Airyscan super‐resolution microscopy of mitochondrial morphology and dynamics in living tumor cells

Kolossov, Vladimir L.; Sivaguru, Mayandi; Huff, Joseph; Luby, Katherine; Kanakaraju, Kaviamuthan; Gaskins, H. Rex

doi:10.1002/jemt.22968

Cited by 26 publications

(23 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To shed light on the phenotype of the V67A and P145A mutations, we applied an imaging approach, using high resolution confocal microscopy (Airyscan) to assess the distribution of both viral and cellular factors during infection [ 65 , 66 ]. In this regard, lipid droplets (LD) are important organelles for the assembly of infectious HCV particles, although their precise role remains to be elucidated [ 44 ].…”

Section: Resultsmentioning

confidence: 99%

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

et al. 2018

View full text Add to dashboard Cite

The NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, we conducted a mutagenic study of 12 absolutely conserved and surface-exposed residues within the context of a JFH-1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. P35A exhibited a modest reduction in infectivity, however V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high resolution confocal microscopy we demonstrate that V67A and P145A disrupted the localisation of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed compared to wildtype HCV. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken together, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. Domain I also contributes to a change in lipid droplet morphology, increasing their size. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.

show abstract

Section: Resultsmentioning

confidence: 99%

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

et al. 2018

View full text Add to dashboard Cite

show abstract

“…Explanation (2) is in principle possible but there are no nanomechanical processes known yet that result in an oval local deformation of an MNT without the involvement of an object inside the MNT causing the deformation. Explanation (3) is most likely since (i) the presence of mitochondria inside MNT bulges was previously shown by several studies [28] [22]; (ii) the MNT bulge diameter values determined in our study (range: 68.0-440.0 nm) correspond to diameters of organelles like mitochondria (typical diameter: 100-1000 nm) [31] [32] [33], peroxisomes (typical diameter in RPE cells: 100-300 nm) [34], or exosomes (typical diameter: 30-100 nm) [35] [36]; (iii) the length of the MNT bulges overlap with the length distributions of mitochondria and peroxisomes [23] [34], and (iv) movements of MNT bulges were reported by other studies (speed: 0.16 µm/s [14], 20.7 ± 2.3 µm/h [37], 0.08 µm/s [19], 0.0259 ± 0.0079 µm/s [38], 0.033-0.059 µm/s [39], 0.045 ± 0.005 µm/s [21]) indicating the presence of a real object inside the MNT.…”

Section: Resultsmentioning

confidence: 54%

Ultrastructural features (bulges) of membrane nanotubes between cone-like photoreceptor cells: An investigation employing scanning electron microscopy

Scholkmann¹,

Seidel²,

Funk³

et al. 2018

Matters

View full text Add to dashboard Cite

Membrane nanotubes (MNTs) are nanotubular cell-to-cell connections enabling cell-tocell signaling and cargo transfer. The presence of local MNT bulges has been reported by several studies. However, a detailed characterization of MNT bulges concerning their geometrical properties has not been done yet. The aim of our study was to analyze MNT from cone-like photoreceptor cells (661 W) using scanning electron microscopy (SEM) in order to characterize MNT bulges, thereby increasing the knowledge of ultrastructural feature of MNTs. Our SEM analysis of two MNTs with multiple bulges revealed that (i) MNT bulges are characterized by a statistically significant local increase in the MNT diameter (125% and 250% for both MNTs analyzed), (ii) the thickness and length of the MNT bulges correspond to dimensions of mitochondria, peroxisomes and exosomes, and (iii) the MNT bulges seem not to be randomly distributed on the MNTs but exhibit a preferred spacing with a different median value for each MNT. Our findings highlight that the ultrastructure of MNT exhibits interesting properties that need to be further investigated.

show abstract

“…SIM has a good imaging speed, can use any labels used in conventional live-cell imaging, and has tolerable light toxicity, similar to conventional live-cell imaging. Airyscan is also widely used for live-cell imaging (Kolossov et al, 2018;Mishra et al, 2019;Scipioni et al, 2018) and here has similar properties compared to SIM. The limitation of both SIM and Airyscan is that the improvement in resolution compared to confocal imaging is small (less than a factor of two).…”

Section: Super-resolution Microscopymentioning

confidence: 99%

Imaging of the immune system – towards a subcellular and molecular understanding

Wen

Fan

Mikulski

et al. 2020

Journal of Cell Science

View full text Add to dashboard Cite

Immune responses involve many types of leukocytes that traffic to the site of injury, recognize the insult and respond appropriately. Imaging of the immune system involves a set of methods and analytical tools that are used to visualize immune responses at the cellular and molecular level as they occur in real time. We will review recent and emerging technological advances in optical imaging, and their application to understanding the molecular and cellular responses of neutrophils, macrophages and lymphocytes. Optical live-cell imaging provides deep mechanistic insights at the molecular, cellular, tissue and organism levels. Live-cell imaging can capture quantitative information in real time at subcellular resolution with minimal phototoxicity and repeatedly in the same living cells or in accessible tissues of the living organism. Advanced FRET probes allow tracking signaling events in live cells. Light-sheet microscopy allows for deeper tissue penetration in optically clear samples, enriching our understanding of the higher-level organization of the immune response. Super-resolution microscopy offers insights into compartmentalized signaling at a resolution beyond the diffraction limit, approaching single-molecule resolution. This Review provides a current perspective on live-cell imaging in vitro and in vivo with a focus on the assessment of the immune system.

show abstract

Airyscan super‐resolution microscopy of mitochondrial morphology and dynamics in living tumor cells

Cited by 26 publications

References 32 publications

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

A role for domain I of the hepatitis C virus NS5A protein in virus assembly

Ultrastructural features (bulges) of membrane nanotubes between cone-like photoreceptor cells: An investigation employing scanning electron microscopy

Imaging of the immune system – towards a subcellular and molecular understanding

Contact Info

Product

Resources

About