Subcellular compartmentalization of CD38 in non hematopoietic cells a study to characterize its functional role in mitochondria 5

* Figure 4.15 CD38 immunoreactivity in mouse cerebellum, labeled with goat polyclonal CD38 antibody-sc-7049.. The postsynaptic densities showed the most intense labeling arrow head foll

*

Figure 4.15 CD38 immunoreactivity in mouse cerebellum, labeled with goat

polyclonal CD38 antibody-sc-7049 The immunoreactivity was present in many dendrites The postsynaptic densities showed the most intense labeling (arrow head) followed by CD38 staining associated with mitochondria Minimal labeling was observed alongside the plasma membrane (B) Negative immunostaining observed in CD38 KO samples (A) -Axon terminal white arrow-CD38 immunoreactive

*

B

A

*

Figure 4.16 CD38 immunoreactivity in mouse cerebellum, labeled with goat

polyclonal CD38 antibody-sc-7049 The labeling occurred diffusely throughout the processes at the granular layer (A), in particular association with mitochondria in varying degree of CD38 staining intensity (B) Notice the the immunostaing is

B

A

Figure 4.17 CD38 immunoreactivity in mouse cerebellum, labeled with goat

polyclonal CD38 antibody-sc-7049 Intense immunolabeling was associated with mitochondria, plasma membrane in the Golgi cell dendrite region (Gd) at molecular layer Varying degree of CD38 immunostaining associated with mitochondria in different region was observed (arrow) The postsynaptic densities showed the most intense labeling (arrowhead)

Scale bar:1µm

Gd

4.2.4.2 Localization of CD38 on mitochondria using immunogold labeling

Following the EM/immunoperoxidase study as mentioned above, independent confirmation of CD38 localization to the outer mitochondrial membrane was sought

by EM/immunogold labeling experiments This has a direct advantage over immunofluoresence or immunoperoxidase in that the labeling of individual samples can be correlated with ultrastructural features, such comparisons are important because the EM/immunogold technique has an excellent signal to noise ratio with practically no background noise, making the categorization of cells as + or – staining

absolutely unambiguous (de Harven et al., 1993) True enough, immunogold labeling

does indeed further confirm the results obtained from immunoperoxidase study

Immunogold localization of CD38 using a 15nm gold conjugate rabbit anti-goat IgG revealed the presence of CD38 in isolated mitochondria from the wild type mice brain tissues The result was examined with the use of sample blanks In the sample blanks, 2 negative controls, a) Non-immune Goat IgG instead of primary goat CD38 antibody and b) isolated mitochondria from CD38 KO mice brain tissues were employed to examine for the specificity of the CD38 antibody in this experiment (data not shown) As expected, there were no gold particles detected in association with the purified mitochondria isolated from CD38 KO mouse brains In electron micrographs of isolated mitochondria from the wild type mice brain tissues, the gold labeling clearly showed that CD38 is indeed present on the mitochondria The

localization on mitochondria by immunoperoxidase labeling with DAB, the results obtained from the immunogold labeling of fractionated mitochondria showed consistent CD38 labeling and distribution pattern on the surface of mitochondria This also showed that the fractionated procedure did not alter the localization of CD38 on the organelle These data further supported the localization of CD38 on the outer mitochondrial membrane which strengthens the interpretation that the association of CD38 with the mitochondria is not due to the extraction procedure Altogether, these results showed specific localization of CD38 on the outer mitochondrial membrane and a specific orientation of the molecule present on the mitochondria

Immunogold localization of nicastrin, an inner mitochondrial resident membrane protein was employed in this study to serve as a comparison to the outer mitochondrial membrane localization of CD38 Here, it was observed that most gold particles labeled the interior of mitochondria; only a few gold particles were on or near the rim of mitochondria (Figure 4.19 A-C) The electron micrographs obtained showed the pattern of internal staining, as opposed to the pattern of staining with CD38 involving the surface of the outer mitochondrial membrane facing the cytosolic side In contrast to the immunogold labeling of CD38 on the outer mitochondria membrane, all gold particles observed were distributed throughout the interior of mitochondria in close association with the inner mitochondria membrane None of the gold particles was found associated on the outer mitochondrial membrane facing the cytosolic side

B