New Algorithm to Determine True Colocalization in Combination with Image Restoration and Time-Lapse Confocal Microscopy to Map Kinases in Mitochondria

The subcellular localization and physiological functions of biomolecules are closely related and thus it is crucial to precisely determine the distribution of different molecules inside the intracellular structures. This is frequently accomplished by fluorescence microscopy with well-characterized markers and posterior evaluation of the signal colocalization. Rigorous study of colocalization requires statistical analysis of the data, albeit yet no single technique has been established as a standard method. Indeed, the few methods currently available are only accurate in images with particular characteristics. Here, we introduce a new algorithm to automatically obtain the true colocalization between images that is suitable for a wide variety of biological situations. To proceed, the algorithm contemplates the individual contribution of each pixel's fluorescence intensity in a pair of images to the overall Pearsońs correlation and Manders' overlap coefficients. The accuracy and reliability of the algorithm was validated on both simulated and real images that reflected the characteristics of a range of biological samples. We used this algorithm in combination with image restoration by deconvolution and time-lapse confocal microscopy to address the localization of MEK1 in the mitochondria of different cell lines. Appraising the previously described behavior of Akt1 corroborated the reliability of the combined use of these techniques. Together, the present work provides a novel statistical approach to accurately and reliably determine the colocalization in a variety of biological images

Effect of <i>Bv</i>PIP2;1 and <i>Bv</i>PIP1;1 co-expression on oocyte plasma membrane permeability (P<i>_f</i>).

<p>Different amounts of cRNA of <i>Bv</i>PIP2;1, <i>Bv</i>PIP1;1 or a mix of <i>Bv</i>PIP2;1 and <i>Bv</i>PIP1;1 (<i>Bv</i>PIP2;1:<i>Bv</i>PIP1;1) were injected in <i>Xenopus</i> oocytes and after three days osmotic water permeability coefficient (P<i>_f</i>) was determined. In brackets is the relative quantity of cRNA injected in each oocyte, being 1 equal to 0,3 ng, and 2 or 3, two or three fold that amount, respectively. A four-fold injection of <i>Bv</i>PIP2;1 (4) was used as a control to show that the expression system was not saturated, NI are non-injected oocytes. Data are expressed as mean values (mean P<i>_f</i> ±SEM, n = 12−15). The figure shows representative data from five independent experiments. Different letters indicate significance between bars (p<0.05). All P<i>_f</i> corresponding to oocytes co-injected with different cRNA ratios of <i>Bv</i>PIP2;1: <i>Bv</i>PIP1;1 were not significantly different from P<i>_f</i> of <i>Bv</i>PIP2;1 (1) injected oocytes.</p

Localization of <i>Bv</i>PIP1;1-ECFP and <i>Bv</i>PIP2;2-EYFP in <i>Xenopus laevis</i> oocytes.

<p>Radial (x–z) confocal images of <i>X</i>. <i>laevis</i> oocytes expressing <i>Bv</i>PIP1;1-ECFP (A) (green) and <i>Bv</i>PIP2;2- EYFP (C) (green), previously injected with TMR-Dextran (red). The oocyte surface is on the right of each image frame and the interior of the oocyte is to the left. Inside each image the enlargement of the indicated square section is shown. Confocal (x–y) images collected at various focal depths into the <i>X</i>. <i>laevis</i> oocyte expressing <i>Bv</i>PIP1;1-ECFP (B) and <i>Bv</i>PIP2;2-EYFP (D) at 1µm steps from outside the oocyte till the cortical granules level, approximately 5 µm from the plasma membrane, are shown.</p

<i>Bv</i>PIP2;1 loop A root mean square fluctuation (RMSF).

<p>The RMSF of loops A are shown in yellow, green, blue and red lines corresponding to chains A, B, C and D respectively in all panels. Panels from A to C represent temporal intervals of the MDS ranging from 0 to 10 ns, 10 to 20 ns and 20 to 30 ns respectively. The inset shows the <i>Bv</i>PIP2;1 model, where green is used to point extracellular elements of the aquaporin, blue to point intracellular elements, purple to mark alpha helixes, bordeaux to distinguish loops B and E embedded in the membrane region and finally grey to show N-t and C-t. The same color pattern is used in the x-axis of panels to discriminate the location of residues of the primary structure in the protein. In the inset a red arrow is used to point loop A. The figure shows valleys in the RMSF which points that the secondary structure remains stable, the peaks represent movable parts of the protein and comparing extracellular elements, loop A is most flexible than loop C along the whole MDS.</p

Osmotic permeability (P<i>_f</i>) of oocytes membranes expressing <i>Bv</i>PIP2;1 mutants (N64H/E65Q or N64I/E65Q) with <i>Bv</i>PIP1;1.

<p><i>Bv</i>PIP2;1 mutants (N64H/E65Q and N64I/E65Q) show high water transport activity indicating that they are functional aquaporins. Both mutants are able to functionally interact with <i>Bv</i>PIP1;1 as they generate oocyte membrane P<i>_f</i> values far superior to those promoted by the mutant alone (*p<0.001). NI are non-injected oocytes. Values are representative data of three independent experiments using different oocyte batches. For each condition mean values are shown as mean P<i>_f</i> ±SEM, n = 12−15.</p

Localization of <i>Bv</i>PIP1;1-ECFP when co-expressed with <i>Bv</i>PIP2;2 or <i>Bv</i>PIP2;1 in <i>Xenopus laevis</i> oocytes.

<p>Radial (x–z) confocal images of <i>X</i>. <i>laevis</i> oocytes co-expressing <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;2 (green) (A) or co-expressing <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;1 (green) (D), both previously injected with TMR-Dextran (red). The oocyte surface is near the top of each image frame and the interior of the oocyte is in the bottom. Inside the image the enlargement of the indicated square section is shown. Stack of confocal (x–y) images were collected at various focal depths into the oocyte and then deconvolved and surface-render reconstructed with Huygens Professional Software. (B) Projections of the z-stack of images acquired with 100 nm step for oocytes co-expressing <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;2 (green) and (E) for oocytes co-expressing <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;1(green). (C) and (F) shows several views of the 3D reconstructed images for oocytes co-expressing <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;2 (green) and <i>Bv</i>PIP1;1-ECFP:<i>Bv</i>PIP2;1(green), respectively. The 0° view corresponds to the cortical granules level inside the oocyte and 180° to the plasma membrane plane (approximately 5 µm from the cortical granules level).</p

<i>Bv</i>PIP2;1 loops A MDS frames superimposition.

<p>The figure shows the MDS for <i>Bv</i>PIP2;1 loops A. Each <i>Bv</i>PIP2;1 monomer is in a different color; chain A is in yellow, chain B is in green, chain C is in blue and chain D is in red. The superimposition of 30 ns MDS of <i>Bv</i>PIP2;1 loops A is shown in a color range, where red is the starting position, white is an intermediate position and blue is the final one.</p