Characterization of MAP1B heavy chain interaction with actin.
Microtubule-associated protein 1B is an essential protein during brain development and neurite outgrowth and was studied by several assays to further characterize actin as a major interacting partner. Tubulin and actin co-immunoprecipitated with MAP1B at similar ratios throughout development. Their identity was identified by mass spectrometry and was confirmed by Western blots. In contrast to previous reports, the MAP1B-actin interaction was not dependent on the MAP1B phosphorylation state, since actin was precipitated from brain tissue throughout development at similar ratios and equal amounts were precipitated before and after dephosphorylation with alkaline phosphatase. MAP1B heavy chain was able to bind actin directly and therefore the N-terminal part of MAP1B heavy chain must also contain an actin-binding site. The binding force of this interaction was measured by atomic force microscopy and values were in the same range as those of MAP1B binding to tubulin or that measured in MAP1B self-aggregation. Aggregation was confirmed by negative staining and electron microscopy. Experiments including COS-7 cells, PC12 cells, cytochalasin D and immunocytochemistry with subsequent confocal laser microscopy, suggested that MAP1B may bind to actin but has no obvious microfilament stabilizing effect. We conclude, that the MAP1B heavy chain has a microtubule-stabilization effect, and contains an actin-binding site that may play a role in the crosslinking of actin and microtubules, a function that may be important in neurite elongation.
Cueille
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Département de Biologie Cellulaire et de Morphologie, Université de Lausanne, 9 rue du bugnon, CH-1005 Lausanne, Switzerland.
Blanc
C Tallichet
CT
Popa-Nita
S
S
Kasas
S
S
Catsicas
S
S
Dietler
G
G
Riederer
B M
BM
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000254:growth & development / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013347:Subcellular Fractions
D000199:Actins; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; C085284:microtubule-associated protein 1B