James McColl

Quantitative single-molecule microscopy reveals that CENP-A(Cnp1) deposition occurs during G2 in fission yeast

The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and ‘counts’ individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-ACnp1 with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-ACnp1 levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-ACnp1 is deposited solely during the G2 phase of the cell cycle.

DySCo: Quantitating Associations of Membrane Proteins Using Two-Color Single-Molecule Tracking

Abstract We present a general method called dynamic single-molecule colocalization for quantitating the associations of single cell surface molecules labeled with distinct autofluorescent proteins. The chief advantages of the new quantitative approach are that, in addition to stable interactions, it is capable of measuring nonconstitutive associations, such as those induced by the cytoskeleton, and it is applicable to situations where the number of molecules is small.

Single molecule characterization of the interactions between amyloid-β peptides and the membranes of hippocampal cells

Oligomers of the 40 and 42 residue amyloid-β peptides (Aβ40 and Aβ42) have been implicated in the neuronal damage and impaired cognitive function associated with Alzheimer’s disease. However, little is known about the specific mechanisms by which these misfolded species induce such detrimental effects on cells. In this work, we use single-molecule imaging techniques to examine the initial interactions between Aβ monomers and oligomers and the membranes of live cells. This highly sensitive method enables the visualization of individual Aβ species on the cell surface and characterization of their oligomerization state, all at biologically relevant, nanomolar concentrations. The results indicate that oligomers preferentially interact with cell membranes, relative to monomers and that the oligomers become immobilized on the cell surface. Additionally, we observe that the interaction of Aβ species with the cell membrane is inhibited by the presence of ATP-independent molecular chaperones. This study demonstrates the power of this methodology for characterizing the interactions between protein aggregates and the membranes of live neuronal cells at physiologically relevant concentrations and opens the door to quantitative studies of the cellular responses to potentially pathogenic oligomers.

The T Cell Receptor Triggering Apparatus Is Composed of Monovalent or Monomeric Proteins

Understanding the component stoichiometry of the T cell antigen receptor (TCR) triggering apparatus is essential for building realistic models of signal initiation. Recent studies suggesting that the TCR and other signaling-associated proteins are preclustered on resting T cells relied on measurements of the behavior of membrane proteins at interfaces with functionalized glass surfaces. Using fluorescence recovery after photobleaching, we show that, compared with the apical surface, the mobility of TCRs is significantly reduced at Jurkat T cell/glass interfaces, in a signaling-sensitive manner. Using two biophysical approaches that mitigate these effects, bioluminescence resonance energy transfer and two-color coincidence detection microscopy, we show that, within the uncertainty of the methods, the membrane components of the TCR triggering apparatus, i.e. the TCR complex, MHC molecules, CD4/Lck and CD45, are exclusively monovalent or monomeric in human T cell lines, implying that TCR triggering depends only on the kinetics of TCR/pMHC interactions. These analyses also showed that constraining proteins to two dimensions at the cell surface greatly enhances random interactions versus those between the membrane and the cytoplasm. Simulations of TCR-pMHC complex formation based on these findings suggest how unclustered TCR triggering-associated proteins might nevertheless be capable of generating complex signaling outputs via the differential recruitment of cytosolic effectors to the cell membrane.

Optical monitoring of stem cell-substratum interactions

Modulation of the coupling of light into a waveguide via a grating, together with a novel approach to analyzing the data, is used to investigate the attachment of human embryonal carcinoma stem cells to three substrata: silica-titania (representative of artificial implants); poly-lysine (a commonly used laboratory cell culture substrate); and mucin (the coating of the mucosae). By considering both in-coupling peak width and position, the secretion of microexudate by the cells, the formation of filopodia, and the overall change in their shape (spreading) can be distinguished. This cannot be achieved by the conventional microscopic imaging approach. Moreover, we obtain the kinetics of these processes with excellent time resolution.

Smad1 transcription factor integrates BMP2 and Wnt3a signals in migrating cardiac progenitor cells

Significance Prospective cardiac cells emerge during gastrulation and undergo long-range migration toward the ventral midline, where they fuse to give rise to a single contractile tube, which subsequently undergoes complex morphogenesis. How cardiac progenitor cells are guided in their movement by extrinsic signals is still enigmatic. We previously identified wingless-type family member (Wnt) 3a as an important guidance signal. Here we used live video microscopy in chick embryos to uncover a role for bone morphogenetic proteins (BMPs) in the control of cardiac progenitor cell migration. Functional approaches, complementation, and rescue experiments reveal cooperation between BMP signalling and the Wnt/glycogen synthase kinase 3 beta pathway: both converge to stabilize activated SMA and MAD related protein. Insights into the molecular integration of signaling pathways in migrating cells affect our understanding of cardiac malformations during embryo development. In vertebrate embryos, cardiac progenitor cells (CPCs) undergo long-range migration after emerging from the primitive streak during gastrulation. Together with other mesoderm progenitors, they migrate laterally and then toward the ventral midline, where they form the heart. Signals controlling the migration of different progenitor cell populations during gastrulation are poorly understood. Several pathways are involved in the epithelial-to-mesenchymal transition and ingression of mesoderm cells through the primitive streak, including fibroblast growth factors and wingless-type family members (Wnt). Here we focus on early CPC migration and use live video microscopy in chicken embryos to demonstrate a role for bone morphogenetic protein (BMP)/SMA and MAD related (Smad) signaling. We identify an interaction of BMP and Wnt/glycogen synthase kinase 3 beta (GSK3β) pathways via the differential phosphorylation of Smad1. Increased BMP2 activity altered migration trajectories of prospective cardiac cells and resulted in their lateral displacement and ectopic differentiation, as they failed to reach the ventral midline. Constitutively active BMP receptors or constitutively active Smad1 mimicked this phenotype, suggesting a cell autonomous response. Expression of GSK3β, which promotes the turnover of active Smad1, rescued the BMP-induced migration phenotype. Conversely, expression of GSK3β-resistant Smad1 resulted in aberrant CPC migration trajectories. De-repression of GSK3β by dominant negative Wnt3a restored normal migration patterns in the presence of high BMP activity. The data indicate the convergence of BMP and Wnt pathways on Smad1 during the early migration of prospective cardiac cells. Overall, we reveal molecular mechanisms that contribute to the emerging paradigm of signaling pathway integration in embryo development.

A Quantitative Comparison of Single-Dye Tracking Analysis Tools Using Monte Carlo Simulations

Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles’ displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.

Single-Molecule Imaging Reveals that Small Amyloid-β1–42 Oligomers Interact with the Cellular Prion Protein (PrPC)

Oligomers of the amyloid‐β peptide (Aβ) play a central role in the pathogenesis of Alzheimer’s disease and have been suggested to induce neurotoxicity by binding to a plethora of cell‐surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single‐molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrPC) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrPC and that the species bound to PrPC are predominantly small oligomers (dimers and trimers). Single‐molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor‐mediated oAβ‐induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.

Hydrodynamic trapping of molecules in lipid bilayers

In this work we show how hydrodynamic forces can be used to locally trap molecules in a supported lipid bilayer (SLB). The method uses the hydrodynamic drag forces arising from a flow through a conical pipette with a tip radius of 1–1.5 μm, placed approximately 1 μm above the investigated SLB. This results in a localized forcefield that acts on molecules protruding from the SLB, yielding a hydrodynamic trap with a size approximately given by the size of the pipette tip. We demonstrate this concept by trapping the protein streptavidin, bound to biotin receptors in the SLB. It is also shown how static and kinetic information about the intermolecular interactions in the lipid bilayer can be obtained by relating how the magnitude of the hydrodynamic forces affects the accumulation of protein molecules in the trap.

Structural hysteresis and hierarchy in adsorbed glycoproteins.

The adsorption and desorption of the giant heavily glycosylated protein mucin from solutions of different bulk concentrations have been followed at the nanometer scale using high resolution molecular microscopy based on optical waveguide lightmode spectroscopy. Modeling the layer as a uniaxial thin film allowed the in situ determination of adsorbed mass, mean layer thickness, and structural anisotropy. These parameters manifest highly significant adsorption-desorption hysteresis, indicating at least two dominant glycoprotein conformational types (i.e., molecular states, structurally and kinetically distinguishable). One of them is proposed to be a conformationally extended state that engenders uniaxial symmetry and dominates layers generated from low bulk concentrations. The revealed structure and the mechanism by which it is formed are postulated to be a general feature of the self-assembly of large glycoproteins. We expect that, inter alia, this knowledge will be relevant for understanding the extraordinary effectiveness of mucin thin films as boundary lubricants.