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. 2011 Mar 30;286(20):18079–18092. doi: 10.1074/jbc.M111.236018

FIGURE 4.

FIGURE 4.

Knockdown of TRAK1 by targeted TRAK1 shRNAi reduces mitochondrial transport in axons of hippocampal neurons. Hippocampal neurons prepared from P0 rat brain were transfected at 3 DIV with either pDsRed1-Mito alone, pDsRed1-Mito + the scrambled shRNAi, pGreenTRAK1scr, or pDsRed1-Mito + pGreenTRAK1 or for the rescue experiments with pTurboFP635-NMito, pTurboFP635-NMito + pRedTRAK1, pTurboFP635-NMito + pRedTRAK1 + pEGFP-rTRAK1silent, or pTurboFP635-NMito + pRedTRAK1 + EGFPTRAK2 and imaged at 6 DIV all as described under “Experimental Procedures.” A and C are representative examples of transfected neurons where Outline refers to an image with saturated fluorescence intensity to show the complete cell outline. In A, ZsGFP shows the green fluorescence enabling identification of transfected neurons, DsRed1-Mito shows the distribution of mitochondria, and Merge shows the merge of ZsGFP + DsRed1-Mito fluorescence. In C, DsRedTRAK1 shows the red fluorescence (colored here blue) for the TRAK1 shRNAi vector, TurboP635-Mito shows the distribution of mitochondria, GFP-TRAK1silent shows the fluorescence due to EGFP-rTRAK1silent, and Merge shows a merge of all three. B and D show a section of an axon at time t = 0 for each condition as labeled with the respective kymographs below. Scale bars are 20 μm. The parameters of mitochondrial dynamics are summarized in Table 2.