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1.
Biophys J ; 116(5): 893-909, 2019 03 05.
Article in English | MEDLINE | ID: mdl-30773293

ABSTRACT

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.


Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Fluorescent Dyes/metabolism , Hemagglutinins, Viral/metabolism , Orthomyxoviridae , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Cell Survival , Mice , Models, Biological , NIH 3T3 Cells
2.
PLoS One ; 11(3): e0147506, 2016.
Article in English | MEDLINE | ID: mdl-27002724

ABSTRACT

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.


Subject(s)
Microscopy/methods , Animals , Fluorescence , Mice , NIH 3T3 Cells
3.
Curr Top Membr ; 75: 59-123, 2015.
Article in English | MEDLINE | ID: mdl-26015281

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.


Subject(s)
Cell Membrane/metabolism , Microfilament Proteins/metabolism , Microscopy/methods , Mitochondria/ultrastructure , Viruses/ultrastructure , Animals , Humans , Models, Biological
4.
Mol Cell Neurosci ; 58: 11-21, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24211701

ABSTRACT

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.


Subject(s)
Growth Cones/metabolism , Neurites/metabolism , Neurogenesis , Tropomyosin/metabolism , Actin Cytoskeleton/metabolism , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Neuroblastoma/metabolism , Protein Isoforms/metabolism , Pseudopodia/metabolism , Rats
5.
Biophys J ; 104(10): 2182-92, 2013 May 21.
Article in English | MEDLINE | ID: mdl-23708358

ABSTRACT

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.


Subject(s)
Actins/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Actin Cytoskeleton/metabolism , Actin Depolymerizing Factors/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/ultrastructure , Influenza A Virus, H2N2 Subtype/chemistry , Influenza A Virus, H2N2 Subtype/metabolism , Mice , NIH 3T3 Cells , Protein Multimerization
6.
J Opt ; 15(9)2013 Sep.
Article in English | MEDLINE | ID: mdl-26185614

ABSTRACT

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.

7.
J Vis Exp ; (82): e50680, 2013 Dec 09.
Article in English | MEDLINE | ID: mdl-24378721

ABSTRACT

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.


Subject(s)
Fluorescence Polarization/instrumentation , Fluorescence Polarization/methods , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Animals , Fluorescent Dyes/chemistry , Mice , Microscopy, Video/instrumentation , Microscopy, Video/methods , NIH 3T3 Cells , Photobleaching
8.
Int Rev Cell Mol Biol ; 298: 33-94, 2012.
Article in English | MEDLINE | ID: mdl-22878104

ABSTRACT

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.


Subject(s)
Actin Cytoskeleton/metabolism , Neurons/metabolism , Tropomyosin/metabolism , Animals , Humans , Microfilament Proteins/metabolism , Models, Biological
9.
Eur J Cell Biol ; 89(7): 489-98, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20223554

ABSTRACT

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.


Subject(s)
Neurogenesis/physiology , Tropomyosin/metabolism , Alternative Splicing/genetics , Alternative Splicing/physiology , Animals , Axons/metabolism , Brain/cytology , Brain/metabolism , Cell Line , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Immunohistochemistry , Mice , Neurogenesis/genetics , Neurons/cytology , Neurons/metabolism , Phenotype , Protein Isoforms/genetics , Protein Isoforms/metabolism , Tropomyosin/genetics
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