Brief Communications in 2009

A modular, automatable system using recombineering to facilitate multigene assembly, called Acembl, provides a streamlined approach for expressing multiprotein complexes in Escherichia coli.

The use of a spatial light modulator for illuminating the sample in structured-illumination microscopy (SIM) increases imaging speed by three orders of magnitude. The resulting 100-nm resolution and 11-Hz frame rate allowed video imaging of tubulin polymerization and depolymerization as well as kinesin movement on microtubules.

A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts.

The combination of 5′ exonuclease, DNA polymerase and ligase with overlapping DNA fragments facilitates the in vitro assembly of large DNA constructs, including an entire bacterial genome.

Several red and orange fluorescent proteins are reported to be photoconvertible. Specifically, three red fluorescent proteins that can be switched to green, and two orange fluorescent proteins that can be switched to far red are reported.

Upon binding multiple fluorophores and being complexed into tetramers, these RNA imaging probes show high sensitivity and can detect single endogenous RNA molecules at low probe concentration.

A fluorescent protein, Sirius, with the most blue-shifted emission spectrum to date, is reported. Sirius allows extended multicolor imaging as well as imaging in acidic environments owing to its pH insensitivity.

As an alternative to the use of radioactively labeled amino acids, incorporation of puromycin into proteins allows evaluation of translation in heterogenous cell populations by flow cytometry analysis after staining with an antibody to puromycin.

Long-term engraftment of hematopoietic stem cells into the bone marrow of a recipient depends on immunological compatibility between donor and host, or ablation of the host's immune system by irradiation. A 'universal recipient' mouse model now shows that mice that lack T, B and NK cells and bear mutations in the tyrosine kinase Kit accept any donor HSC without irradiation.

Rats are desensitized to xenografts of human neural or embryonic stem cell–derived cells by exposure to the xenogeneic cells during the neonatal period. Brain grafts survive in immunocompetent rats without chronic immunosuppression, allowing long-term studies.

The base-calling algorithm SNPSeeker detects single-nucleotide polymorphisms with frequencies that are below the error rate of the sequencing platform. It is thus well suited to analyze data from large pooled samples and find rare variants that may contribute to diseases or complex traits.

Complex three-dimensional structures on cellular surfaces are often damaged during high-resolution imaging of live cells. Now, hopping probe scanning ion conductance microscopy—which uses a hopping nanopipette that 'hops' instead of 'sliding'—protects surface structures from probe-induced damage.

Sialic acid–containing cell-surface glycoproteins can be chemically labeled with a biotin tag under mild conditions. The method is highly efficient and uses commercially available reagents; it should be useful for studying glycoprotein trafficking as well as in glycoproteomics applications.

A map of single nucleotide polymorphisms (SNPs), also called a haplotype map, is very informative for mapping complex trait loci, but obtaining haplotypes over long genomic distances is very challenging. The combination of dye-labeling each SNP on PCR fragments with total internal reflection microscopy will allow the reading of long-range haplotypes with relative ease.

Solid-state NMR spectroscopy is used to elucidate structural details about proteins that cannot be easily studied by X-ray crystallography, but because the technique is not very sensitive, large sample amounts are required, limiting its biological application. A combination of optimizations now increases the sensitivity of solid-state NMR spectroscopy by up to 5-fold.

Absolute quantitative information about the stoichiometry of protein complex components can be obtained with a modified affinity purification–mass spectrometry method, as demonstrated for the human protein phosphatase 2A network.

As cells move over a substrate, they need to first sever their contact with the matrix by detaching focal adhesions. A setup that allows spatially and temporally controlled release of focal adhesions now facilitates the quantitative measurement of cell movement across a substrate.

An improved version of the green-to-red photoconverting EosPF is presented. mEos2 is a functional fusion partner for many cellular proteins and retains the high localization precision of EosFP in super-resolution imaging. Also in this issue, Subach et al. present an inducible mCherry variant for super-resolution imaging.

A combination of multiphoton laser scanning microscopy and second harmonic generation (SHG) imaging is used to track tumor associated fibroblasts and extracellular matrix remodeling in living mice. The SHG signal is used for image registration over several days.

Conventional in situ hybridization protocols lead to loss of microRNAs, which diffuse out of the formaldehyde-fixed sample owing to their small size. Adding a carbodiimide that stably links the microRNA with the protein matrix around it prevents this diffusion and allows detection of miRNAs at very low expression levels.