Category Archives: membranes

Poking holes into membranes to label proteins for live imaging

There are two major way to label inner proteins, structures or organnelles for live cell imaging. The most common method is fusing the studied protein to a fluorescent protein. A second approach is the addition of labeling agents from outside the cells. However, many labels cannot penetrate through the cell membrane. This is true to some, but not all dyes, but more importantly, to larger agents, such as antibodies or DNA/RNA oligos. To allow these agents to enter cells, researchers can use microinjection, electroporation, bead-loading, or transfection (e.g. of short oligos).

In a paper just published in eLife, a new technique is described to form temporary holed in the cell membrane. These holes allow delivery of any labeling agent into cells. Continue reading

New data on SmartFlare – do they detect mRNA?

Almost exactly a year ago, I wrote a post regarding my concerns with SmartFlare, supposedly a novel method for live imaging of RNA in cells.

In a nutshell, SmartFlare are gold nanoparticles covered in oligos specific to a certain mRNA of interest. Supposedly, cells internalize these particles and, once the mRNA hybridize to the oligo, a complementary fluorecently labeled oligo is being unquenchhed and “flares”, indicating the present of said mRNA.

You can read about my concerns in that older post, but apparently I wasn’t the only one concerned about their validity.

Raphaël Lévy from U. of Liverpool (UK) was concerned as well. He endeavored into an open science project to try and answer his concerns (which is why I allow myself to openly review his paper).

Continue reading

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.

Continue reading