The F‐actin cytoskeleton of neurons following microinjection with the small GTPase V14 RhoA

Abstract

A recently refined technique for microinjecting proteins into small cells (Bird et al. J. Anat.198, 2001) has been used to examine changes in the morphology and F‐actin cytoskeleton of cultured spinal cord neurons following microinjection of IgG alone or in conjunction with V14 Rhoa, a small gtpase known to cause changes in the actin cytoskeleton.Dissociated cell cultures from embryonic chick spinal cord (E7) were plated out onto glass coverslips in Ham’s F12 culture medium, incubated at 37 °C and re‐fed at intervals of 72 h. After 10 d in culture iindividual neurons were microinjected with V14RhoA (1 mg/mL), and/or IgG (to identify injected cells). The cultures were incubated at 37 °C for a further 20 min before fixation in 3.7% formaldehyde. The cells were washed permeabilised with 0.2% Triton X‐100 in PBS and then washed prior to immersion in buffer containing TRITC‐labelled phalloidin and FITC‐labelled anti rat IgG. The cultures were mounted in Mowiol containing a grain of antiquench and viewed on a confocal laser microscope.Microinjected cells showed spread of injectate throughout the cell body and into the proximal parts of the major dendrites. On the rare occasions when the nucleus was injected IgG did not escape into the cytoplasm. In control cells the F‐actin was concentrated close to the cell membrane in cell bodies and in specific parts of the growth cone. When cells were injected with V14RhoA there was an increase in the number of actin containing microspikes on the cell soma and major dendrites close to the origin of the presumptive axon. Near the substrate increased numbers of podosomes were also observed. More varicosities were found in both axons and dendrites after injection with V14RhoA.The changes in the F‐actin cytoskeleton after V14RhoA injections were observed within 30 min of the injections. Although some of the changes were similar to those observed in fibroblasts others, such as the presence of varicosities, were unique to neurons.