Laurent A. Bentolila

Particle Size, Surface Coating, and PEGylation Influence the Biodistribution of Quantum Dots in Living Mice

This study evaluates the influence of particle size, PEGylation, and surface coating on the quantitative biodistribution of near-infrared-emitting quantum dots (QDs) in mice. Polymer- or peptide-coated 64Cu-labeled QDs 2 or 12 nm in diameter, with or without polyethylene glycol (PEG) of molecular weight 2000, are studied by serial micropositron emission tomography imaging and region-of-interest analysis, as well as transmission electron microscopy and inductively coupled plasma mass spectrometry. PEGylation and peptide coating slow QD uptake into the organs of the reticuloendothelial system (RES), liver and spleen, by a factor of 6-9 and 2-3, respectively. Small particles are in part renally excreted. Peptide-coated particles are cleared from liver faster than physical decay alone would suggest. Renal excretion of small QDs and slowing of RES clearance by PEGylation or peptide surface coating are encouraging steps toward the use of modified QDs for imaging living subjects.

Fluorescent Imaging of Single Nanoparticles and Viruses on a Smart Phone

Optical imaging of nanoscale objects, whether it is based on scattering or fluorescence, is a challenging task due to reduced detection signal-to-noise ratio and contrast at subwavelength dimensions. Here, we report a field-portable fluorescence microscopy platform installed on a smart phone for imaging of individual nanoparticles as well as viruses using a lightweight and compact opto-mechanical attachment to the existing camera module of the cell phone. This hand-held fluorescent imaging device utilizes (i) a compact 450 nm laser diode that creates oblique excitation on the sample plane with an incidence angle of ~75°, (ii) a long-pass thin-film interference filter to reject the scattered excitation light, (iii) an external lens creating 2× optical magnification, and (iv) a translation stage for focus adjustment. We tested the imaging performance of this smart-phone-enabled microscopy platform by detecting isolated 100 nm fluorescent particles as well as individual human cytomegaloviruses that are fluorescently labeled. The size of each detected nano-object on the cell phone platform was validated using scanning electron microscopy images of the same samples. This field-portable fluorescence microscopy attachment to the cell phone, weighing only ~186 g, could be used for specific and sensitive imaging of subwavelength objects including various bacteria and viruses and, therefore, could provide a valuable platform for the practice of nanotechnology in field settings and for conducting viral load measurements and other biomedical tests even in remote and resource-limited environments.

microPET-Based Biodistribution of Quantum Dots in Living Mice

This study evaluates the quantitative biodistribution of commercially available CdSe quantum dots (QD) in mice. Methods: 64Cu-Labeled 800- or 525-nm emission wavelength QD (21- or 12-nm diameter), with or without 2,000 MW (molecular weight) polyethylene glycol (PEG), were injected intravenously into mice (5.55 MBq/25 pmol QD) and studied using well counting or by serial microPET and region-of-interest analysis. Results: Both methods show rapid uptake by the liver (27.4–38.9 %ID/g) (%ID/g is percentage injected dose per gram tissue) and spleen (8.0–12.4 %ID/g). Size has no influence on biodistribution within the range tested here. Pegylated QD have slightly slower uptake into liver and spleen (6 vs. 2 min) and show additional low-level bone uptake (6.5–6.9 %ID/g). No evidence of clearance from these organs was observed. Conclusion: Rapid reticuloendothelial system clearance of QD will require modification of QD for optimal utility in imaging living subjects. Formal quantitative biodistribution/imaging studies will be helpful in studying many types of nanoparticles, including quantum dots.

Quantum Dots for In Vivo Small-Animal Imaging

Nanotechnology is poised to transform research, prevention, and treatment of cancer through the development of novel diagnostic imaging methods and targeted therapies. In particular, the use of nanoparticles for imaging has gained considerable momentum in recent years. This review focuses on the growing contribution of quantum dots (QDs) for in vivo imaging in small-animal models. Fluorescent QDs, which are small nanocrystals (1–10 nm) made of inorganic semiconductor materials, possess several unique optical properties best suited for in vivo imaging. Because of quantum confinement effects, the emission color of QDs can be precisely tuned by size from the ultraviolet to the near-infrared. QDs are extremely bright and photostable. They are also characterized by a wide absorption band and a narrow emission band, which makes them ideal for multiplexing. Finally, the large surface area of QDs permits the assembly of various contrast agents to design multimodality imaging probes. To date, biocompatible QD conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Here we consider these novel breakthroughs in light of their potential clinical applications and discuss how QDs might offer a suitable platform to unite disparate imaging modalities and provide information along a continuum of length scales.

WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer

Wnt/β‐catenin signalling has been suggested to be active in basal‐like breast cancer. However, in highly aggressive metastatic triple‐negative breast cancers (TNBC) the role of β‐catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active β‐catenin in human TNBC and predicts survival‐outcome of patients with both TNBC and basal‐like tumours. We provide evidence that transgenic murine Wnt10b‐driven tumours are devoid of ERα, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical β‐catenin signalling leading to up‐regulation of HMGA2. Treatment of mouse and human triple‐negative tumour cells with two Wnt/β‐catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse‐free‐survival and metastasis in TNBC patients.

Correlative nanomechanical profiling with super-resolution F-actin imaging reveals novel insights into mechanisms of cisplatin resistance in ovarian cancer cells

UNLABELLED The exact molecular mechanisms of ovarian cancer platinum resistance are not well understood, and biomarkers to reliably predict ovarian cancer resistance to platinum and other chemotherapeutic agents are lacking. Biomechanics of cisplatin-treated ovarian cancer cells were measured quantitatively at nanoscale level using atomic force microscopy. We demonstrate that cisplatin modulates the cellular nanomechanics of ovarian cancer cells; sensitive cells show dose-dependent increase in cell stiffness, which is effected by disrupting the F-actin polymerization. In contrast, resistant cells show no significant changes in cell stiffness upon cisplatin treatment. Further, stimulated emission depletion, an emerging super-resolution microscopy, shows that at the molecular level, F-actin is indeed remodeled considerably in cisplatin-sensitive and cisplatin-resistant cells. These findings reveal a direct role of the actin remodeling mechanism in cisplatin resistance of ovarian cancer cells, suggesting potential future applications of nanomechanical profiling as a marker for cancer drug sensitivity. FROM THE CLINICAL EDITOR In this paper, nanomechanical profiling and an emerging super-resolution microscopy method was utilized to decipher the mechanisms of cisplatin resistance in ovarian cancer cells, paving the way to future studies of this and similar other problems with drug resistance in cancer biology.

Direct Synthesis of Lamin A, Bypassing Prelamin A Processing, Causes Misshapen Nuclei in Fibroblasts but No Detectable Pathology in Mice

Lamin A, a key component of the nuclear lamina, is generated from prelamin A by four post-translational processing steps: farnesylation, endoproteolytic release of the last three amino acids of the protein, methylation of the C-terminal farnesylcysteine, and finally, endoproteolytic release of the last 15 amino acids of the protein (including the farnesylcysteine methyl ester). The last cleavage step, mediated by ZMPSTE24, releases mature lamin A. This processing scheme has been conserved through vertebrate evolution and is widely assumed to be crucial for targeting lamin A to the nuclear envelope. However, its physiologic importance has never been tested. To address this issue, we created mice with a “mature lamin A-only” allele (LmnaLAO), which contains a stop codon immediately after the last codon of mature lamin A. Thus, LmnaLAO/LAO mice synthesize mature lamin A directly, bypassing prelamin A synthesis and processing. The levels of mature lamin A in LmnaLAO/LAO mice were indistinguishable from those in “prelamin A-only” mice (LmnaPLAO/PLAO), where all of the lamin A is produced from prelamin A. LmnaLAO/LAO exhibited normal body weights and had no detectable disease phenotypes. A higher frequency of nuclear blebs was observed in LmnaLAO/LAO embryonic fibroblasts; however, the mature lamin A in the tissues of LmnaLAO/LAO mice was positioned normally at the nuclear rim. We conclude that prelamin A processing is dispensable in mice and that direct synthesis of mature lamin A has little if any effect on the targeting of lamin A to the nuclear rim in mouse tissues.

Single-step multicolor fluorescence in situ hybridization using semiconductor quantum dot-DNA conjugates.

We report a rapid method for the direct multicolor imaging of multiple subnuclear genetic sequences using novel quantum dot-based fluorescence in situ hybridization (FISH) probes (QD-FISH). Short DNA oligonucleotides were attached on QDs and used in a single hybridization/detection step of target sites in situ. QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and showed good targeting of dense chromatin domains with minimal steric hindrances. We further demonstrated that QD’s broad absorption spectra allowed different colored probes specific for distinct subnuclear genetic sequences to be simultaneously excited with a single excition wavelength and imaged free of chromatic aberrations in a single exposure. Thus, these results demonstrate that QD-FISH probes are very effective in multicolor FISH applications. This work also documents new possibilities of using QD-FISH probes detection down to the single molecule level.

THE TWO ISOFORMS OF MOUSE TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE DIFFER IN BOTH THE ABILITY TO ADD N REGIONS AND SUBCELLULAR LOCALIZATION

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy‐terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site‐specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3′ end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half‐life of the TdTL protein in these cells is 2‐fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.

Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

ABSTRACT The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii. IMPORTANCE The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma. The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.