Category Archives: Immunofluorescence

Quantum dots for Immunofluorescence — Rapha-z-lab

An important post on quantum dots:

Guest post by Dave Mason In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in […]

via Quantum dots for Immunofluorescence — Rapha-z-lab

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).

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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

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.

Imaging gene expression – methods & protocols

A new book in the “Methods in molecular biology” series, recently published, contains 23 imaging protocols in three major research areas: gene expression & RNA dynamics, genome & chromatin dynamics, and nuclear process & structures.

book cover: Imaging Gene Expression

This is a fairly good overview of the field and can help both beginners and researchers looking for new ideas.

The book can be freely downloaded.

Here’s the contents of the book:

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High res imaging of DNA double strand breaks: Clearing the nucleus or marking the spot

DNA can be damaged in many ways. Consequently, there are numerous mechanisms to repair it. It is a fascinating field full of innovative concepts (“DNA repair” was my favorite course during my undergrad studies).  Double strand breaks (DSBs) are considered the most genotoxic, which is why many DNA damaging drugs and treatments intended to treat cancer are intended to create DSBs. On the other hand, DSBs can lead to chromosome translocations which can promote cancer, and can actually be viewed as a hallmark of cancer cells. DSBs also occur naturally during recombination events at Meiosis, and are important intermediates in immune system development.

DSBs are recognized by repair complexes that act to mark and repair the damage. Though the sequence of event has been studied biochemically, knowledge on the in vivo temporal and spatial arrangement has been limited due to lack of good high-res methods to visualize DSBs and repair proteins at DBS sites.

Two papers recently published take two different approaches to get  high-resolution images of events at DSBs.

The first paper, from Stephen Jackson’s lab, implemented a simple method to reduce the background fluorescence in the nucleus, thus increasing the signal/noise ratio. They study the non-homologous end joining (NHEJ) process which initiates with the Ku complex recognizing the DSB, followed by recruitment of the DNA dependent protein kinase (DNA-PK) and ending with ligation by XRCC4. DNA-PK phosphorylates the histone H2AX near DSB (gamma-H2AX). This is a known marker for DSB.

They used laser illumination to create a streak of DSB, and immunofluorescence (IF) against gamma-H2AX and Ku. They used a buffer called CSK that is known to release soluble proteins, thus removing background. Though the gamma-H2AX appears as a clear streak, Ku is all over the nucleus. Ku is also an RNA binding protein. So they added an RNase to the CSK wash. Miraculously, only DNA-bound Ku was now visible.

Look at the image – it looks amazing!

Hi-res image of Ku at DSB

Addition of RNase to the CSK buffer during IF allows for high-resolution imaging of DNA repair proteins. Source: Britton et al (2013) JCB vol 202(3):579-595.

Using this simple upgrade to the protocol, which removed most of the background, and combined with 3D structured illumination (3D-SIM) super resolution microscopy*, they were able to study co-localization (here’s a video), measure distances and study composition of different components of the repair complex. They also used Ku foci count to asses the effect of different drugs on DSBs repair efficiency.

I found this paper important mostly because  this simple improvement in their protocol yielded a vast improvement in imaging. This technique could be useful for studying many other nuclear processes which involve proteins that associate with both DNA and RNA (e.g. transcription complexes).

The second paper, from Tom Misteli’s lab, took a very different approach. They wanted to see sites of chromosomal translocations  following DSBs. First, instead of getting random, or multiple DSBs using drugs, radiation or laser (as in Jackson’s paper), they introduced restriction sites for the rare-cutting IsceI restriction enzyme. One site, on chromosome 7, was surrounded by multiple repeats (256) of the LacO sequence. Other sites, on chromosomes 1 & 10, were surrounded by 96 TetO repeats. The LacO and TetO arrays can be viewed by fusion of GFP to the Lac repressor (which binds LacO) and mCherry to the Tet repressor which binds TetO. Thus, each potential DSB site is marked by a fluorescent array in distinct colors. Expression of ISceI enzyme from a plasmid induces DSB. They then followed the temporal position of the green and red spots, until they get co-localization of the colors – meaning chromosomal translocation has occurred.

Time-Lapse microscopy of cells after transfection with ISceI enzyme, showing the translocation of two distinct sites (marked by GFP and mCherry) into a co-localized spot. Source: Roukus et al. (2013) Science vol 341:660-664.

Time-Lapse microscopy of cells after transfection with ISceI enzyme, showing the translocation of two distinct sites (marked by GFP and mCherry) into a co-localized spot. Source: Roukus et al. (2013) Science vol 341:660-664.

Like in Jackson’s paper, they used this system to look at temporal and spatial events, including recruitment of proteins (tagged with a third color: BFP), and to study the effect of different mechanisms that perturb the DSB repair machinery.

In conclusion: both approaches yielded beautiful and informative images about DSBs behavior and repair. Jackson’s method has a more global and immediate application, since it can be used to study other nuclear processes. Misteli’s system requires a lot of “genetic engineering” but can provide a more precise temporal resolution (since it enables live-cell imaging, unlike Jackson’s modified IF which only uses fixed cells). Misteli’s method can also provide insight into specificity (studying DSBs at specific genomic locations). These arrays can also be used to study chromosome dynamics (i.e. movements within the nucleus, during cell division, or transport of specific genomic regions during transcription induction e.g. to the nuclear pore complex). I am actually using such an array myself, to mark the location of my gene of interest.

* 3D-SIM uses laser light that passes through an optical grating. A series of images created by these striped patterns, generated at high spatial frequency, can then be processed by a computer algorithm into a high resolution imaging. See ref. below for more details.

ResearchBlogging.orgBritton S, Coates J, & Jackson SP (2013). A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair. The Journal of cell biology, 202 (3), 579-95 PMID: 23897892
Roukos V, Voss TC, Schmidt CK, Lee S, Wangsa D, & Misteli T (2013). Spatial dynamics of chromosome translocations in living cells. Science (New York, N.Y.), 341 (6146), 660-4 PMID: 23929981
Schermelleh L, Heintzmann R, & Leonhardt H (2010). A guide to super-resolution fluorescence microscopy. The Journal of cell biology, 190 (2), 165-75 PMID: 20643879

Nuclear pore complex at super resolution

It is very difficult to decipher the spatial arrangement of proteins in a given complex. The most common methods are X-ray crystallography (which is very difficult to achieve for single proteins, let alone complexes), cryo-electron microscopy  and electron tomography (ditto) or  biochemical approaches (e.g. which protein domains interact with each other).  A paper published in today’s Science, shows a different approach, using super-resolution microscopy.

The Nuclear Pore Complex (NPC) is a large complex that enables movement of large particles in and out of the nucleus. Though electron tomography previously showed the general structure of NPC, the spatial order of individual proteins was unclear. Here, the researchers used stochastic optical reconstruction microscopy (STORM), a super resolution microscopy method, of either antibody or nanobody labeled samples. The idea of STORM is to get to the point where fluorophores begin to photobleach, and then take a thousands of images very fast (here they used 100 frames/second). the idea is that only a few fluorophores will lit up in each image, thus reducing background.

NPC viewed by regular confocal microscopy (A) or STORM (B). Scale bars indicate 3 μm and 300 nm, respectively. (C)- images after quality control. (D) - average "ring". (E) Normalized mean radius. (F) Precision of determining the radial position was estimated by cross-validation, performed by averaging 17 sets of 500 pores each (red line marks the median). The standard deviation of the distribution is 0.1 nm. The whiskers on the box plot encompass 99.3% of the distribution.  Source: Szymborska et al. (2013) Science: Vol. 341  no. 6146  pp. 655-658

NPCs viewed by regular confocal microscopy (A) or STORM (B). Scale bars indicate 3 μm and 300 nm, respectively. (C)- images after quality control. (D) – average “ring”. (E) Normalized mean radius. (F) Precision of determining the radial position was estimated by cross-validation, performed by averaging 17 sets of 500 pores each (red line marks the median). The standard deviation of the distribution is 0.1 nm. The whiskers on the box plot encompass 99.3% of the distribution. Source: Szymborska et al. (2013) Science: Vol. 341 no. 6146 pp. 655-658

Furthermore, for each protein, they were able to average thousands of NPCs from the same cell, thus improving statistical analysis compared to cryo-EM (which usually compares only a few good images). They claim to had a standard deviation of ~0.1nm(!).

Since the NPC is ring shaped, they calculated the radius of the ring for each protein they tested. Thus they were able to spatially align the proteins based on their distance from the center of the ring. systematic labeling of different components allowed them to create a model of the NPC scaffold structure.

The potential for their method is big, particularly for structures that are abundant in cells (ribosomes, transcription pre-initiation complex, gated channels, photosynthetic centers – choose your favorite complex) to allow for good statistical analysis.

Though in this paper they used fixed cells, it would be most interesting to implement similar methods to live cells, and view structural changes in real time.

ResearchBlogging.orgSzymborska A, de Marco A, Daigle N, Cordes VC, Briggs JA, & Ellenberg J (2013). Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging. Science (New York, N.Y.), 341 (6146), 655-658 PMID: 23845946

Ries J, Kaplan C, Platonova E, Eghlidi H, & Ewers H (2012). A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nature methods, 9 (6), 582-4 PMID: 22543348

Schermelleh L, Heintzmann R, & Leonhardt H (2010). A guide to super-resolution fluorescence microscopy. The Journal of cell biology, 190 (2), 165-75 PMID: 20643879