Tag Archives: MitoTracker

Tracking membranes by imaging – mCLING and surface glycans

Living cells exhibit many types of membranes which participate in most biological precesses, one way or another. Imaging membranes is usually acheived by two types of reagents: chemical dyes or fluorescent proteins that are targeted to the membrane itself or inside an organelle.

The chemical dyes are usually targeted to an organelle based on a specific chemical property of that organelle.

For example:

Rhodamine 123, tetramethylrosamine, and Mitotracker  are dyes that preferentially target mitochondria, due to its membrane potential. Mitotracker has thiol groups that allow it to bind to matrix proteins, thus making it more resistant to disruption of the membrane potential (e.g. by fixation).

Lysotracker are lypophilic, mildly basic dyes, which accumulate in the acidic lysosomes.

ER-tracker is a BODIPY (boron-dipyrromethene; a group of relatively pH insensitive dyes that are almost all water insoluble) based dyes which are linked to glibenclamide – a sulfonylurease – which binds to ATP sensitive Potassium channels exclusively resident in the ER membrane.

Long chain carbocyanines like DiL, DiO and DiD are lipophylic fluorescent molecules, which are weakly fluorescent in water, but highly fluorescent when incorporetaed into membranes, particularly the plasma membrane.

FM lipophylic styryl dyes bind the plasma membranes in a reversible manner and are also incorporated into internal vesicles.

On the other hand, fluorescent proteins (FP) are targeted to membranes or organelles by fusing them to either whole proteins that localize to a specific organelle, or to short peptides that carry a localization signal. Thus, a nuclear localization signal (NLS) targets the to the nucleus, mitochondrial targeting signal (MTS) to the mitochondria and a palmitoylation signal to the plasma membrane and endocytic vesicle.

There are advantages and disadvantages to each system, relating to ease of use, specificity, photostability etc… I do not want to go into that.

Here, I would like to mention two new methods to image the plasma membrane.

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Eliminating mutated mitochondria during in-vitro fertilization

There are several genetic diseases which originate not from mutations in the nuclear genome but mutations in the mitochondrial genome. In humans, the threshold for disease occurrence is if 60% of the mitochondria has mutated mitochondrial DNA (mtDNA) (a mixed mitochondrial origin is called heteroplasmy). There is currently no cure, and no good way to prevent these genetic diseases.

Since the source of the mitochondria is solely from the oocyte, a lot of effort is invested in trying to get rid of mutated mitochondria by in-vitro fertilization (IVF) procedures – thus preventing the disease in the offspring. Two methods have been tried so far – pronuclear transfer (PNT) and spindle-chromosome transfer (ST).  The idea is to extract the nuclear genetic material of the oocyte or zygote and transfer it to an enucleated oocyte or zygote with healthy mitochondria. However, in both cases, there is still carry-over of some mutated mitochondria to levels that can be as high as 44%.

In this new paper published in Cell, a group of researchers from China suggest a different approach. Continue reading