Artificial laminin polymers assembled in acidic pH mimic basement membrane organization.
Natural laminin matrices are formed on cell membranes by a cooperative process involving laminin self-polymerization and binding to cognate cellular receptors. In a cell-free system, laminin can self-polymerize, given that a minimal critical concentration is achieved. We have previously described that pH acidification renders self-polymerization independent of protein concentration. Here we studied the ultrastructure of acid-induced laminin polymers using electron and atomic force microscopies. Polymers presented the overall appearance of natural matrices and could be described as homogeneous polygonal sheets, presenting struts of 21 +/- 5 and 86 +/- 3 nm of height, which approximately correspond to the sizes of the short and the long arms of the molecule, respectively. The addition of fragment E3 (the distal two domains of the long arm) did not affect the polymerization in solution nor the formation of adsorbed matrices. On the other hand, the addition of fragment E1', which contains two intact short arms, completely disrupted polymerization. These results indicate that acid-induced polymers, like natural ones, involve only interactions between the short arms. The electrostatic surface map of laminin alpha1 LG4-5 shows that acidification renders the distal end in the long arms exclusively positive, precluding homophylic interactions between them. Therefore, acidification reproduces in vitro, and at a physiological protein concentration, what receptor interaction does in the cellular context, namely, it prevents the long arm from disturbing formation of the homogeneous matrix involving the short arms only. We propose that acid-induced polymers are the best tool to study cellular response to laminin in the future.
Barroso
Madalena Martins Sant'ana
MM
Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Freire
Elisabete
E
Limaverde
Gabriel S C S
GS
Rocha
Gustavo Miranda
GM
Batista
Evander J O
EJ
Weissmüller
Gilberto
G
Andrade
Leonardo Rodrigues
LR
Coelho-Sampaio
Tatiana
T
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007797:Laminin / Q000737:chemistry*; D051379:Mice; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008954:Models, Biological; D008968:Molecular Conformation; D011108:Polymers / Q000737:chemistry*; D055672:Static Electricity; D013997:Time Factors
D007797:Laminin; D011108:Polymers; C102645:laminin alpha 2; C084098:laminin A