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Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

Lyn RK, Hope G, Sherratt AR, McLauchlan J, Pezacki JP - PLoS ONE (2013)

Bottom Line: Expression of core protein's lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules.Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core.These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Canada, Ottawa, Ontario, Canada ; Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada.

ABSTRACT
Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein's lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.

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DII-corewt coated LD velocities measured in naïve Huh-7 cells and Huh-7 cells stably expressing an HCV subgenomic replicon.(A–B) Average representative measurement of a much larger data set, LD velocities in retrograde or anterograde directed transport are measured in Huh-7 cells expressing (A) DII-corewt and (B) mock transfected. The velocity amplitudes at each time point are divided into parameters of, low, medium, and high velocities for both directions. The pink parameter line is indicated by a paused event, which was determined by obtaining the average speed of LDs from nocadazole treated Huh-7 cells. (C–D) The frequency of low (15.7 nm/sec –50 nm/sec), medium (50.1 nm/sec –180 nm/sec), and high velocity (>180.1 nm/sec) measurements, expressed as a percentage, in both directions, are plotted after particle tracking LDs in DII-corewt expressing (C) Huh-7 cells, and (D) Huh-7 cells harbouring an HCV subgenomic replicon. The velocities are measured for DII-corewt coated LDs in DII-corewt expressing cells, and LDs from mock cells not expressing DII-corewt. The ratios above each set of columns are calculated by dividing the frequency for each velocity interval of DII-corewt coated LDs by their respective mock LDs.
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pone-0078065-g004: DII-corewt coated LD velocities measured in naïve Huh-7 cells and Huh-7 cells stably expressing an HCV subgenomic replicon.(A–B) Average representative measurement of a much larger data set, LD velocities in retrograde or anterograde directed transport are measured in Huh-7 cells expressing (A) DII-corewt and (B) mock transfected. The velocity amplitudes at each time point are divided into parameters of, low, medium, and high velocities for both directions. The pink parameter line is indicated by a paused event, which was determined by obtaining the average speed of LDs from nocadazole treated Huh-7 cells. (C–D) The frequency of low (15.7 nm/sec –50 nm/sec), medium (50.1 nm/sec –180 nm/sec), and high velocity (>180.1 nm/sec) measurements, expressed as a percentage, in both directions, are plotted after particle tracking LDs in DII-corewt expressing (C) Huh-7 cells, and (D) Huh-7 cells harbouring an HCV subgenomic replicon. The velocities are measured for DII-corewt coated LDs in DII-corewt expressing cells, and LDs from mock cells not expressing DII-corewt. The ratios above each set of columns are calculated by dividing the frequency for each velocity interval of DII-corewt coated LDs by their respective mock LDs.

Mentions: To investigate DII-core’s induced suppression of the mean LD speed, we explored the frequency of low to high instantaneous velocities of DII-corewt coated LDs compared to LDs in mock cells. Since dynein and kinesin motors mediate cargo transport in opposite directions, measured LD velocities can provide information about whether the mobility of DII-corewt coated LDs travel more frequently towards one direction, and thus, reveal differential activity between the two motors. The trajectories of individual LDs were tracked using the center of the nucleus as a fixed point relative to the position of the LD. LD travel runs that were directed towards the MTOC (retrograde manner) were identified as negative displacement, while LDs that moved away from the MTOC (anterograde motion), were identified as having positive displacement (Figure 4A–B). The differential velocity profiles were then segregated into low (15.7–50 nm/sec), medium (50–180 nm/sec), and high velocity (>180 nm/sec) travel runs (Figure 4C–D). LD particle tracking in both directions revealed that the frequency of high and medium velocity travel runs for DII-corewt coated LDs was lower when compared to LDs from the mock sample (Figure 4C). This is represented as a ratio for the frequency for DII-corewt coated LDs divided by LDs from the mock, with similar ratios determined for both directions. For example, at high velocity travel runs, the ratios were calculated to be 0.47 for the anterograde direction, and 0.48 for the retrograde direction (Figure 4C). The differential frequencies for the high and medium velocities were also consistent with DII-corewt coated LDs in Huh-7 cells expressing a subgenomic replicon of HCV (Figure 4D). Therefore, the shorter travel distances of DII-corewt coated LDs is reflected in the lower frequency of high velocity travel runs, and is independent of the presence of non-structural HCV proteins that are involved in membranous web formation and viral replication.


Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

Lyn RK, Hope G, Sherratt AR, McLauchlan J, Pezacki JP - PLoS ONE (2013)

DII-corewt coated LD velocities measured in naïve Huh-7 cells and Huh-7 cells stably expressing an HCV subgenomic replicon.(A–B) Average representative measurement of a much larger data set, LD velocities in retrograde or anterograde directed transport are measured in Huh-7 cells expressing (A) DII-corewt and (B) mock transfected. The velocity amplitudes at each time point are divided into parameters of, low, medium, and high velocities for both directions. The pink parameter line is indicated by a paused event, which was determined by obtaining the average speed of LDs from nocadazole treated Huh-7 cells. (C–D) The frequency of low (15.7 nm/sec –50 nm/sec), medium (50.1 nm/sec –180 nm/sec), and high velocity (>180.1 nm/sec) measurements, expressed as a percentage, in both directions, are plotted after particle tracking LDs in DII-corewt expressing (C) Huh-7 cells, and (D) Huh-7 cells harbouring an HCV subgenomic replicon. The velocities are measured for DII-corewt coated LDs in DII-corewt expressing cells, and LDs from mock cells not expressing DII-corewt. The ratios above each set of columns are calculated by dividing the frequency for each velocity interval of DII-corewt coated LDs by their respective mock LDs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815211&req=5

pone-0078065-g004: DII-corewt coated LD velocities measured in naïve Huh-7 cells and Huh-7 cells stably expressing an HCV subgenomic replicon.(A–B) Average representative measurement of a much larger data set, LD velocities in retrograde or anterograde directed transport are measured in Huh-7 cells expressing (A) DII-corewt and (B) mock transfected. The velocity amplitudes at each time point are divided into parameters of, low, medium, and high velocities for both directions. The pink parameter line is indicated by a paused event, which was determined by obtaining the average speed of LDs from nocadazole treated Huh-7 cells. (C–D) The frequency of low (15.7 nm/sec –50 nm/sec), medium (50.1 nm/sec –180 nm/sec), and high velocity (>180.1 nm/sec) measurements, expressed as a percentage, in both directions, are plotted after particle tracking LDs in DII-corewt expressing (C) Huh-7 cells, and (D) Huh-7 cells harbouring an HCV subgenomic replicon. The velocities are measured for DII-corewt coated LDs in DII-corewt expressing cells, and LDs from mock cells not expressing DII-corewt. The ratios above each set of columns are calculated by dividing the frequency for each velocity interval of DII-corewt coated LDs by their respective mock LDs.
Mentions: To investigate DII-core’s induced suppression of the mean LD speed, we explored the frequency of low to high instantaneous velocities of DII-corewt coated LDs compared to LDs in mock cells. Since dynein and kinesin motors mediate cargo transport in opposite directions, measured LD velocities can provide information about whether the mobility of DII-corewt coated LDs travel more frequently towards one direction, and thus, reveal differential activity between the two motors. The trajectories of individual LDs were tracked using the center of the nucleus as a fixed point relative to the position of the LD. LD travel runs that were directed towards the MTOC (retrograde manner) were identified as negative displacement, while LDs that moved away from the MTOC (anterograde motion), were identified as having positive displacement (Figure 4A–B). The differential velocity profiles were then segregated into low (15.7–50 nm/sec), medium (50–180 nm/sec), and high velocity (>180 nm/sec) travel runs (Figure 4C–D). LD particle tracking in both directions revealed that the frequency of high and medium velocity travel runs for DII-corewt coated LDs was lower when compared to LDs from the mock sample (Figure 4C). This is represented as a ratio for the frequency for DII-corewt coated LDs divided by LDs from the mock, with similar ratios determined for both directions. For example, at high velocity travel runs, the ratios were calculated to be 0.47 for the anterograde direction, and 0.48 for the retrograde direction (Figure 4C). The differential frequencies for the high and medium velocities were also consistent with DII-corewt coated LDs in Huh-7 cells expressing a subgenomic replicon of HCV (Figure 4D). Therefore, the shorter travel distances of DII-corewt coated LDs is reflected in the lower frequency of high velocity travel runs, and is independent of the presence of non-structural HCV proteins that are involved in membranous web formation and viral replication.

Bottom Line: Expression of core protein's lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules.Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core.These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases.

View Article: PubMed Central - PubMed

Affiliation: National Research Council of Canada, Ottawa, Ontario, Canada ; Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada.

ABSTRACT
Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein's lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.

Show MeSH
Related in: MedlinePlus