Assessment of focal complexes in B35 Neuroblastoma growthcones following Lovastatin treatment

Proper development of the nervous system requires efficient and precise axon growth and guidance, growths requiring dynamic focal complex adhesion and de-adhesion in the neuronal growth cone. The formation of focal complexes is regulated in part by guanine triphosphatases (GTPases) of the Rho family, which signal bi-directionally to focal complex constituents. Rho GTPases are also regulated through binding of guanosine triphosphate (GTP), a process facilitated by membrane targeting through geranylgeranylation (a specific type of prenylation) at their C terminus. Prior work in our laboratory demonstrated that decreasing geranylgeranylation with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase inhibitor, lovastatin, decreases neurite initiation and increases neurite branching and cell body rounding. These effects were reversed by co-treatment with geranylgeraniol, a precursor for geranylgeranylation. In the current work, we determined whether manipulating protein prenylation decreases the association of focal complex proteins (paxillin, talin or vinculin) with β1-integrin in B35 growth cones. B35 neuroblastoma cells were grown on uncoated glass coverslips, or coverslips coated with 25 μg/ml laminin or collagen. Cells were maintained in media were either not treated, or treated with lovastatin (LOV, 20 μM) alone or in combination with geranylgeraniol (GGOH, 10 μM). Focal complex formation in growth cones on the different substrata was assessed using co-immunoprecipitation and double-label immunocytochemistry. Culturing on different substrata did not alter the amount of paxillin or talin that associated with 1 integrin. However, vinculin co-localization with 1 integrin was decreased for cells plated on collagen or laminin; and co-localization with talin was increased in cells cultured on laminin, compared to cells plated on uncoated coverslips. For cells plated on collagen, GGOH decreased the amount of paxillin, but not talin or vinculin, that co-localized with β1 integrin compared to control cells maintained in reduced serum medium or treated with LOV. For cells cultured on their native, secreted substratum (undefined) or laminin, manipulating geranylgeranylation did not alter the association of focal complex proteins with 1 integrin in whole cell lysates, growth cones or cell bodies. However, treatment to alter protein geranylgeranylation (LOV, GGOH, LOV+GGOH) decreased the amount of co-localization of paxillin and talin, and GGOH treatment decreased the amount of vinculin co-localized with 1 integrin in growth cones, compared to cells in reduced serum medium (SCM) for cells cultured on collagen. Together, we interpret these data to indicate that inhibiting protein prenylation decreases growth cone focal adhesions, likely leading to decreased neurite initiation. Understanding the molecular mechanisms that regulate Rho GTPase control of neuronal process extension will facilitate our understanding of nervous system development, as well as identify potential therapeutic targets for treating developmental, traumatic and degenerative neural lesions.