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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 LDs are particle tracked using simultaneous TPF and DIC microscopy.This is a representative image of DII-corewt expressed in Huh-7 cells. Three individual LDs with dissimilar environments were selected (A–C, white arrows), and their trajectories were measured to calculate the overall distances traveled. (D) A larger DIC image of (B) includes boxes to identify each LD trajectory (inset 1–3). The value above each box (D) indicates their overall travel distances for (1) DII-corewt coated LD, (2) non DII-corewt coated LD within the same cell, (3) and a LD in an adjacent cell not expressing DII-corewt. Each LD trajectory is magnified to demonstrate the LD track with selective freeze frame time-intervals representing the LD position at their indicated times. Due to frequent bidirectional movements, the displayed trajectories represent a general movement path, and does not portray total distance. All of the LDs are tracked according to the same start and end time. All scale bars represent 10 µm.
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pone-0078065-g002: DII-corewt coated LDs are particle tracked using simultaneous TPF and DIC microscopy.This is a representative image of DII-corewt expressed in Huh-7 cells. Three individual LDs with dissimilar environments were selected (A–C, white arrows), and their trajectories were measured to calculate the overall distances traveled. (D) A larger DIC image of (B) includes boxes to identify each LD trajectory (inset 1–3). The value above each box (D) indicates their overall travel distances for (1) DII-corewt coated LD, (2) non DII-corewt coated LD within the same cell, (3) and a LD in an adjacent cell not expressing DII-corewt. Each LD trajectory is magnified to demonstrate the LD track with selective freeze frame time-intervals representing the LD position at their indicated times. Due to frequent bidirectional movements, the displayed trajectories represent a general movement path, and does not portray total distance. All of the LDs are tracked according to the same start and end time. All scale bars represent 10 µm.

Mentions: Since DII-corewt behaves similarly to naïve full-length core protein [45], we assessed whether the interaction between DII-corewt and LDs affected LD mobility. DII-corewt expressing cells contain populations of naïve and DII-corewt-bound LDs. By simultaneous TPF and differential interference contrast (DIC) imaging, the trajectories of LDs from both populations can be tracked by following LD movements with and without overlap of the DII-corewt GFP tag. It is important to note that LD mobility may potentially be affected by factors including cell passage number, biological replicate and cell confluency. To circumvent this, in every experiment that was conducted, the LD measurements acquired from Huh-7 cells expressing DII-corewt were directly compared with LD measurements from a mock sample of the same biological replicate. We have previously shown that LDs in full-length core expressing cells were motile, but travel at half the speeds by comparison to mock LDs [17]. With GFP-tagged DII-corewt expressing cells, we observed a similar pattern, and showed that DII-corewt coated LDs traveled with an average speed of approximately 40.3 nm/sec compared to LDs in mock-treated Huh-7 cells, which traveled at 67.2 nm/sec (Table 1). To compare these values, we divided the average speeds of DII-corewt coated LDs by LDs in mock cells and observed a ratio of 0.60. To illustrate these changes more clearly, a representative image was captured from a time-course movie (Figure 2A–C, arrowheads) that tracked spatially unique LDs under different expression conditions within the same field of view. For example, the general trajectories of LD mobility for individual DII-corewt coated and non-coated LDs in the same cell are illustrated (Figure 2D, box 1 vs box 2, inset 1 vs inset 2). As expected, non-coated LDs travelled a longer distance. Additionally, LDs in an adjacent non-expressing cell traveled further than LDs that are bound to DII-corewt (Figure 2D, box 3 and inset 3). Furthermore, the presence of HCV non-structural proteins, which are recruited to LDs during the viral lifecycle and are required to form the membranous web [54], do not affect the ability of DII-corewt to induce changes in LD speeds and travel distances (Figure S1).


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 LDs are particle tracked using simultaneous TPF and DIC microscopy.This is a representative image of DII-corewt expressed in Huh-7 cells. Three individual LDs with dissimilar environments were selected (A–C, white arrows), and their trajectories were measured to calculate the overall distances traveled. (D) A larger DIC image of (B) includes boxes to identify each LD trajectory (inset 1–3). The value above each box (D) indicates their overall travel distances for (1) DII-corewt coated LD, (2) non DII-corewt coated LD within the same cell, (3) and a LD in an adjacent cell not expressing DII-corewt. Each LD trajectory is magnified to demonstrate the LD track with selective freeze frame time-intervals representing the LD position at their indicated times. Due to frequent bidirectional movements, the displayed trajectories represent a general movement path, and does not portray total distance. All of the LDs are tracked according to the same start and end time. All scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815211&req=5

pone-0078065-g002: DII-corewt coated LDs are particle tracked using simultaneous TPF and DIC microscopy.This is a representative image of DII-corewt expressed in Huh-7 cells. Three individual LDs with dissimilar environments were selected (A–C, white arrows), and their trajectories were measured to calculate the overall distances traveled. (D) A larger DIC image of (B) includes boxes to identify each LD trajectory (inset 1–3). The value above each box (D) indicates their overall travel distances for (1) DII-corewt coated LD, (2) non DII-corewt coated LD within the same cell, (3) and a LD in an adjacent cell not expressing DII-corewt. Each LD trajectory is magnified to demonstrate the LD track with selective freeze frame time-intervals representing the LD position at their indicated times. Due to frequent bidirectional movements, the displayed trajectories represent a general movement path, and does not portray total distance. All of the LDs are tracked according to the same start and end time. All scale bars represent 10 µm.
Mentions: Since DII-corewt behaves similarly to naïve full-length core protein [45], we assessed whether the interaction between DII-corewt and LDs affected LD mobility. DII-corewt expressing cells contain populations of naïve and DII-corewt-bound LDs. By simultaneous TPF and differential interference contrast (DIC) imaging, the trajectories of LDs from both populations can be tracked by following LD movements with and without overlap of the DII-corewt GFP tag. It is important to note that LD mobility may potentially be affected by factors including cell passage number, biological replicate and cell confluency. To circumvent this, in every experiment that was conducted, the LD measurements acquired from Huh-7 cells expressing DII-corewt were directly compared with LD measurements from a mock sample of the same biological replicate. We have previously shown that LDs in full-length core expressing cells were motile, but travel at half the speeds by comparison to mock LDs [17]. With GFP-tagged DII-corewt expressing cells, we observed a similar pattern, and showed that DII-corewt coated LDs traveled with an average speed of approximately 40.3 nm/sec compared to LDs in mock-treated Huh-7 cells, which traveled at 67.2 nm/sec (Table 1). To compare these values, we divided the average speeds of DII-corewt coated LDs by LDs in mock cells and observed a ratio of 0.60. To illustrate these changes more clearly, a representative image was captured from a time-course movie (Figure 2A–C, arrowheads) that tracked spatially unique LDs under different expression conditions within the same field of view. For example, the general trajectories of LD mobility for individual DII-corewt coated and non-coated LDs in the same cell are illustrated (Figure 2D, box 1 vs box 2, inset 1 vs inset 2). As expected, non-coated LDs travelled a longer distance. Additionally, LDs in an adjacent non-expressing cell traveled further than LDs that are bound to DII-corewt (Figure 2D, box 3 and inset 3). Furthermore, the presence of HCV non-structural proteins, which are recruited to LDs during the viral lifecycle and are required to form the membranous web [54], do not affect the ability of DII-corewt to induce changes in LD speeds and travel distances (Figure S1).

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