Fluorescence Microscopy Techniques Research Articles

Elastic contact between rough surfaces: Bridging the gap between theory and experiment

Friction is generated where solids touch, thus quantitative understanding of the microscopic contact area is crucial to predict macroscopic friction. All surfaces are rough, and typically small-scale roughness rather than macroscopic geometry sets the contact area. Rough contact theories have been developed that predict the contact area, using the surface topography and the mechanical properties of the solids as input. However, the validity of these theories remains under debate. Systematic comparison between theory and experiment has been lacking, due to the experimental difficulty to access the contact area, especially at small length scales where idealised assumptions of contact models may not hold. Here, we use a state-of-the-art fluorescence microscopy technique to directly access the contact area of a glass-glass contact with nanometric out-of-plane resolution. We systematically vary the roughness of the contact and carefully measure the surface topography across length scales. Comparison of high-resolution contact experiments to different contact theories enables us to unambigously conclude that only elastic Persson theory quantitatively describes experimental observations.

Performance of sulfided NiMo catalyst supported on pillared bentonite Al and Ti under hydrodeoxygenation reaction of guaiacol

Bio-crude oil is known to be sustainable, eco-environmentally, and an alternative energy source produced by biomass pyrolysis. However, its quality remains relatively low due to a higher oxygen concentration compared to liquid fuels from fossils. Therefore, an upgrading process is necessary through the catalytic hydrodeoxygenation (HDO) process. This work synthesized pillared bentonite using Al and Ti metals as the pillaring agent to produce Al-PILC and Ti-PILC as catalyst support for sulfided NiMo. Their catalytic activity in HDO reaction using guaiacol as a model compound of bio-crude oil were also evaluated. Characterization of the bentonite-pillared materials, including Al-PILC, Mo/Al-PILC, NiMo/Al-PILC, Ti-PILC, Mo/Ti-PILC, and NiMo/Ti-PILC, was performed using Surface Area Analyzer, X-ray Diffractometer (XRD), Temperature-Programmed Desorption of ammonia (NH3-TPD), X-Ray Fluorescence (XRF), and Scanning Electron Microscope (SEM) techniques. The characterization results confirm the pillarization process of bentonite using Al and Ti metals as the pillaring agent, and the preparation of the NiMo catalyst using the stepwise impregnation method was successfully prepared. The NiMo/Ti-PILC catalyst performs a superior conversion value on the HDO guaiacol reaction than other catalysts. A well dispersion of Mo and Ni metals on the surface support (NiMo/Ti-PILC), thus creating numerous active sites of the catalyst after the sulfidation. Variations in time and temperature during the HDO guaiacol reaction significantly affected the conversion.

Rapid examination of lung tissues by nonlinear microscopy.

Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

Large-scale annotated dataset for cochlear hair cell detection and classification

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5–15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Host-Pathogen Interactions and Correlated Factors That Are Affected in Replicative-Aged Cryptococcus neoformans.

Cryptococcus neoformans is a facultative intracellular fungal pathogen. Ten-generation-old (10GEN) C. neoformans cells are more resistant to phagocytosis and killing by macrophages than younger daughter cells. However, mechanisms that mediate this resistance and intracellular parasitism are poorly understood. Here, we identified important factors for the intracellular survival of 10GEN C. neoformans, such as urease activity, capsule synthesis, and DNA content using flow cytometry and fluorescent microscopy techniques. The real-time visualization of time-lapse imaging was applied to determine the phagosomal acidity, membrane permeability, and vomocytosis (non-lytic exocytosis) rate in J774 macrophages that phagocytosed C. neoformans of different generational ages. Our results showed that old C. neoformans exhibited higher urease activity and enhanced Golgi activity. In addition, old C. neoformans were more likely to be arrested in the G2 phase, resulting in the occasional formation of aberrant trimera-like cells. To finish, the advanced generational age of the yeast cells slightly reduced vomocytosis events within host cells, which might be associated with increased phagolysosome pH and membrane permeability. Altogether, our results suggest that old C. neoformans prevail within acidic phagolysosomes and can manipulate the phagosome pH. These strategies may be used by old C. neoformans to resist phagosomal killing and drive cryptococcosis pathogenesis. The comprehension of these essential host-pathogen interactions could further shed light on mechanisms that bring new insights for novel antifungal therapeutic design.

Sol-Gel Spin Coated Transparent Single-TiO2 and SiO2 Thin Film on Glass for Opto-electronic Applications

Great attention has been given on the sol-gel synthesis and spin-coating deposition techniques for the fabrication of various thin films. Because of its simple technique to fabricate dielectric (metal oxide) films using tetraethyl orthosilicate and titanium isopropoxide precursors. In this work, transparent single layer of SiO2 and TiO2 thin film was deposited on a glass substrate and characterized by FTIR, UV-visible spectroscopy, fluorescence and scanning electron microscopy techniques. The single layer thin film was mechanically stable and strong by adhering to glass substrates. The FTIR transmittance spectrum evidenced the existence of Si-O-Si and Ti-O-Ti at ~1100 and 1400 cm-1. The UV-visible absorbance spectra of single layer of SiO2 and TiO2 thin films showed strong absorption at below 00 nm. The excitation wavelength at 380 nm, the fluorescence spectra of both thin films showed the highest emission spectrum in between 733-798 nm. The SEM studies confirmed the smooth surface in both SiO2 and TiO2 layers. Further, it could be useful for various applications such as back reflector in solar cells, anti-reflection coatings, hydrophobic and anti-fogging materials.

Direct observation of a crescent-shape chromosome in expanded Bacillus subtilis cells

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins – the key drivers of chromosomal organization – along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Clinical application of the HM-1000 image processing for HER2 fluorescence in situ hybridization signal quantification in breast cancer

BackgroundAccurate quantification of human epidermal growth factor receptor 2 (HER2) gene amplification is important for predicting treatment response and prognosis in patients with breast cancer. Fluorescence in situ hybridization (FISH) is the gold standard for the diagnosis of HER2 status, particularly in cases with equivocal status on immunohistochemistry (IHC) staining, but has some limitations of non-classical amplifications and such cases are diagnosed basing on additional IHC and FISH. This study investigated the clinical utility of a novel super-resolution fluorescence microscopy technique for the better FISH signal visualization and HER2 FISH classification.MethodsFourteen breast cancer tissue samples were retrospectively collected between September 2018 and February 2022, and FISH HER2 signal quantification was evaluated by determining the HER2/chromosome 17 centromere (CEP17) ratio and the number of HER2 signals per nucleus in super- versus conventional-resolution images.ResultsSuper-resolution images maintained the same overall HER2 diagnosis from routine, but HER2 FISH amplification changed negative to monosomy in two cases. Two Letrozole non-response relapses coincided to monosomy samples. The median number of HER2 signals per nucleus was 7.5 in super-resolution images and 4.0 in conventional-resolution images in HER2-positive samples and 2.8 and 2.1 signals per nucleus, respectively, in HER2-negative samples.ConclusionsSuper-resolution images improved signal visualization, including a significant difference in the number of countable HER2 and CEP17 signals in a single nucleus compared with conventional-resolution images. Increased accuracy of signal quantification by super-resolution microscopy may provide clinicians with more detailed information regarding HER2 FISH status that allows to better FISH classification such as HER2-low samples.

Quantifying nanoscopic alterations associated with mitochondrial dysfunction using three-dimensional single-molecule localization microscopy.

Mitochondrial morphology provides unique insights into their integrity and function. Among fluorescence microscopy techniques, 3D super-resolution microscopy uniquely enables the analysis of mitochondrial morphological features individually. However, there is a lack of tools to extract morphological parameters from super-resolution images of mitochondria. We report a quantitative method to extract mitochondrial morphological metrics, including volume, aspect ratio, and local protein density, from 3D single-molecule localization microscopy images, with single-mitochondrion sensitivity. We validated our approach using simulated ground-truth SMLM images of mitochondria. We further tested our morphological analysis on mitochondria that have been altered functionally and morphologically in controlled manners. This work sets the stage to quantitatively analyze mitochondrial morphological alterations associated with disease progression on an individual basis.

Foraminifera as a model of eukaryotic genome dynamism.

In contrast to the canonical view that genomes cycle only between haploid and diploid states, many eukaryotes have dynamic genomes that change content throughout an individual's life cycle. However, the few detailed studies of microeukaryotic life cycles render our understanding of eukaryotic genome dynamism incomplete. Foraminifera (Rhizaria) are an ecologically important, yet understudied, clade of microbial eukaryotes with complex life cycles that include changes in ploidy and genome organization. Here, we apply fluorescence microscopy and image analysis techniques to over 2,800 nuclei in 110 cells to characterize the life cycle of Allogromia laticollaris strain Cold Spring Harbor (CSH), one of few cultivable foraminifera species. We show that haploidy and diploidy are brief moments in the A. laticollaris life cycle and that A. laticollaris nuclei endoreplicate up to 12,000 times the haploid genome size. We find that A. laticollaris reorganizes a highly endoreplicated nucleus into thousands of haploid genomes through a non-canonical mechanism called Zerfall, in which the nuclear envelope degrades and extrudes chromatin into the cytoplasm. Based on these findings, along with changes in nuclear architecture across the life cycle, we believe that A. laticollaris uses spatio-temporal mechanisms to delineate germline and somatic DNA within a single nucleus. The analyses here extend our understanding of the genome dynamics across the eukaryotic tree of life.IMPORTANCEIn traditional depictions of eukaryotes (i.e., cells with nuclei), life cycles alternate only between haploid and diploid phases, overlooking studies of diverse microeukaryotic lineages (e.g., amoebae, ciliates, and flagellates) that show dramatic variation in DNA content throughout their life cycles. Endoreplication of genomes enables cells to grow to large sizes and perhaps to also respond to changes in their environments. Few microeukaryotic life cycles have been studied in detail, which limits our understanding of how eukaryotes regulate and transmit their DNA across generations. Here, we use microscopy to study the life cycle of Allogromia laticollaris strain CSH, an early-diverging lineage within the Foraminifera (an ancient clade of predominantly marine amoebae). We show that DNA content changes significantly throughout their life cycle and further describe an unusual process called Zerfall, by which this species reorganizes a large nucleus with up to 12,000 genome copies into hundreds of small gametic nuclei, each with a single haploid genome. Our results are consistent with the idea that all eukaryotes demarcate germline DNA to pass on to offspring amidst more flexible somatic DNA and extend the known diversity of eukaryotic life cycles.

Disruption Mechanisms of Enveloped Viruses by Ionic and Nonionic Surfactants.

The world has witnessed multiple pandemics and endemics caused by enveloped viruses in the past century. To name a few, the ongoing COVID-19 pandemic and other pandemics/endemics caused by coronaviruses, influenza viruses, HIV-1, etc. The external and topical applications of surfactants have been effective in limiting the spread of viruses. While it is well-known that surfactants inactivate virus particles (virions), the mechanism of action of surfactants against enveloped virions has not yet been established. In this work, we have evaluated the surfactant-induced disruption mechanism of a cocktail of enveloped viruses containing particles of mumps, measles, and rubella viruses. We applied the total internal reflection fluorescence microscopy technique to trace the temporal changes in the fluorescence signal from single virions upon the addition of a surfactant solution. We report that surfactants solubilize either the viral lipid membrane, proteins, or both. Ionic surfactants, depending on their charge and interaction type with the viral lipids and proteins, can cause bursting or perforation of the viral envelope, whereas a nonionic surfactant can cause either symmetric expansion or perforation of the viral envelope depending on the surfactant concentration.

A decellularized matrix enriched collagen microscaffold for a 3D in vitro liver model.

The development of liver scaffolds retaining their three-dimensional (3D) structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of an alginate-based platform using a combination of decellularized matrices and collagen to preserve the functionality of liver cells. The scaffolds were characterized using SEM and fluorescence microscopy techniques. The proliferation and functional behaviours of hepatocellular carcinoma HuH7 cells were observed. It was found that the decellularized skin scaffold with collagen was better for maintaining the growth of cells in comparison to other decellularized matrices. In addition, we observed a significant increase in the functional profile once exogenous collagen was added to the liver matrix. Our study also suggests that a cirrhotic liver model should have a different matrix composition as compared to a healthy liver model. When primary rat hepatocytes were used for developing a healthy liver model, the proliferation studies with hepatocytes showed a decellularized skin matrix as the better option, but the functionality was only maintained in a decellularized liver matrix with addition of exogenous collagen. We further checked if these platforms can be used for studying drug induced toxicity observed in the liver by studying the activation of cytochrome P450 upon drug exposure of the cells growing in our model. We observed a significant induction of the CYP1A1 gene on administering the drugs for 6 days. Thus, this platform could be used for drug-toxicity screening studies using primary hepatocytes in a short span of time. Being a microscaffold based system, this platform offers some advantages, such as smaller volumes of samples, analysing multiple samples simultaneously and a minimal amount of decellularized matrix in the matrix composition, making it an economical option compared to a completely dECM based platform.

Physiological quality, lignin and the ultrastructural characterization of soybean seeds

In soybeans, the integument or seed coat is an important modulator between the external and internal environment. It plays a fundamental role in seed vitality, and its lignin content may influence the seed quality. The objective of this experiment was to evaluate the quality of soybean seeds from a partial diallel and their reciprocals and its relationship with lignin content, seed coat thickness, and deposition location. The seeds were also evaluated for physiological quality through germination and accelerated aging tests. The lignin content was quantified by absorbance, and the integument thickness was analyzed and measured using scanning electron microscopy. The most contrasting cultivars for lignin content were analyzed using fluorescence microscopy and histochemical techniques. Cultivars and their reciprocals differed in seed physiological quality. We found differences in the genotypes for integument thickness. Using histochemical techniques, autofluorescence was observed in the same regions identified as lignified. Positive staining for lignin was observed in the hilum region. Among the genotypes, we found variations in the physiological quality of seeds (germination and accelerated aging test), lignin content, and integument thickness.

IU-ExM: nanoscopy of organelles and tissues with iterative ultrastructure expansion microscopy

Expansion microscopy (ExM) is a highly effective technique for super-resolution fluorescence microscopy that enables imaging of biological samples beyond the diffraction limit with conventional fluorescence microscopes. Despite the development of several enhanced protocols, ExM has not yet demonstrated the ability to achieve the precision of nanoscopy techniques such as Single Molecule Localization Microscopy (SMLM). Here, to address this limitation, we have developed an iterative ultrastructure expansion microscopy (iU-ExM) approach that achieves SMLM-level resolution. With iU-ExM, it is now possible to visualize the molecular architecture of gold-standard samples, such as the eight-fold symmetry of nuclear pores or the molecular organization of the conoid in Apicomplexa. With its wide-ranging applications, from isolated organelles to cells and tissue, iU-ExM opens new super-resolution avenues for scientists studying biological structures and functions.

Probes for noninvasive biological visualization and biosensing of cancer cells

The early detection of tumors and precancerous conditions is vital for cancer diagnosis. Advances in fluorescence microscopic techniques and materials synthesis processes have revolutionized biomarker detection and image-guided cancer surveillance. In particular, novel materials-based diagnostic tools and innovative therapies have facilitated a precise understanding of biological processes at the molecular level. This critical review presents an overview of bioimaging probes, including functionalized chromophoric systems, non-functionalized chromophoric systems, and nanoscale biosensors. Technical challenges and future directions related to these approaches are considered.

Isthmin-1 attenuates allergic Asthma by stimulating adiponectin expression and alveolar macrophage efferocytosis in mice

BackgroundAllergic asthma is a common respiratory disease that significantly impacts human health. Through in silico analysis of human lung RNASeq, we found that asthmatic lungs display lower levels of Isthmin-1 (ISM1) expression than healthy lungs. ISM1 is an endogenous anti-inflammatory protein that is highly expressed in mouse lungs and bronchial epithelial cells, playing a crucial role in maintaining lung homeostasis. However, how ISM1 influences asthma remains unclear. This study aims to investigate the potential involvement of ISM1 in allergic airway inflammation and uncover the underlying mechanisms.MethodsWe investigated the pivotal role of ISM1 in airway inflammation using an ISM1 knockout mouse line (ISM1−/−) and challenged them with house dust mite (HDM) extract to induce allergic-like airway/lung inflammation. To examine the impact of ISM1 deficiency, we analyzed the infiltration of immune cells into the lungs and cytokine levels in bronchoalveolar lavage fluid (BALF) using flow cytometry and multiplex ELISA, respectively. Furthermore, we examined the therapeutic potential of ISM1 by administering recombinant ISM1 (rISM1) via the intratracheal route to rescue the effects of ISM1 reduction in HDM-challenged mice. RNA-Seq, western blot, and fluorescence microscopy techniques were subsequently used to elucidate the underlying mechanisms.ResultsISM1−/− mice showed a pronounced worsening of allergic airway inflammation and hyperresponsiveness upon HDM challenge. The heightened inflammation in ISM1−/− mice correlated with enhanced lung cell necroptosis, as indicated by higher pMLKL expression. Intratracheal delivery of rISM1 significantly reduced the number of eosinophils in BALF and goblet cell hyperplasia. Mechanistically, ISM1 stimulates adiponectin secretion by type 2 alveolar epithelial cells partially through the GRP78 receptor and enhances adiponectin-facilitated apoptotic cell clearance via alveolar macrophage efferocytosis. Reduced adiponectin expression under ISM1 deficiency also contributed to intensified necroptosis, prolonged inflammation, and heightened severity of airway hyperresponsiveness.ConclusionsThis study revealed for the first time that ISM1 functions to restrain airway hyperresponsiveness to HDM-triggered allergic-like airway/lung inflammation in mice, consistent with its persistent downregulation in human asthma. Direct administration of rISM1 into the airway alleviates airway inflammation and promotes immune cell clearance, likely by stimulating airway adiponectin production. These findings suggest that ISM1 has therapeutic potential for allergic asthma.Graphical abstract

Improving optical sectioning with spinning disk structured illumination microscopy.

A new fluorescence microscopy technique for optical sectioning was investigated. This technique combined Spinning Disk microscopy (SD) with Structured Illumination Microscopy (SIM), resulting in more background removal than either method. Spinning Disk Structured Illumination Microscopy (SD-SIM) resulted in higher signal-to-background ratios. The method detected and quantified a dendritic spine neck that was impossible to detect with either SIM or SD alone.

The amount of Nck rather than N-WASP correlates with the rate of actin-based motility of Vaccinia virus.

Vaccinia virus is a large double-stranded DNA virus and a close relative of Mpox and Variola virus, the causative agent of smallpox. During infection, Vaccinia hijacks its host's transport systems and promotes its spread into neighboring cells by recruiting a signaling network that stimulates actin polymerization. Over the years, Vaccinia has provided a powerful model to understand how signaling networks regulate actin polymerization. Nevertheless, we still lack important quantitative information about the system, including the precise number of viral and host molecules required to induce actin polymerization. Using quantitative fluorescence microscopy techniques, we have determined the number of viral and host signaling proteins accumulating on virions during their egress. Our analysis has uncovered two unexpected new aspects of this process: the number of viral proteins in the virion is not fixed and the velocity of virus movement depends on the level of a single adaptor within the signaling network.