Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.
MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.
Olson
K R
KR
Department of Biology, University of Rochester, New York 14627, USA.
McIntosh
J R
JR
Olmsted
J B
JB
D000818:Animals; D015415:Biomarkers; D002451:Cell Compartmentation; D002478:Cells, Cultured; D006224:Cricetinae; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000502:physiology; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008870:Microtubules / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013329:Structure-Activity Relationship
D015415:Biomarkers; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins