Category Archives: development

Visualizing translation: insert TRICK pun here

Unlike transcription, it is much harder to image translation at the single molecule level. The reasons are numerous. For starters, transcription sites (TS) are fairly immobile, whereas mRNAs, ribosomes and proteins move freely in the cytoplasm, often very fast. Then there are only a few TS per nucleus, but multiple mRNAs are translating in the cytoplasm. Next, there’s the issue of signal to noise – at the transcription site, the cell often produces multiple RNAs, thus any tagging on the RNA is amplified at the transcription site.  Last, it is fairly easy to detect the transcription product – RNA – at a single-molecule resolution due to multiple tagging on a single molecule (either by FISH or MS2-like systems). However, it is much more difficult to detect a single protein, be it by fluorescent protein tagging, or other ways (e.g. FabLEMs).

The rate of translation is ~5 amino acids  per second, less than 4 minutes to a protein 1000 amino-acids long. This is faster than the folding and maturation rate of most of even the fastest-folding fluorescent proteins. This means that by the time the protein fluoresce, it already left the ribosome. However, attempts were made in the past with some success.

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FISEB 2014 meeting -day 1

FISEB meeting happens every three years, and it includes participants from 28 different experimental biology societies in Israel. It is the best meeting to learn about biological-medical research performed in Israel at all fields and doctrines.

4 days, 8-10 parallel sessions, hundreds of lectures, >1000 posters, >2200 participants.

The first day started by a plenary lecture by Aryeh Warshel, Nobel lauret. He is really far from my field, and his lecture was very much confusing to me. But he has nice cartoons 🙂 The bottom line – enzymes are able to catalyze reactions due to electrostatic connections that are maintained stable (unlike in water).

From the afternoon sessions, I chose “signaling pathways & networks”. Relevant to this blog:

Yoav Henis from Tel-Aviv Uni. talked about oligomerization of TGF-beta receptors. he used a method he calls “co-patching”, which is essentially IF with two different antibodies for two receptor subunits. homodimerization will yield single color “patch” whereas heterodimerization will yield an overlap of both colors (co-patch). He then looked at the % of co-patch with different receptor subunits with/without ligand, or with mutants.

Maya Schulinder from Weizmann Institute talked about the contacts between mitochondria and other organelles (ER, vacuole) in yeast. These contacts are important for lipid metabolism. She new about the mito-ER contact but found there must be a second contact (bypass mechanism). She used an interesting screen method to find the bypass mechanism to the mito-ER contact: she expressed one of the contact protein as a GFP fusion. She expected that if the bypass mechanism and the mito-ER contact “share the load” of lipid metabolism, then deletion of the bypass will increase the number of the mito-ER contacts to compensate. Using automation, she imaged 6200 deletion mutants (from the yeast deletion library) each expressing this GFP fusion. As expected, she found 4 candidates which turned out to be very interesting.

Roni Seger from Weizmann showed that targeting the nuclear localization signal of ERK can be a novel cure for certain pathologies, including certain types of cancer.

On the other hand, Maya Zigler from the Hebrew Uni. suggested another new idea to cure cancer – by inducing the surrounding immune cells to destroy the tumor.

Ido Amit from Weizmann as well told us that we may not really know all the different types of cells that exist. What most people do, particularly in immunology, is rely on one or two known “markers” and use FACS or other methods to sort the cells based on these markers. However, some of the markers overlap. and there may be cells for which we do not have any markers and they “disappear” in the crowd of unsorted cells. or, the could be further sub-types we do not know about. So he approached the problem in an unbiased way – he took all the cells in the spleen, and did single cell RNA seq to individual cells from the spleen. Thus, each cell type has several hundred/thousand “markers” based on gene expression profiles. Not only did this method agree with the common FACS sorting markers, but he identified several sub-types unknown before.  Expect his paper this month in Science. His paper just got published in Science.

Finally, Yaron Shav-tal from Bar-Ilan Uni. used the MS2 system to study how perturbing the signaling pathway of serum stimulation affects transcription of beta-actin gene. As per usual – very neat job and interesting results.

Open source microscope & software: openSPIM & Fiji

Just hears a great talk today by Pavel Tomancak from Max-Planck institute. He’s doing amazing work in systematic imaging of fly RNAome & proteome during development. Check out his website (click his name above).

He also talked about Fiji, which an open source software that is just like ImageJ, but with supporting community that develops new scripts & applications.

A most unusual “open source” development that he is very proud about is the openSPIM.

SPIM – Selective Plane Illumination Microscopy – is a microscopy method in which a laser beam sends a narrow light sheet to the specimen, and the objective is at 90 degree from the light sheet (there are SPIM developments with up to 4 objectives that can image from all 4 sides. See here). This type of microscopy is good for imaging thick live specimens (e.g. whole worm, fly or fish embryo etc..). Due to the thickness of the sample, wide field imaging will cause too much background.

So, there are good SPIM microscopes that you can buy from companies like Zeiss. but he developed the openSPIM, which is a build-it-yourself  SPIM microscope, that actually fits into a suitcase (he took it to South-Africa with him to show college & high-school kids). He claims that non-specialist can construct it in one hour. All the details (parts, assembly instructions (“IKEA/Lego style”) are found at the website. The cost, he estimates, is ~40,000$ (with the camera being about half the cost). He claims that the openSPIM is comparable, in quality of images, to commercial SPIM microscopes from 5 years ago.  Pretty good for standard imaging.

I like the idea.

Cells reach out their “hands” to create new limbs

Communication between cells takes many forms. There could be communication by sending out microvesicles with important messages inside, by sending out free molecules (like hormones) or by special structures (e.g. synapses).

Sonic hedgehog (SHH) is a signaling protein that is important for the development of vertebrate limbs. It was thought to be release from a small group of cells at the posterior end of the limb bud, and is recognized by receptors on cells a long distance away.

Not this Sonic Hedgehog… (image taken from http://sonic.wikia.com)

A new paper publish in Nature from Maria Barna’s lab shows that SHH actually remains bound on the external side of the cells that produce it. The cells simply send very long thin protrusions (here named filopodia) that reach all the way to similar filopodia of the receiving cells.

I think that not only the story is very novel and interesting, but the images are very pretty.

Several “technical” issues:

In order to study the SHH signaling in live chick embryos, they designed a custom made live in ovo microscopy system: a temperature controlled plate; on it an egg container chamber, and an objective that is dipping into the yoke.

They show that standard fixation methods (e.g formaldehyde) destroy these filopodia. Also, a volume marker (in this case sfGFP) that just fills the cytoplasm does not give a strong enough signal to detect these filopodia (possibly since they are very thin, and packed with actin filaments  and other proteins, so there’s very little free volume left).

So, they used palmitoylated fluorescent proteins, pmeGFP (green) and pmKate2 (red) that target them to the plasma membrane. This enabled them to visualize these very thin and long filopodia. Here’s a video movie from their paper.

They use a variety of cytoskeletal proteins fused to EGFP or to mKate2 to learn about the structure of these filopodia. Their conclusion is that these structures contain only a specialized form of actin filaments.

They show beautifully that SHH (fused to EGFP) travels to the tip of these filopodia:

They used a split GFP technology to show that SHH is actually found on the outside of the cell membrane.  In split GFP, two fusion proteins are produced, each one is fused to “half” of a GFP protein (its not exactly half but let not go into that now). If the two fused proteins are in close proximity, the two halves associate to produce an GFP that fluoresce irreversibly. The two separate halves do no fluoresce. So one half was fused to SHH and the other was anchored to the extracellular leaflet of the plasma membrane, and when both were expressed, they got green fluorescence.

In total – a very nice and pretty paper.
ResearchBlogging.orgSanders TA, Llagostera E, & Barna M (2013). Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature, 497 (7451), 628-32 PMID: 23624372

Nature methods special issue: focus on bioimage informatics (Part III)

Continuing with the Brief communications section:
Rapid, accurate particle tracking by calculation of radial symmetry centers

Tracking single particles is a major challenge, since in many cases the particles are smaller than the pixel size. Several image analysis methods have been developed to analyze subpixel localization of particles. Here, Raghuveer Parthasarathy describes a new approach to calculate subpixel localization of particles, using radial symmetry analysis. Although the accuracy of his algorithm is similar to that of other algorithms (Gaussian fittings such as Non-Linear Least Square minimization – NLLS and Maximum likelihood estimations –MLE), his calculations are ~100 faster than other algorithms.

For those of you who do single particle analysis and super-resolution microscopy, this algorithm may be very helpful.

Rational design of true monomeric and bright photoactivatable fluorescent proteins

As already mentioned in the previous post, photoactivated localization microscopy (PALM) and Stochastic optical reconstruction microscopy (STORM) are two of the techniques used in super-resolution microscopy. In many cases PALM/STORM use photoactivatable fluorescent proteins (PA-FPs). The efficiency of super-resolution microscopy relies on the properties of these PA-FPs, such as brightness, photostability, pH stability, oligomeric state, maturation rate, photoswitching/activation yields etc…

EoSFP, which was cloned in 2004 from the scleractinian coral Lobophyllia hemprichii and furhter engineered, is a green-to-red photoswitchable protein with the highest photon output of all PA-FPs. Upon UV irradiation, it permanently switches its emission peak from 516 to 581 (excitation is at 505).  Monomeric form, mEoSFP and mEos2 were developed (mEoSFP is less used, since its chromofore does not maturate at 37°C, limiting the use to non-mammalian cells).

Here, the authors claim than mEoS2 forms oligomers at high concentrations, which may limit the use of this protein as a fusion partner to the studied protein, and can also skew super-resolution analysis that assumes only monomeric mEoS2 forms. Therefore, the authors solved the crystal structure of mEoS2. Based on the structure, they developed improved, true monomeric variants (mEoS3.1 & mEoS3.2), which are also brighter and mature faster.

Confocal images of HEK293 cells transiently transfected with plasmids encoding indicated fusions and imaged at the middle layer (top) or near the plasma membrane (bottom). See the differnces between mEoS2 and mEoS3! Source: Zhang et al. Nature Methods 9,727–729(2012)

In the supplementary data of this paper you will find a lot of data on the different mEoS variants (not only 2, 3.1 and 3.2 but others as well).

The lesson to be learned here – the properties of the fluorescent protein that you are using to tag your protein of interest may affect the properties of the studied fusion protein/organelle/cell and these factors should always be taken into consideration.

Tracking mitochondria dynamics in live HeLa cells. The large box: Mitochondria in HeLa cells tagged with mEoS3.2-mito prior to photoswitching. Rectangle – area of UV illumination to switch color. boxes on left: time-lapse of mEoS3.2-mito only in the activated region. Source: Zhang et al. Nature Methods 9,727–729(2012)

Multiview light-sheet microscope for rapid in toto imaging

Embryogenesis and morphogenesis are highly dynamic processes that are difficult to image since it involves multicellular samples in the millimeter range. In such cases, it is difficult to image subcellular processes on the one hand, and get a clear 3-D view of the entire sample (which need to be properly rotated). Some techniques that allow sample rotation exist, and an emerging method called selective plane illumination microscopy (SPIM), are helpful in following such processes on whole embryos. However, samples are required to rotate in several angles, often not keeping with the same axis, and the time resolution required for each rotation sometimes exceeds the biological dynamics. Here, research from the lab of Lars Hufnagel developed a new microscopy system, which they term MuVi-SPIM, consisting of four arms (with objectives) that can perform as illuminating or detecting objective. This allows rapid four 3D-view imaging of the sample. Very nifty!

3D image reconstruction of a Drosophila embryo expressing the membrane marker Gap43-mCherry in cycle 14. Alternating green and magenta colors correspond to the image contributions from the eight different views. Inset shows a close-up view of the image fusion on the boundary between two different views. Source: Krzic et al. Nature Methods 9,730–733(2012)

The next parts will review the four articles in this issue.

ResearchBlogging.orgParthasarathy R (2012). Rapid, accurate particle tracking by calculation of radial symmetry centers. Nature methods PMID: 22688415
Zhang M, Chang H, Zhang Y, Yu J, Wu L, Ji W, Chen J, Liu B, Lu J, Liu Y, Zhang J, Xu P, & Xu T (2012). Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nature methods, 9 (7), 727-729 PMID: 22581370
Krzic U, Gunther S, Saunders TE, Streichan SJ, & Hufnagel L (2012). Multiview light-sheet microscope for rapid in toto imaging. Nature methods, 9 (7), 730-733 PMID: 22660739