Alioscka A Sousa

National Institutes of Health, Maryland, United States

Are you Alioscka A Sousa?

Claim your profile

Publications (44)167.17 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, "analog" sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-μm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon-vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates.
    The Journal of neuroscience : the official journal of the Society for Neuroscience. 07/2014; 34(27):8948-62.
  • Source
    Biophysical Journal 01/2013; 104(2):146A. · 3.67 Impact Factor
  • Alioscka A Sousa, Michael J Kruhlak
    [Show abstract] [Hide abstract]
    ABSTRACT: To dissect the astonishing complexity of the biomolecular machinery functioning within a cell, imaging has been an integral tool in biology, allowing researches to "view" the detailed molecular biology responsible for coordinating cellular life. To visualize the molecular components of cellular structures requires, in particular, imaging techniques capable of reaching nanoscale spatial resolutions. Such nanoimaging techniques are the focus of this volume. Chapters in the current volume are divided into four parts and include specialized techniques in the areas of light, electron, and scanning probe microscopy, as well as methodologies employing combinatorial and complementary imaging approaches.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 950:1-10. · 1.29 Impact Factor
  • Biophysical Journal 01/2013; 104(2):353-. · 3.67 Impact Factor
  • Alioscka A Sousa, Richard D Leapman
    [Show abstract] [Hide abstract]
    ABSTRACT: Scanning transmission electron microscopy (STEM) in the dark-field mode of operation is a technique regularly used to record high-contrast images from isolated macromolecular assemblies at nanometer resolution. Dark-field STEM images are unique in that they can be readily quantified to provide information on the mass of individual molecular complexes. Importantly, because STEM images contain simultaneous mass and overall molecular shape information, the concept of "mass mapping" can be realized to provide distinctive measurements of the mass per area of planar assemblies or the mass per length of filamentous structures. In this chapter we describe how the STEM technique can be applied to generate characteristic measurements of mass per length from isolated Alzheimer's amyloid fibrils.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 950:195-207. · 1.29 Impact Factor
  • Biophysical Journal 01/2013; 104(2):514-. · 3.67 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We examined the structure and biomineralization of prismatic magnetosomes in the magnetotactic marine vibrio Magnetovibrio blakemorei strain MV-1 and a non-magnetotactic mutant derived from it, using a combination of cryo-electron tomography and freeze-fracture. The vesicles enveloping the Magnetovibrio magnetosomes were elongated and detached from the cell membrane. Magnetosome crystal formation appeared to be initiated at a nucleation site on the membrane inner surface. Interestingly, while scattered filaments were observed in the surrounding cytoplasm, their association with the magnetosome chains could not be unequivocally established. Our data suggests fundamental differences between prismatic and octahedral magnetosomes in their mechanisms of nucleation and crystal growth as well as in their structural relationships with the cytoplasm and plasma membrane.
    Journal of Structural Biology 12/2012; · 3.36 Impact Factor
  • Microscopy and Microanalysis 07/2012; 18(S2):542-543. · 2.50 Impact Factor
  • Microscopy and Microanalysis 07/2012; 18(S2):1596-1597. · 2.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Morphology of aggregation intermediates, polymorphism of amyloid fibrils and aggregation kinetics of the "Arctic" mutant of the Alzheimer's amyloid β-peptide, Aβ((1-40))(E22G), in a physiologically relevant Tris buffer (pH 7.4) were thoroughly explored in comparison with the human wild type Alzheimer's amyloid peptide, wt-Aβ((1-40)), using both in situ atomic force and electron microscopy, circular dichroism and thioflavin T fluorescence assays. For arc-Aβ((1-40)) at the end of the 'lag'-period of fibrillization an abrupt appearance of ∼3nm size 'spherical aggregates' with a homogeneous morphology, was identified. Then, the aggregation proceeds with a rapid growth of amyloid fibrils with a variety of morphologies, while the spherical aggregates eventually disappeared during in situ measurements. Arc-Aβ((1-40)) was also shown to form fibrils at much lower concentrations than wt-Aβ((1-40)): ⩽2.5μM and 12.5μM, respectively. Moreover, at the same concentration, 50μM, the aggregation process proceeds more rapidly for arc-Aβ((1-40)): the first amyloid fibrils were observed after c.a. 72h from the onset of incubation as compared to approximately 7days for wt-Aβ((1-40)). Amyloid fibrils of arc-Aβ((1-40)) exhibit a large variety of polymorphs, at least five, both coiled and non-coiled distinct fibril structures were recognized by AFM, while at least four types of arc-Aβ((1-40)) fibrils were identified by TEM and STEM and their mass-per-length statistics were collected suggesting supramolecular structures with two, four and six β-sheet laminae. Our results suggest a pathway of fibrillogenesis for full-length Alzheimer's peptides with small and structurally ordered transient spherical aggregates as on-pathway immediate precursors of amyloid fibrils.
    Journal of Structural Biology 06/2012; 180(1):174-89. · 3.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a major component of postsynaptic densities (PSDs) involved in synaptic regulation. It has been previously shown that upon activity CaMKII from the spine reversibly aggregates at the cytoplasmic surfaces of PSDs, where it encounters various targets for phosphorylation. Targets for CaMKII are also present within the PSD, but there has been no reliable method to pinpoint whether, or where, CaMKII is located inside the PSD. Here we show that CaMKII can be mapped molecule-by-molecule within isolated PSDs using negative stain electron microscopy tomography. CaMKII molecules found in the core of the PSD may represent a pool distinct from the CaMKII residing at the cytoplasmic surface. J. Comp. Neurol. 520:4218-4225, 2012. © 2012 Wiley Periodicals, Inc.
    The Journal of Comparative Neurology 05/2012; 520(18):4218-25. · 3.66 Impact Factor
  • Alioscka A Sousa, Richard D Leapman
    [Show abstract] [Hide abstract]
    ABSTRACT: The design of the scanning transmission electron microscope (STEM), as conceived originally by Crewe and coworkers, enables the highly efficient and flexible collection of different elastic and inelastic signals resulting from the interaction of a focused probe of incident electrons with a specimen. In the present paper we provide a brief review for how the STEM today can be applied towards a range of different problems in the biological sciences, emphasizing four main areas of application. (1) For three decades, the most widely used STEM technique has been the mass determination of proteins and other macromolecular assemblies. Such measurements can be performed at low electron dose by collecting the high-angle dark-field signal using an annular detector. STEM mass mapping has proven valuable for characterizing large protein assemblies such as filamentous proteins with a well-defined mass per length. (2) The annular dark-field signal can also be used to image ultrasmall, functionalized nanoparticles of heavy atoms for labeling specific amino-acid sequences in protein assemblies. (3) By acquiring electron energy loss spectra (EELS) at each pixel in a hyperspectral image, it is possible to map the distributions of specific bound elements like phosphorus, calcium and iron in isolated macromolecular assemblies or in compartments within sectioned cells. Near single atom sensitivity is feasible provided that the specimen can tolerate a very high incident electron dose. (4) Electron tomography is a new application of STEM that enables three-dimensional reconstruction of micrometer-thick sections of cells. In this technique a probe of small convergence angle gives a large depth of field throughout the thickness of the specimen while maintaining a probe diameter of <2nm; and the use of an on-axis bright-field detector reduces the effects of beam broadening and thus improves the spatial resolution compared to that attainable by STEM dark-field tomography.
    Ultramicroscopy 05/2012; · 2.47 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and how they behave in the body. Here we report the synthesis of ultrasmall, uniform 144-atom AuNPs protected by p-mercaptobenzoic acid followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH-coated nanoparticles (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles' intracellular fate, the cell-penetrating peptide TAT is attached noncovalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)-TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells.
    Small 04/2012; 8(14):2277-86. · 7.82 Impact Factor
  • Biophysical Journal 01/2012; 102(3):393-. · 3.67 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Monodisperse Au(25)L(18)(0) (L = S(CH(2))(2)Ph) and [n-Oct(4)N(+)][Au(25)L(18)(-)] clusters were synthesized in tetrahydrofuran. An original strategy was then devised to oxidize them: in the presence of bis(pentafluorobenzoyl) peroxide, the neutral or the negatively charged clusters react as efficient electron donors in a dissociative electron-transfer (ET) process, in the former case yielding [Au(25)L(18)(+)][C(6)F(5)CO(2)(-)]. As opposed to other reported redox methods, this dissociative ET approach is irreversible, easily controllable, and clean, particularly for NMR purposes, as no hydrogen atoms are introduced. By using this approach, the -1, 0, and +1 charge states of Au(25)L(18) could be fully characterized by (1)H and (13)C NMR spectroscopy, using one- and two-dimensional techniques, in various solvents, and as a function of temperature. For all charge states, the NMR results and analysis nicely match recent structural findings about the presence of two different ligand populations in the capping monolayer, each resonance of the two ligand families displaying distinct NMR patterns. The radical nature of Au(25)L(18)(0) is particularly evident in the (1)H and (13)C NMR patterns of the inner ligands. The NMR behavior of radical Au(25)L(18)(0) was also simulated by DFT calculations, and the interplay between theory and experiments revealed a fundamental paramagnetic contribution coming from Fermi contact shifts. Interestingly, the NMR patterns of Au(25)L(18)(-) and Au(25)L(18)(+) were found to be quite similar, pointing to the latter cluster form as a diamagnetic species.
    Analytical Chemistry 06/2011; 83(16):6355-62. · 5.70 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.
    Journal of Neuroscience 04/2011; 31(17):6329-38. · 6.91 Impact Factor
  • Source
    M A Aronova, A A Sousa, R D Leapman
    [Show abstract] [Hide abstract]
    ABSTRACT: Electron energy loss spectroscopy (EELS) was used to obtain information about the radiation chemistry of frozen aqueous specimens in the electron microscope by observing the hydrogen and oxygen K-edges. Measurements on frozen solutions of 30% hydrogen peroxide revealed the presence of molecular oxygen identified by a distinct 531-eV peak at the O K-edge even for electron doses below 100 e/nm². The molecular oxygen content of irradiated H₂O₂ solution was determined by least squares fitting of O K-edge reference spectra from water and gas-phase oxygen. It was found that the fraction of molecular oxygen to water oxygen was in the range 0.03-0.05. EELS from pure frozen water showed no features attributable to molecular oxygen or molecular hydrogen (K edge at ~13 eV) even at high electron doses above 10⁵ e/nm². Spectra from frozen sucrose and protein solutions and their mixtures, however, did show evolution of a molecular hydrogen peak at ~13 eV for doses above 10⁵ e/nm², consistent with previous measurements and indicative of hydrogen bubble formation. Molecular oxygen was not observed in any of the frozen solutions of organic compounds indicating that oxygen is not a major product of free radical decay, in contrast to molecular hydrogen formation.
    Micron 04/2011; 42(3):252-6. · 1.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently portable Raman probes have emerged along with a variety of applications, including carbon nanotube (CNT) characterization. Aqueous dispersed CNTs have shown promise for biomedical applications such as drug/gene delivery vectors, photo-thermal therapy, and photoacoustic imaging. In this study we report the simultaneous detection and irradiation of carbon nanotubes in 2D monolayers of cancer cells and in 3D spheroids using a portable Raman probe. A portable handheld Raman instrument was utilized for dual purposes: as a CNT detector and as an irradiating laser source. Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) were dispersed aqueously using a lipid-polymer (LP) coating, which formed highly stable dispersions both in buffer and cell media. The LP coated SWCNT and MWCNT aqueous dispersions were characterized by atomic force microscopy, transmission electron microscopy, dynamic light scattering, Fourier transform infrared spectroscopy and Raman spectroscopy. The cellular uptake of the LP-dispersed SWCNTs and MWCNTs was observed using confocal microscopy, and fluorescein isothiocyanate (FITC)-nanotube conjugates were found to be internalized by ovarian cancer cells by using Z-stack fluorescence confocal imaging. Biocompatibility of SWCNTs and MWCNTs was assessed using a cell viability MTT assay, which showed that the nanotube dispersions did not hinder the proliferation of ovarian cancer cells at the dosage tested. Ovarian cancer cells treated with SWCNTs and MWCNTs were simultaneously detected and irradiated live in 2D layers of cancer cells and in 3D environments using the portable Raman probe. An apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay carried out after laser irradiation confirmed that cell death occurred only in the presence of nanotube dispersions. We show for the first time that both SWCNTs and MWCNTs can be selectively irradiated and detected in cancer cells using a simple handheld Raman instrument. This approach could potentially be used to treat various diseases, including cancer.
    Theranostics 01/2011; 1:310-21. · 7.81 Impact Factor
  • Microscopy and Microanalysis 01/2011; 17:972-973. · 2.50 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To study the distribution and clearance of polyethylene glycol (PEG)-ylated single-walled carbon nanotube (SWCNTs) as drug delivery vehicles for the anticancer drug cisplatin in mice. PEG layers were attached to SWCNTs and dispersed in aqueous media and characterized using dynamic light scattering, scanning transmission electron microscopy and Raman spectroscopy. Cytotoxicity was assessed in vitro using Annexin-V assay, and the distribution and clearance pathways in mice were studied by histological staining and Raman spectroscopy. Efficacy of PEG-SWCNT-cisplatin for tumor growth inhibition was studied in mice. PEG-SWCNTs were efficiently dispersed in aqueous media compared with controls, and did not induce apoptosis in vitro. Hematoxylin and eosin staining, and Raman bands for SWCNTs in tissues from several vital organs from mice injected intravenously with nanotube bioconjugates revealed that control SWCNTs were lodged in lung tissue as large aggregates compared with the PEG-SWCNTs, which showed little or no accumulation. Characteristic SWCNT Raman bands in feces revealed the presence of bilary or renal excretion routes. Attachment of cisplatin on bioconjugates was visualized with Z-contrast scanning transmission electron microscopy. PEG-SWCNT-cisplatin with the attached targeting ligand EGF successfully inhibited growth of head and neck tumor xenografts in mice. PEG-SWCNTs, as opposed to control SWCNTs, form more highly dispersed delivery vehicles that, when loaded with both cisplatin and EGF, inhibit growth of squamous cell tumors.
    Nanomedicine 12/2010; 5(10):1535-46. · 5.26 Impact Factor