Imaging the Motility of Inositol Trisphosphate Receptors in Intact Mammalian Cells using Single Particle Tracking Photoactivated Localization Microscopy (Sptpalm)

ABSTRACT The antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria. IMPORTANCE As so-called “superbug” bacteria resistant to commonly prescribed antibiotics have become a global threat to public health in recent years, noble metals, such as silver, in various forms have been attracting broad attention due to their antimicrobial activities. However, most of the studies in the existing literature have relied on the traditional bioassays for studying the antimicrobial mechanism of silver; in addition, temporal resolution is largely missing for understanding the effects of silver on the molecular dynamics inside bacteria. Here, we report our study of the antimicrobial effect of silver ions at the nanoscale on the diffusive dynamics of histone-like nucleoid-structuring (H-NS) proteins in live bacteria using single-particle-tracking photoactivated localization microscopy. This work highlights the importance of dynamic study of single proteins in live cells for understanding the functions of antimicrobial agents in bacteria.

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High-Speed Localization Microscopy and Single-Particle Tracking

Encyclopedia of Cell Biology ◽

10.1016/b978-0-12-394447-4.20096-5 ◽

2016 ◽

pp. 121-128

Author(s):

M. El Beheiry ◽

M. Dahan

Keyword(s):

Particle Tracking ◽

Single Particle ◽

High Speed ◽

Single Particle Tracking ◽

Localization Microscopy

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Single-particle tracking localization microscopy reveals nonaxonemal dynamics of intraflagellar transport proteins at the base of mammalian primary cilia

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0654 ◽

2019 ◽

Vol 30 (7) ◽

pp. 828-837 ◽

Cited By ~ 5

Author(s):

T. Tony Yang ◽

Minh Nguyet Thi Tran ◽

Weng Man Chong ◽

Chia-En Huang ◽

Jung-Chi Liao

Keyword(s):

Particle Tracking ◽

Single Particle ◽

Primary Cilia ◽

Protein Complexes ◽

Retinal Pigment ◽

Intraflagellar Transport ◽

Vital Role ◽

Single Particle Tracking ◽

Retinal Pigment Epithelial Cells ◽

Localization Microscopy

Primary cilia play a vital role in cellular sensing and signaling. An essential component of ciliogenesis is intraflagellar transport (IFT), which is involved in IFT protein recruitment, axonemal engagement of IFT protein complexes, and so on. The mechanistic understanding of these processes at the ciliary base was largely missing, because it is challenging to observe the motion of IFT proteins in this crowded region using conventional microscopy. Here, we report short-trajectory tracking of IFT proteins at the base of mammalian primary cilia by optimizing single-particle tracking photoactivated localization microscopy for IFT88-mEOS4b in live human retinal pigment epithelial cells. Intriguingly, we found that mobile IFT proteins “switched gears” multiple times from the distal appendages (DAPs) to the ciliary compartment (CC), moving slowly in the DAPs, relatively fast in the proximal transition zone (TZ), slowly again in the distal TZ, and then much faster in the CC. They could travel through the space between the DAPs and the axoneme without following DAP structures. We further revealed that BBS2 and IFT88 were highly populated at the distal TZ, a potential assembly site. Together, our live-cell single-particle tracking revealed region-dependent slowdown of IFT proteins at the ciliary base, shedding light on staged control of ciliary homeostasis.

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Single-particle tracking of Nucleotide Excision Repair proteins inside living bacteria

10.1101/293910 ◽

2018 ◽

Author(s):

Alicja Piotrowska ◽

Mathew Stracy ◽

Pawel Zawadzki

Keyword(s):

Particle Tracking ◽

Single Particle ◽

Excision Repair ◽

Ccd Camera ◽

Single Particle Tracking ◽

Point Of View ◽

Photoactivated Localization Microscopy ◽

Nucleotide Excision ◽

Multiple Frames

Single-particle tracking (SPT) combined with Photoactivated Localization Microscopy (sptPALM) provides an opportunity to perform complex molecular biology experiments inside living cells. By tracking the motion of DNA repair proteins in vivo, information can be extracted not only about their diffusion, but also about the kinetics and spatial distribution of DNA binding1–3. From a methodological point of view, a Total Internal Reflection Microscope equipped with a sensitive detector (usually an EM-CCD camera4) is commonly used, allowing detection of individual fluorophores. The signal from individual emitters can be analysed and the position of a given fluorophore established with high accuracy (up to a single nm) by Gaussian fitting. To determine the mobility of each fluorophore, the positions of individual molecules are linked into trajectories over multiple frames using a tracking algorithm5.

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Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM

10.1101/475939 ◽

2018 ◽

Cited By ~ 3

Author(s):

Elke De Zitter ◽

Daniel Thédié ◽

Viola Mönkemöller ◽

Siewert Hugelier ◽

Joël Beaudouin ◽

...

Keyword(s):

Particle Tracking ◽

Single Particle ◽

Fluorescent Proteins ◽

Single Particle Tracking ◽

Dark State ◽

Localization Microscopy ◽

Mechanistic Investigation ◽

Fluorescent State

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of significantly longer tracks in sptPALM with minimum effort.

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