Buttner et al. showed that depletion of mitochondria DNA in yeast inhibits ROS formation and cell apoptosis induced by a-synuclein, further suggesting a direct functional connection between a-synuclein and mitochondria. More than those, a series of articles indicates that asynuclein may directly interact with mitochondria. We reported that, in addition to its predominantly cytosolic and vesicular localization, a fraction of a-synuclein localizes in the mitochondria under physiological condition, which is confirmed by many other studies. Nevertheless, the normal function and pathogenic role of mitochondrial a-synuclein need further investigation. Recently, Kamp et al. reported that a-synuclein has an inhibitory function on membrane fusion, and it binds to mitochondria and directly leads to mitochondrial fragmentation when overexpressed in cell cultures and Caenorhabditis elegans. In addition, Nakamura et al. described that the effect is not accompanied by changes in the morphology of other organelles, including endoplasmic reticulum and lysosomes. This may reveal a novel model of mitochondrial dynamic regulation,Nedaplatin yet some questions remains unanswered: Since recombinant asynuclein induces fission of artificial membranes, why does it have no influence on other lipid membranes in cells, such as ER and lysosomes, except mitochondria? Therefore, more direct evidence is needed to show the role of a-synuclein levels in mitochondrial morphology. Mitochondria and a-synuclein were double-stained in this study, which not only allows us to compare mitochondrial morphology and a-synuclein expression and distribution among three distinct cell lines, including Hela, SH-SY5Y and PC12 cells, but also to directly assess mitochondrial morphological changes in cells following a-synuclein overexpression. Our results indicate that a-synuclein expression levels has little influence on mitochondrial morphology in normal cells, but knockdown of asynuclein prevents MPP + -induced mitochondrial fragmentation in SH-SY5Y and PC12 cells. These data imply that a-synuclein plays no role in mitochondrial morphology under normal condition,Terutroban but may have some effect on that at the presence of certain environment factors. To observe the effects of a-synuclein overexpression on mitochondrial morphology, we need to track down a-synuclein overexpressed cells. However, some traditional approaches may be unsuitable for a-synuclein, such as fluorescent protein tags used in many papers. As a typical natively unfolded protein, a-synuclein lacks of rigid and ordered structure under physiological conditions in vitro, and its structure depends extremely on its environment and accommodates a number of unrelated conformations, which might affect its functions accordingly. If asynuclein was tagged with a fluorescent protein, whose molecular weight is even bigger than a-synuclein, we could not guarantee that this would not affect its structure or functions. Similar problems may remain when a fluorescent protein is co-transfected as an independent reporter. Another option is to transfect cells with untagged full length human SNCA cDNA, stain live cells with MitoTracker, fix them and then process for immunofluorescence staining of a-synuclein, since MitoTracker Red CMXRos stains mitochondria in live cells and is well-retained in fixed, permeabilized cells. Mitochondrial length is usually used to assess mitochondrial morphology in live cells, thus firstly, we need to test whether mitochondrial length would be affected by cell fixation. SH-SY5Y, PC12 or Hela cells were stained with MitoTracker-Red CMXRos and then observed on live cell imaging system using a 1006objective. Images of the same cells were taken immediately before and 30 min after fixation with 4% paraformaldehyde to detect mitochondrial morphogenetic alterations, one image often covers 1–2 SH-SY5Y or Hela cells or 4–8 PC12 cells, and the experiment was carried out five times for each cell line.