Multipotent luminal mammary cancer stem cells model tumor heterogeneity.

INTRODUCTION: The diversity of human breast cancer subtypes has led to the hypothesis that breast cancer is actually a number of different diseases arising from cells at various stages of differentiation. The elusive nature of the cell(s) of origin thus hampers approaches to eradicate the disease. METHODS: Clonal cell lines were isolated from primary transgenic polyomavirus middle T (PyVmT) luminal tumors. Mammary cancer stem cell (MaCSC) properties were examined by immunofluorescence, flow cytometry, differentiation assays and in vivo tumorigenesis. RESULTS: Clonal cell lines isolated from primary PyVmT mouse mammary luminal tumors can differentiate into luminal, myoepithelial, alveolar and adipocyte lineages. Upon orthotopic injection, progeny of a single cell follow a pattern of progression from ductal carcinoma in situ, to adenoma, adenocarcinoma and epithelial metastasis that recapitulates the transgenic model. Tumors can evolve in vivo from hormone receptor-positive to hormone receptor-negative Her2-positive, or triple negative CD44hi basal-like and claudin-low tumors. Contrary to the current paradigm, we have defined a model in which multiple tumor subtypes can originate from a single multipotent cancer stem cell that undergoes genetic and/or epigenetic evolution during tumor progression. As in human tumors, the more aggressive tumor subtypes express nuclear p53. Tumor cell lines can also be derived from these more advanced tumor subtypes. CONCLUSIONS: Since the majority of human tumors are of the luminal subtype, understanding the cell of origin of these tumors and how they relate to other tumor subtypes will impact cancer therapy. Analysis of clonal cell lines derived from different tumor subtypes suggests a developmental hierarchy of MaCSCs, which may provide insights into the progression of human breast cancer.

Optically monitoring voltage in neurons by photo-induced electron transfer through molecular wires.

Fluorescence imaging is an attractive method for monitoring neuronal activity. A key challenge for optically monitoring voltage is development of sensors that can give large and fast responses to changes in transmembrane potential. We now present fluorescent sensors that detect voltage changes in neurons by modulation of photo-induced electron transfer (PeT) from an electron donor through a synthetic molecular wire to a fluorophore. These dyes give bigger responses to voltage than electrochromic dyes, yet have much faster kinetics and much less added capacitance than existing sensors based on hydrophobic anions or voltage-sensitive ion channels. These features enable single-trial detection of synaptic and action potentials in cultured hippocampal neurons and intact leech ganglia. Voltage-dependent PeT should be amenable to much further optimization, but the existing probes are already valuable indicators of neuronal activity.

Ratiometric activatable cell-penetrating peptides provide rapid in vivo readout of thrombin activation.

In real time: thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.

Improving the photostability of bright monomeric orange and red fluorescent proteins.

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

Structure-function analysis of the maize bulliform cell cuticle and its potential role in dehydration and leaf rolling.

The hydrophobic cuticle of plant shoots serves as an important interaction interface with the environment. It consists of the lipid polymer cutin, embedded with and covered by waxes, and provides protection against stresses including desiccation, UV radiation, and pathogen attack. Bulliform cells form in longitudinal strips on the adaxial leaf surface, and have been implicated in the leaf rolling response observed in drought-stressed grass leaves. In this study, we show that bulliform cells of the adult maize leaf epidermis have a specialized cuticle, and we investigate its function along with that of bulliform cells themselves. Bulliform cells displayed increased shrinkage compared to other epidermal cell types during dehydration of the leaf, providing a potential mechanism to facilitate leaf rolling. Analysis of natural variation was used to relate bulliform strip patterning to leaf rolling rate, providing further evidence of a role for bulliform cells in leaf rolling. Bulliform cell cuticles showed a distinct ultrastructure with increased cuticle thickness compared to other leaf epidermal cells. Comparisons of cuticular conductance between adaxial and abaxial leaf surfaces, and between bulliform-enriched mutants versus wild-type siblings, showed a correlation between elevated water loss rates and presence or increased density of bulliform cells, suggesting that bulliform cuticles are more water-permeable. Biochemical analysis revealed altered cutin composition and increased cutin monomer content in bulliform-enriched tissues. In particular, our findings suggest that an increase in 9,10-epoxy-18-hydroxyoctadecanoic acid content, and a lower proportion of ferulate, are characteristics of bulliform cuticles. We hypothesize that elevated water permeability of the bulliform cell cuticle contributes to the differential shrinkage of these cells during leaf dehydration, thereby facilitating the function of bulliform cells in stress-induced leaf rolling observed in grasses.

Characterization of engineered channelrhodopsin variants with improved properties and kinetics.

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

OBJECTIVE: Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. METHODS: K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPPNleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPPNleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. RESULTS: In chronic arthritis, RACPPNleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPPNleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPPNleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. CONCLUSION: RACPPNleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPPNleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression.

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species.

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane.

Fluorescently labeled peptide increases identification of degenerated facial nerve branches during surgery and improves functional outcome.

Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL), complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41) can improve intraoperative visualization of chronically (up to 9 months) denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair.

Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides.

Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathologic examination. We report a family of novel ratiometric activatable cell-penetrating peptides, which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 re-emission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 and 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40-fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information about cancer invasion.

Fluorescent peptides highlight peripheral nerves during surgery in mice.

Nerve preservation is an important goal during surgery because accidental transection or injury leads to significant morbidity, including numbness, pain, weakness or paralysis. Nerves are usually identified by their appearance and relationship to nearby structures or detected by local electrical stimulation (electromyography), but thin or buried nerves are sometimes overlooked. Here, we use phage display to select a peptide that binds preferentially to nerves. After systemic injection of a fluorescently labeled version of the peptide in mice, all peripheral nerves are clearly delineated within 2 h. Contrast between nerve and adjacent tissue is up to tenfold, and useful contrast lasts up to 8 h. No changes in behavior or activity are observed after treatment, indicating a lack of obvious toxicity. The fluorescent probe also labels nerves in human tissue samples. Fluorescence highlighting is independent of axonal integrity, suggesting that the probe could facilitate surgical repair of injured nerves and help prevent accidental transection.

Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome.

Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M(-1) cm(-1); and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.

A guide to choosing fluorescent proteins.

The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.

Evolution of new nonantibody proteins via iterative somatic hypermutation.

B lymphocytes use somatic hypermutation (SHM) to optimize immunoglobulins. Although SHM can rescue single point mutations deliberately introduced into nonimmunoglobulin genes, such experiments do not show whether SHM can efficiently evolve challenging novel phenotypes requiring multiple unforeseeable mutations in nonantibody proteins. We have now iterated SHM over 23 rounds of fluorescence-activated cell sorting to create monomeric red fluorescent proteins with increased photostability and far-red emissions (e.g., 649 nm), surpassing the best efforts of structure-based design. SHM offers a strategy to evolve nonantibody proteins with desirable properties for which a high-throughput selection or viable single-cell screen can be devised.

A monomeric red fluorescent protein.

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.