Quantitative analysis of protein dynamics during asymmetric cell division.
In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis.
Mayer
Bernd
B
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Dr.-Bohr-Gasse 3-5, 1030 Vienna, Austria.
Emery
Gregory
G
Berdnik
Daniela
D
Wirtz-Peitz
Frederik
F
Knoblich
Juergen A
JA
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology*; D003593:Cytoplasm / Q000378:metabolism; D004330:Drosophila / Q000378:metabolism* / Q000502:physiology; D029721:Drosophila Proteins / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins; D007605:Juvenile Hormones / Q000378:metabolism*; D008465:Mechanoreceptors / Q000166:cytology*; D018613:Microscopy, Confocal; D011493:Protein Kinase C / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D002352:Carrier Proteins; D029721:Drosophila Proteins; D007605:Juvenile Hormones; C115164:PON protein, Drosophila; D025521:Tumor Suppressor Proteins; C081435:l(2)gl protein, Drosophila; C060606:numb protein, Drosophila; D049452:Green Fluorescent Proteins; C115015:PKC-3 protein; D011493:Protein Kinase C