Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

United States Army Medical Research Institute of Infectious Diseases (USAMRIID)

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.

Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis.

In addition to their role in providing ATP for cellular functions via oxidative phosphorylation, mitochondria also play a critical role in initiating and/or regulating apoptosis through the release of proteins such as cytochrome c from intermembrane and intracristal compartments. The mechanism by which these proteins are able to cross the outer mitochondrial membrane has been a subject of controversy. This paper will review some recent results that demonstrate that inner mitochondrial membrane remodeling does occur during apoptosis in HeLa cells but does not appear to be a requirement for release of cytochrome c from intracristal compartments. Inner membrane remodeling does appear to be related to fragmentation of the mitochondrial matrix, and the form of the remodeling suggests a topological mechanism for inner membrane fission and fusion.

Correlated light and electron microscopy illuminates the role of mitochondrial inner membrane remodeling during apoptosis.

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of "virtual" 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts.

Chapter 2 Correlated light and electron microscopy/electron tomography of mitochondria in situ.

Pathogenic mycobacteria, which cause multiple diseases including tuberculosis, secrete factors essential for disease via the ESX-1 protein export system and are partially protected from host defenses by their lipid-rich cell envelopes. These pathogenic features of mycobacterial biology are believed to act independently of each other. Key ESX-1 components include three ATPases, and EccA1 (Mycobacterium marinum MMAR_5443; M.Â tuberculosis Rv3868) is the least characterized. Here we show that M.Â marinum EccA1's ATPase activity is required for ESX-1-mediated protein secretion, and surprisingly for the optimal synthesis of mycolic acids, integral cell-envelope lipids. Increased mycolic acid synthesis defects, observed when an EccA1-ATPase mutant is expressed in an eccA1-null strain, correlate with decreased inÂ vivo virulence and intracellular growth. These data suggest that two mycobacterial virulence hallmarks, ESX-1-dependent protein secretion and mycolic acid synthesis, are critically linked via EccA1.

EccA1, a component of the Mycobacterium marinum ESX-1 protein virulence factor secretion pathway, regulates mycolic acid lipid synthesis.

Les mycobact&eacute;ries pathog&egrave;nes, qui causent les maladies multiples, y compris la tuberculose, s&eacute;cr&egrave;tent des facteurs essentiels pour la maladie via le syst&egrave;me d&#39;exportation de prot&eacute;ine ESX-1 et sont partiellement prot&eacute;g&eacute;s contre les d&eacute;fenses de l&#39;h&ocirc;te de leurs enveloppes de cellules riches en lipides. Ces caract&eacute;ristiques pathog&egrave;nes de biologie mycobact&eacute;rie sont cens&eacute;s agir ind&eacute;pendamment les uns des autres. Les composants principaux ESX-1 incluent trois ATPases et EccA1 (Mycobacterium marinum MMAR_5443 ; M. tuberculosis Rv3868) est la moins caract&eacute;ris&eacute;e. Ici, nous montrons que l&#39;activit&eacute; ATPasique M. marinum de EccA1 est n&eacute;cessaire pour la s&eacute;cr&eacute;tion de prot&eacute;ine induite par l&#39;ESX-1 et &eacute;tonnamment la synth&egrave;se optimale des acides mycoliques, lipides de cellule-enveloppe int&eacute;grale. D&eacute;fauts de synth&egrave;se de l&#39;acide mycolique accrue, observ&eacute;es quand un mutant EccA1-ATPase est exprim&eacute; dans une souche eccA1 null, sont en corr&eacute;lation avec une diminution de virulence in vivo et croissance intracellulaire. Ces donn&eacute;es sugg&egrave;rent que les deux caract&eacute;ristiques de virulence mycobact&eacute;riennes, s&eacute;cr&eacute;tion de prot&eacute;ines ESX-1-d&eacute;pendante et synth&egrave;se de l&#39;acide mycolique, sont gravement li&eacute;s via EccA1.

EccA1, un composant du sentier de la Mycobacterium marinum ESX-1 prot&eacute;ine virulence facteur s&eacute;cr&eacute;tion, r&eacute;gule la synth&egrave;se des lipides acides mycoliques.

Micobatteri patogeni, che causano malattie multiple, tra cui tubercolosi, secernono fattori essenziali per la malattia attraverso il sistema di esportazione della proteina ESX-1 e sono parzialmente protetta da difese ospite da loro buste delle cellule ricche di lipidi. Si ritiene che queste caratteristiche patogene di biologia micobatterica agire indipendentemente una da altra. Chiave ESX-1 componenti includono tre ATPasi ed EccA1 (Mycobacterium marinum MMAR_5443; M. tuberculosis Rv3868) &egrave; il meno caratterizzato. Qui vi mostriamo che attivit&agrave; ATPasi M. marinum di EccA1 &egrave; necessaria per la secrezione della proteina ESX-1-mediata e sorprendentemente la sintesi ottima di micolico acidi, lipidi integrale cella-busta. Difetti di sintesi acido micolico aumentata, osservati quando un mutante EccA1-ATPasi &egrave; espressa in un ceppo di eccA1-null, correlano con diminuita virulenza in vivo e crescita intracellulare. Questi dati suggeriscono che le due caratteristiche di virulenza micobatterica, secrezione della proteina ESX-1-dipendente e sintesi di acido micolico, criticamente sono collegati tramite EccA1.

EccA1, un componente del Mycobacterium marinum ESX-1 proteina virulenza fattore secrezione percorso, regola la sintesi di lipidi acido micolico.

Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5-1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Fixation-resistant photoactivatable fluorescent proteins for CLEM.

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection.

Transmission electron microscopy can be used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. In a transmission electron microscope (TEM), the image is created by passing an electron beam through a specimen with contrast generated by electron scattering from dense elements in the specimen. Viruses do not normally contain dense elements, so a negative stain that places dense heavy metal salts around the sample is added to create a dark border. To prepare a virus sample for a negative stain transmission electron microscopy, a virus suspension is applied to a TEM grid specimen support, which is a 3mm diameter fragile specimen screen coated with a few nanometers of plastic film. Then, deionized (dI) water rinses and a negative stain solution are applied to the grid. All infectious viruses must be handled in a biosafety cabinet (BSC) and many require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL) 3 and 4 is especially challenging because the support grids are small, fragile, and easily moved by air currents. In this study we evaluated a new device for negative staining viruses called mPrep/g capsule. It is a capsule that holds up to two TEM grids during all processing steps and for storage after staining is complete. This study reports that the mPrep/g capsule method is valid and effective to negative stain virus specimens, especially in high containment laboratory environments.

Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining.

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM data sets for aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. The choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replication creates high-contrast, 3D images of the cytoplasmic surface of the plasma membrane but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (∼10-50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2-7 d, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology.

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples.

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter.

Mei G. Sun

Pathology Division

United States Army Medical Research Institute of Infectious Diseases (USAMRIID)

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Mei G. Sun

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Pathology Division

United States Army Medical Research Institute of Infectious Diseases (USAMRIID)

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Pathology Division