Domain-Specific Activation of Neuronal Migration and Neurite Outgrowth-Promoting Activities of Laminin

The ECM glycoprotein laminin has profound and varied actions on neurons in vitro. Little is known about how laminin's multiple domains and receptor-binding sites interact in determining its overall effects. Here, it is shown that laminin's ability to promote migration of olfactory epithelium neuronal cells maps to distal long arm domain E8 and is mediated by alpha 6 beta 1 integrin. Surprisingly, treatment of laminin with antibodies against its short arms (domains E1' or P1') uncovered a new neuronal migration-promoting activity, mediated by a beta 1 integrin other than alpha 6 beta 1. Laminin treated with anti-short arm antibodies also promoted beta 1 integrin-dependent neurite outgrowth from late embryonic retinal neurons, which are normally unresponsive to laminin. These "antibody-induced" migration and neurite outgrowth activities mapped to laminin's distal long arm, far from the site(s) of antibody binding. Evidence is presented that the induced activities are not actually cryptic in laminin, but are suppressed by an activity that is located in laminin's P1' domain and that may be lacking in the laminin homolog merosin.

. The data in Figure  2 show that the GoH3 monoclonal antibody, which binds to and blocks the function of ah-containing integrins (Sonnenberg et al., 1988) Figure 1. The effect of GoH3 is an approximately 50% decrease in cell migration.
This result contrasts markedly with the complete blockade of migration that is observed when CoH3 is added to explants plated on an untreated laminin substratum (i.e., the experiment shown in Figure 2; data from a repetition of that experiment, but carried out in parallel with the one shown above, were essentially identical to those shown in Figure 2). antibodies did not promote cell migration.
In addi- level of cell migration than laminin. This is illustrated in Figure  5, which also compares the effects of the GoH3 antibody on cell migration on laminin and merosin substrata.
As shown, cell migration on merosin substrata was only partially inhibited by GoH3, even at antibody concentrations 5times the level that inhibited all cell migration on laminin (see Figure  2). Nevertheless, all the migration-promoting activity of h 600 Distance (pm> In an experiment conducted in parallel with the one shown in Figure 2, OE explants were plated on coverslips treated with merosin (60 pglml) and cultured in the presence of GoH3 or control IgC. By comparing these data with Figure 2, it can be seen that merosin promotes considerably more neuronal migration than laminin and that migration on merosin is only partly inhibitable by GoH3. In both these regards, the effects of merosin resemble those of anti-PI'-treated laminin (see Figures Figure 1. The data indicate that anti-E8 antibodies block the additional neuronal migration-promoting activity induced in laminin by anti-PI' antibodies.  Figure  7B); that application of anti-PI' antibodies to BSA or El' substrata is insufficient to generate substrata that promote neurite outgrowth ( Figure  7C); that 8, integrins are required for the neurite outgrowth response to anti-PI'-treated laminin ( Figure  7D); and that a6 integrins are not required for this response ( Figure  7E). integrin function (see Figure  2).

Discussion
In the   CoH3 prevented virtually all cell migration on a laminin substratum (see Figure 2), it only partially reduced (by about two-thirds) the migration on an E8 substratum.
such activities when separated from the rest of the laminin molecule ( Figure  9; Table  4).  (Table  3), by proteolytic cleavage between laminin's short arms and long arm (see Figure  9, C8-9), or by proteolytic cleavage in the middle of the long arm (see E8  et al., 1990;Sanes et al., 1990;Kallunki et al., 1992;Vailly et al., 1994)  (CD-l random-bred mice; Charles River), were prepared and cultured in serum-free, low calcium medium (complete LCM) as described (Calof and Lander, 1991 and its area (in urn*) was determined.
The distance that each cell had migrated was taken as the straightline distance between the center of its cell body and the nearest edge of the explant; this measurement was made for every migratory cell of eachexplantthatwasevaluated.All migratorycellswerecounted for explants totaling a minimum area of 300,000 urn* (usually 4 or more explants).
For conditions in which migration was abundant, as with a laminin substratum, this corresponded to measuring the migratory distances of more than 600 total cells for a given condition. Cell