detected within retinal ganglion cell axons following intravitreal injection of 35S-methioinine. Microinjected biotinylated subunits were prominent within axonal neurites of NB2a/d1 cells and cultured dorsal root ganglion neurons substantially before they were retained following Triton-extraction. Prevention of biotinylated subunit, but not dextran tracer, translocation into neurites by nocodazole confirmed that microinjected subunits did not enter axons merely due to diffusion or injection-based pressure. Immuno-EM confirmed the association of biotin label with axonal NFs. These findings point towards multiple populations of NF subunits within axons and leave open the possibility that axonal NFs may be more dynamic than previously considered.

Modeling the Role of Myosin 1c in Neuronal Growth Cone Turning

We addressed the mechanical basis for how embryonic chick dorsal root ganglion growth cones turn on a uniform substrate of laminin-1. Turning is significantly correlated with lamellipodial area but not with filopodial length. We assessed the lamellipodial contribution to turning by asymmetric micro-CALI of myosin isoforms that causes localized lamellipodial expansion (myosin 1c) or filopodial retraction (myosin V). Episodes of asymmetric micro-CALI of myosin 1c (or myosin 1c and V together) caused significant turning of the growth cone. In contrast, repeated micro-CALI of myosin V or irradiation without added antibody did not turn growth cones. These findings argue that lamellipodia and not filopodia are necessary for growth cone turning. To model the role of myosin 1c on growth cone turning, we fitted the measured trajectories from asymmetric micro-CALI of myosin 1c-treated and untreated growth cones to the persistent random walk model. The first parameter in this equation, root-mean-square speed, is indistinguishable between the two data sets whereas the second parameter, the persistence of motion, is significantly increased (2.5-fold) as a result of asymmetric inactivation of myosin 1c by micro-CALI. This analysis demonstrates that growth cone turning results from an increase in the persistence of directional motion rather than a change in speed. Taken together, our results suggest that myosin 1c is a molecular correlate for directional persistence underlying growth cone motility.

Spectrin-actin interaction is required for neurite extension in NB 2a/dl neuroblastoma cells

Spectrin is an actin‐binding membrane skeleton protein involved in the maintenance of cell shape and generation of distinct membrane protein domains. Actin binds to the N‐terminal domain of β‐spectrin. To examine the function of spectrin‐actin interaction in neurons, we sought to disrupt this interaction in differentiating NB 2a neuroblastoma cells by microinjecting an N‐terminal domain‐specific anti‐β‐spectrin antibody. We found that microinjection of the affinity‐purified N‐terminal domain‐specific anti‐β‐spectrin inhibited the extension of the neurites in NB 2a/dl cells. The microinjected cells remained flat, and put out many filopodia‐like processes; but these processes failed to extend when the cells were induced to differentiate in the presence of dbc AMP or in serum‐free medium. The N‐terminal domain‐specific anti‐β‐spectrin also inhibited the binding of spectrin to actin. By contrast, the microinjection of monospecific anti‐α‐spectrinG did not inhibit neurite extension. These results suggest that β‐spectrin‐actin interaction may be required for neurite extension, which is critical for development of polarity in nerve cells.

Chromophore assisted laser inactivation of cellular proteins

A molecular understanding of biology requires that we establish the in situ functions of the proteins in cellular processes. To address this, we developed chromophore- assisted laser inactivation (CALI) for probing the in vivo function of proteins. CALI inactivates specific proteins in living cells by using non-blocking antibodies conjugated with malachite green (MG) dye. MG absorbs 620 nm laser light (which is not absorbed by cells) to generate short lived free radicals with limited range of oxidative damage (15 angstroms) around the dye. This inactivates the bound protein without significantly affecting its neighbors. CALI has been applied to 40 proteins and achieved specific inactivation in almost all those tested. We have developed micro-CALI which uses a focused laser beam (10 micrometers ) to acutely inactivate specific proteins within cells. We have used this to address the molecular mechanisms of neuronal growth cone motility and has implicated a diversity of proteins (e.g. molecular motors, cytoskeletal, and signaling molecules) in discrete steps of growth cone motility. We hope that micro-CALI will be a useful research tool for addressing dynamic processes in biology and medicine.