Nikki M. Curthoys

Actin Mediates the Nanoscale Membrane Organization of the Clustered Membrane Protein Influenza Hemagglutinin

The influenza viral membrane protein hemagglutinin (HA) is required at high concentrations on virion and host-cell membranes for infectivity. Because the role of actin in membrane organization is not completely understood, we quantified the relationship between HA and host-cell actin at the nanoscale. Results obtained using superresolution fluorescence photoactivation localization microscopy (FPALM) in nonpolarized cells show that HA clusters colocalize with actin-rich membrane regions (ARMRs). Individual molecular trajectories in live cells indicate restricted HA mobility in ARMRs, and actin disruption caused specific changes to HA clustering. Surprisingly, the actin-binding protein cofilin was excluded from some regions within several hundred nanometers of HA clusters, suggesting that HA clusters or adjacent proteins within the same clusters influence local actin structure. Thus, with the use of imaging, we demonstrate a dynamic relationship between glycoprotein membrane organization and the actin cytoskeleton at the nanoscale.

Tropomyosins induce neuritogenesis and determine neurite branching patterns in B35 neuroblastoma cells.

BACKGROUND The actin cytoskeleton is critically involved in the regulation of neurite outgrowth. RESULTS The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner. CONCLUSIONS Our data indicate that tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth. SIGNIFICANCE Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury. SUMMARY The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.

New aspects of tropomyosin-regulated neuritogenesis revealed by the deletion of Tm5NM1 and 2

Previous studies have shown that the overexpression of tropomyosins leads to isoform-specific alterations in the morphology of subcellular compartments in neuronal cells. Here we have examined the role of the most abundant set of isoforms from the gamma-Tm gene by knocking out the alternatively spliced C-terminal exon 9d. Despite the widespread location of exon 9d-containing isoforms, mice were healthy and viable. Compensation by products containing the C-terminal exon 9c was seen in the adult brain. While neurons from these mice show a mild phenotype at one day in culture, neurons revealed a significant morphological alteration with an increase in the branching of dendrites and axons after four days in culture. Our data suggest that this effect is mediated via altered stability of actin filaments in the growth cones. We conclude that exon 9d-containing isoforms are not essential for survival of neuronal cells and that isoform choice from the gamma-Tm gene is flexible in the brain. Although functional redundancy does not exist between tropomyosin genes, these results suggest that significant redundancy exists between products from the same gene.

Super-Resolution Imaging of Molecular Emission Spectra and Single Molecule Spectral Fluctuations.

Localization microscopy can image nanoscale cellular details. To address biological questions, the ability to distinguish multiple molecular species simultaneously is invaluable. Here, we present a new version of fluorescence photoactivation localization microscopy (FPALM) which detects the emission spectrum of each localized molecule, and can quantify changes in emission spectrum of individual molecules over time. This information can allow for a dramatic increase in the number of different species simultaneously imaged in a sample, and can create super-resolution maps showing how single molecule emission spectra vary with position and time in a sample.

Functional diversity of actin cytoskeleton in neurons and its regulation by tropomyosin.

Neurons comprise functionally, molecularly, and spatially distinct subcellular compartments which include the soma, dendrites, axon, branches, dendritic spines, and growth cones. In this chapter, we detail the remarkable ability of the neuronal cytoskeleton to exquisitely regulate all these cytoplasmic distinct partitions, with particular emphasis on the microfilament system and its plethora of associated proteins. Importance will be given to the family of actin-associated proteins, tropomyosin, in defining distinct actin filament populations. The ability of tropomyosin isoforms to regulate the access of actin-binding proteins to the filaments is believed to define the structural diversity and dynamics of actin filaments and ultimately be responsible for the functional outcome of these filaments.

Bleed-through correction for rendering and correlation analysis in multi-colour localization microscopy.

Multi-colour localization microscopy has enabled sub-diffraction studies of colocalization between multiple biological species and quantification of their correlation at length scales previously inaccessible with conventional fluorescence microscopy. However, bleed-through, or misidentification of probe species, creates false colocalization and artificially increases certain types of correlation between two imaged species, affecting the reliability of information provided by colocalization and quantified correlation. Despite the potential risk of these artefacts of bleed-through, neither the effect of bleed-through on correlation nor methods of its correction in correlation analyses has been systematically studied at typical rates of bleed-through reported to affect multi-colour imaging. Here, we present a reliable method of bleed-through correction applicable to image rendering and correlation analysis of multi-colour localization microscopy. Application of our bleed-through correction shows our method accurately corrects the artificial increase in both types of correlations studied (Pearson coefficient and pair correlation), at all rates of bleed-through tested, in all types of correlations examined. In particular, anti-correlation could not be quantified without our bleed-through correction, even at rates of bleed-through as low as 2%. Demonstrated with dichroic-based multi-colour FPALM here, our presented method of bleed-through correction can be applied to all types of localization microscopy (PALM, STORM, dSTORM, GSDIM, etc.), including both simultaneous and sequential multi-colour modalities, provided the rate of bleed-through can be reliably determined.

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

Dances with Membranes: Breakthroughs from Super-resolution Imaging.

Abstract Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology. The development of super-resolution microscopy has provided powerful tools for quantification of membrane organization at the level of individual proteins and lipids, and many of these tools are compatible with living cells. Previously inaccessible questions are now being addressed, and the field of membrane biology is developing rapidly. This chapter discusses how the development of super-resolution microscopy has led to fundamental advances in the field of biological membrane organization. We summarize the history and some models explaining how proteins are organized in cell membranes, and give an overview of various super-resolution techniques and methods of quantifying super-resolution data. We discuss the application of super-resolution techniques to membrane biology in general, and also with specific reference to the fields of actin and actin-binding proteins, virus infection, mitochondria, immune cell biology, and phosphoinositide signaling. Finally, we present our hopes and expectations for the future of super-resolution microscopy in the field of membrane biology.

Tropomyosins in Neuronal Morphogenesis and Development

This chapter discusses different aspects of Tropomyosins (Tms) in neurons. Section 18.1 is a brief outline of the Tm isoforms found in brain, Section 18.2 a detailed description of the expression patterns of these isoforms throughout development in neurons and the brain, Section 18.3 summarises the actin binding proteins (ABPs) which are localised in neurons and which interact with these Tm isoforms, Section 18.4 discusses the growth cone, and how Tm isoforms and ABPs may work together to regulate structure and function in this compartment, Section 18.5 examines the structure of the synapse, and which Tm isoforms and ABPs are implicated in synapse morphology and function, Section 18.6 is a brief overview of the implications of Tms in neurological disorders, and Section 18.7 summarises the reasons why understanding Tm isoform functions in neurons can aid in our understanding of the processes controlling neuronal morphogenesis and development. Tm isoforms each have specific spatial distributions across tissue, cells and subcellular compartments. These distributions are temporally regulated, with expression levels and localisation of Tms changing throughout development. In brain, the repertoire of Tm isoforms expressed changes with maturation, and a number of changes in the expression levels and localisation of specific Tms are associated with the defined cellular processes of neuronal morphogenesis and differentiation. By mapping the expression of Tms in brain and in neurons, and the developmental shifts in expression, a composite picture is formed of how different Tms can augment actin filament function throughout neuronal maturation.

Clean localization super-resolution microscopy for 3D biological imaging

We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3Dimage. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.