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Many studies continue to map when and where proteins are co-localized. Our study asks when and where they interact.

 

 

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Voxel Dynamics at the protein network level influence how neurons are interconnected, which in turn drives diverse behaviors and stores unique memories of an individual. Using FRET, we began to map dynamic interaction between proteins within living animals. The unit of image data is voxel, the smallest three-dimensional volume resolvable wtih a fluorescence microscope.

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:embryo.psd

 

 

SIngle neuron analysis Protein interactions are quantified within the aCC motoneuron of intact Drosophila embryos. A typical voxel has at least 1,000 times the volume of the FRET detection limti (FRET is expected once every 1,000 encounters by a pure chance). At each voxel, we determine the FRET (defined as how much EGFP-tagged Cdc42 is present, multiplied by how much of it is in association with the mCherry-tagged partner protein).

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_1.psd

 

 

FRET at dendrite Cdc42 interacts with Par6 and WASp at dendrite. Cdc42 mean association probability and s.e.m. with microtubule-stabilizing Par6 and actin-organizing WASp in N aCC motoneuron samples. Asterisk indicates a statistically significant difference from the neurite background at P < 0.01 with a two-tailed t-test. The aCC motoneuron occurs in every half-segment. It connects a neuropil in the CNS to a muscle in the body wall. Approximately 1,000 voxels from a single aCC motoneuron are divided into twelve subcellular compartments: cell body (1, 2), dendrite (dark 4,5,6), neurite within the CNS (3,7,8,9), axon outside the CNS (10, 11), and axon terminal that innervates the target muscle (12).

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_2.psd

 

 

Restricted interaction Cdc42 interacts with Par6 and WASp only at the specific time and place within the aCC motoneuron. Asterisks indicate significant deviation from the background at P < 0.001. Mean concentration and association probability of Cdc42 from (N) aCC samples at three time points in animals co-expressing Cdc42 with Par6 (eve-GAL4 / + ; UAS-EGFP::Cdc42 UAS-mCherry::Par6 / +) and animals co-expressing Cdc42 with WASp (eve-GAL4 / + ; UAS-EGFP::Cdc42 UAS-mCherry::WASp / +). Co-expression of EGFP-labeled Cdc42 and unfused mCherry (eve-GAL4/ + ; UAS-EGFP::Cdc42 UAS-mCherry / +) serves as a control. Sample size in parenthesis indicates the number of aCC motoneurons examined.

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_3.psd

 

 

Not mere co-localization Only a limited percentage of the voxels during the entire period exhibits FRET above 2.5%.

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_4.psd

 

 

Coordination of partners Sample three-dimensional reconstructions of aCC dendritic compartments at 65% embryonic stage. Where dendrites emerge (open arrows), Cdc42 physically associates with Par6 and WASp at the shaft (5) and tip (6), respectively. Scale bar 1 micron for each axis.

 

 

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Signal and growth Five minutes or less is required for a visible morphological change to occur. Virtually simultaneously, Cdc42 increases interactions with Par6 and WASp at the shaft (5) and tip (6), respectively.

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_6.psd

 

 

Mutat Cdc42 At a low level expression (eve-GAL4 / + ; UAS-EGFP::Cdc42V12 UAS-mCherry::Par6 / + or eve-GAL4 / + ; UAS-EGFP::Cdc42V12 UAS-mCherry::WASp / +), mutant Cdc42 interacts with Par6 and WASp in virtually all subcellular compartments. Asterisk (]) indicates a statistically significant difference from the neurite background at P < 0.01 with a two-tailed t-test. In the dendritic subcellular compartments at 70% embryonic stage, however, the spatiotemporal interaction patterns of Par6 and WASp were very similar to those with the wild type Cdc42, i.e., with Par6 and WASp at the shaft (5) and tip (6), respectively. As a control, the mutant Cdc42 interaction pattern with the CRIB domain is shown on the left most. (Note: The aCC motoneuron exhibited no aberrant morphologies with the mutant Cdc42 expressed at this expression dosage. However, if expressed at considerably higher dosages, this mutant Cdc42 induces dramatic phenotypes.)

 

 

Description: Macintosh HD:Users:akira:Desktop:FILE:WEB:free:what_7.psd

 

 

Unrestricted interaction A large percentage of the voxels during the entire period exhibits FRET above 2.5%.

 

 

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