Cell attachment and neurite stability in NG108-15 cells: what is the role of microtubules?

Undifferentiated NG108-15 cells forming rapid-onset neurites were acutely exposed to nocodazole or trypsin. Resorption, cell rounding and detachment were delayed or prevented by 5'-deoxy,5'-methylthioadenosine (MTA), which selectively enhanced the strength of attachment responses. However, taxol, which stabilized microtubules, did not protect cell shape appreciably when trypsin or mechanical stimuli were used to decrease the strength of attachment. Together with numerous control experiments, this evidence suggests that the mechanical properties of microtubules do not contribute acutely to maintaining cell shape, though microtubules may play an indirect regulatory role (e.g. through their interactions with actin and substratum attachment sites). Patterns of trypsin-induced resorption resembled those seen 'spontaneously' in NG108-15 cells growing on laminin, and in fibroblastic CHO cells, suggesting that these results may be both physiologically relevant and applicable widely to many cell types.     

Effect of laminin and cytoskeletal agents on neurite formation by NG108-15 cells

Laminin promotes attachment and process formation in the neuroblastoma X glioma hybrid cell line NG108-15. As cells attached to laminin, they flattened and remained dispersed rather than associated in clumps. Process formation was observed within 1 hr after exposure to laminin and was dose dependent. Cycloheximide, an inhibitor of protein synthesis, did not block laminin-mediated attachment and neurite formation. Addition of drugs that depolymerize the cytoskeleton led to different behaviors for cells grown on plastic compared with those in the presence of laminin. Cells on plastic treated with either vinblastine or cytochalasin neither flattened nor grew processes. Cells plus laminin and vinblastine retracted processes, but remained flat, suggesting that laminin-induced processes can be destabilized by disrupting microtubules. Cells sequentially treated with laminin and cytochalasin produced processes that were thin and highly branched. Cells in high concentrations of cytochalasin on a laminin substrate formed aberrant processes even when their soma did not flatten. Since laminin counteracted the effect of cytochalasin on process outgrowth but did not alter the effect of cytochalasin on flattening of the cell body, different mechanisms mediated by microfilaments may be involved in cell flattening and in process formation.     

Role of laminin in stimulating rapid-onset neurites in NG108-15 cells: relative contribution of attachment and motility responses.

The present paper analyzed how attachment and motility responses contribute to the initial formation of rapid-onset neurites in undifferentiated NG108-15 cells. Attachment responses played a positive role in stabilizing neurites against slippage, but exerted a negative effect upon active motility--which appears to be the major rate-determining event in initial outgrowth. The role of laminin in this paradigm was elucidated by analyzing the effects of polyglutamate and other agents (bovine serum albumin and polyethylene glycol chain derivatives) which reduced the number of passive substratum attachment sites, but which did not affect cells directly nor affect the binding of laminin to the substratum. Polyglutamate decreased attachment and spreading of cells, yet greatly accelerated the initial laminin-dependent formation of rapid-onset neurites, and greatly enhanced active neurite remodelling and cell translocation as well. Together with data presented in previous studies in this series, these findings indicate that laminin stimulates rapid-onset neurites primarily by stimulating active motility responses. Insofar as rapid-onset neurites appear to provide a valid model of how neuronal growth cones are regulated, it is likely that these findings will be relevant to developing primary neurons as well.