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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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Tracking LD mobility at distinct locations of the cell.While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-coreG161A is a representative image acquired from a large data set. Huh-7 cells expressing DII-coreG161A is shown as (A) a merged image of DIC and TPF, and (B) TPF. DII-coreG161A coated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C) LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-coreG161A coated LD for each of the segregated region, and the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the perinuclear region with higher levels of DII-coreG161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent 10 µm.
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pone-0078065-g005: Tracking LD mobility at distinct locations of the cell.While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-coreG161A is a representative image acquired from a large data set. Huh-7 cells expressing DII-coreG161A is shown as (A) a merged image of DIC and TPF, and (B) TPF. DII-coreG161A coated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C) LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-coreG161A coated LD for each of the segregated region, and the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the perinuclear region with higher levels of DII-coreG161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent 10 µm.

Mentions: We have previously used live-cell imaging by CARS and DIC microscopy to visualize the ability of full-length core protein to induce LD migration towards the perinuclear region associated with HCV replication and assembly [17]. Based on these data and published work by Boulant et al., it was suggested that core may directly or indirectly favor a molecular motor imbalance by perturbing the mechanics of one motor over the other [35]. Since expression of full-length and DII-core induces LD migration towards the perinuclear region, a molecular motor imbalance should drive a greater frequency of travel runs in the retrograde direction. For this reason, we counted the total frequency of travel runs for one direction that combined low, medium, and high velocity travel runs. However, the frequency of travel runs for wt and mutant DII-core coated LDs were similar in both directions over our four minute time course (Figure S3E). Finally, directionality of LD travel was assessed against cytoplasmic location, relative to the nucleus, since DII-core coated LDs were also observed to be scattered throughout the cell (Figure S4). Cells were divided into regions, as shown in Figure 5C, with regions identified as close to the perinuclear region (close), middle of the cytoplasm (mid), and in the cell periphery (far). However, a trend was not observed for wild-type and mutant DII-core coated LD velocities. This suggests that at time of analysis, movement of DII-core coated LDs travel equally in both directions and is unrelated to its location in the cell, except when the LDs reach the perinuclear region. Although our time measurements last approximately four minutes, we have included a large data set and statistics measured from all regions of the cell. Importantly, we wanted to measure the movement of LDs at a particular stage during core expression, before core induces LD accumulation in the perinuclear region. While it is difficult to normalize our data to 48–72 hours during the time span of infection, LD mobility measurements required video-rate imaging that is attainable over a shorter time course with averaging of many trials.


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)

Tracking LD mobility at distinct locations of the cell.While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-coreG161A is a representative image acquired from a large data set. Huh-7 cells expressing DII-coreG161A is shown as (A) a merged image of DIC and TPF, and (B) TPF. DII-coreG161A coated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C) LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-coreG161A coated LD for each of the segregated region, and the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the perinuclear region with higher levels of DII-coreG161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078065-g005: Tracking LD mobility at distinct locations of the cell.While all of the mutants were tracked accordingly, Huh-7 cells expressing DII-coreG161A is a representative image acquired from a large data set. Huh-7 cells expressing DII-coreG161A is shown as (A) a merged image of DIC and TPF, and (B) TPF. DII-coreG161A coated LDs are selected, and indicated by the arrows, to demonstrate fluorescence overlap between TPF and DIC. (C) LDs localized at different areas within the transfected cell (green outline) were segregated into regions relative to the center of the nucleus, such as close (orange shading), mid (blue), and far (no shading). Each black arrow represents a DII-coreG161A coated LD for each of the segregated region, and the velocities were measured for each direction in the close (D), mid (E), far (F) regions. The red arrow selects for a region of dense LDs in the perinuclear region with higher levels of DII-coreG161A. (G) The velocity of the LD, identified by the red arrow was measured. All scale bars represent 10 µm.
Mentions: We have previously used live-cell imaging by CARS and DIC microscopy to visualize the ability of full-length core protein to induce LD migration towards the perinuclear region associated with HCV replication and assembly [17]. Based on these data and published work by Boulant et al., it was suggested that core may directly or indirectly favor a molecular motor imbalance by perturbing the mechanics of one motor over the other [35]. Since expression of full-length and DII-core induces LD migration towards the perinuclear region, a molecular motor imbalance should drive a greater frequency of travel runs in the retrograde direction. For this reason, we counted the total frequency of travel runs for one direction that combined low, medium, and high velocity travel runs. However, the frequency of travel runs for wt and mutant DII-core coated LDs were similar in both directions over our four minute time course (Figure S3E). Finally, directionality of LD travel was assessed against cytoplasmic location, relative to the nucleus, since DII-core coated LDs were also observed to be scattered throughout the cell (Figure S4). Cells were divided into regions, as shown in Figure 5C, with regions identified as close to the perinuclear region (close), middle of the cytoplasm (mid), and in the cell periphery (far). However, a trend was not observed for wild-type and mutant DII-core coated LD velocities. This suggests that at time of analysis, movement of DII-core coated LDs travel equally in both directions and is unrelated to its location in the cell, except when the LDs reach the perinuclear region. Although our time measurements last approximately four minutes, we have included a large data set and statistics measured from all regions of the cell. Importantly, we wanted to measure the movement of LDs at a particular stage during core expression, before core induces LD accumulation in the perinuclear region. While it is difficult to normalize our data to 48–72 hours during the time span of infection, LD mobility measurements required video-rate imaging that is attainable over a shorter time course with averaging of many trials.

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