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Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

Ghosh M, Roy K, Das Mukherjee D, Chakrabarti G, Roy Choudhury K, Roy S - PLoS Negl Trop Dis (2014)

Bottom Line: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit.To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages.Such defects may contribute to ineffective intracellular signaling and other cellular functions.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.

ABSTRACT

Background: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.

Methodology/principal findings: We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting.

Conclusions/significances: To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.

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Related in: MedlinePlus

Lateral mobility of PLCδ1.Fluorescence recovery after photo bleaching was measured in plcδ1-gfp transfected RAW 264.7 cells. The diffusion Coefficient (D) of PLCδ1-GFP for N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was measured using equation (1.3). The data represents average of 7 cells with ±SD. *** represents p<0.0005 and ‘ns’ means not significant.
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pntd-0003367-g005: Lateral mobility of PLCδ1.Fluorescence recovery after photo bleaching was measured in plcδ1-gfp transfected RAW 264.7 cells. The diffusion Coefficient (D) of PLCδ1-GFP for N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was measured using equation (1.3). The data represents average of 7 cells with ±SD. *** represents p<0.0005 and ‘ns’ means not significant.

Mentions: The lateral mobility of PLCδ1 wasstudied in live cells at 37°C.The cells were bleached and fluorescence recovery of the bleached region was measured up to 30 s (S3A Figure). It was observed that the extent of recovery was similar in N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL (S4 Figure) though the rate of recovery was different. The analysis of fluorescence recovery kinetics showed that the diffusion coefficient of PLCδ1 in N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was 1.4±0.2, 2.5±0.5, 1.6±0.3 and 2.6±0.5 µm2 s−1 respectively (Fig. 5). The photobleach effect was specific because in the unrelated region, mean fluorescence intensity was constant. To show that the recovery was predominantly from the plasma membrane, cytosolic protein was also bleached as a control. It was observed that the recovery of PLCδ1 was very poor almost not detectable (S3B Figure).


Leishmania donovani infection enhances lateral mobility of macrophage membrane protein which is reversed by liposomal cholesterol.

Ghosh M, Roy K, Das Mukherjee D, Chakrabarti G, Roy Choudhury K, Roy S - PLoS Negl Trop Dis (2014)

Lateral mobility of PLCδ1.Fluorescence recovery after photo bleaching was measured in plcδ1-gfp transfected RAW 264.7 cells. The diffusion Coefficient (D) of PLCδ1-GFP for N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was measured using equation (1.3). The data represents average of 7 cells with ±SD. *** represents p<0.0005 and ‘ns’ means not significant.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256160&req=5

pntd-0003367-g005: Lateral mobility of PLCδ1.Fluorescence recovery after photo bleaching was measured in plcδ1-gfp transfected RAW 264.7 cells. The diffusion Coefficient (D) of PLCδ1-GFP for N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was measured using equation (1.3). The data represents average of 7 cells with ±SD. *** represents p<0.0005 and ‘ns’ means not significant.
Mentions: The lateral mobility of PLCδ1 wasstudied in live cells at 37°C.The cells were bleached and fluorescence recovery of the bleached region was measured up to 30 s (S3A Figure). It was observed that the extent of recovery was similar in N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL (S4 Figure) though the rate of recovery was different. The analysis of fluorescence recovery kinetics showed that the diffusion coefficient of PLCδ1 in N-MΦ, I-MΦ, I-MΦ-CL and I-MΦ-AL was 1.4±0.2, 2.5±0.5, 1.6±0.3 and 2.6±0.5 µm2 s−1 respectively (Fig. 5). The photobleach effect was specific because in the unrelated region, mean fluorescence intensity was constant. To show that the recovery was predominantly from the plasma membrane, cytosolic protein was also bleached as a control. It was observed that the recovery of PLCδ1 was very poor almost not detectable (S3B Figure).

Bottom Line: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit.To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages.Such defects may contribute to ineffective intracellular signaling and other cellular functions.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.

ABSTRACT

Background: The protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.

Methodology/principal findings: We determined the lateral mobility of a membrane protein in normal, LD infected and liposome treated LD infected cells using GFP-tagged PLCδ1 as a probe. The mobility of PLCδ1 was computationally analyzed from the time lapse experiment using boundary distance plot and radial profile movement. Our results showed that the lateral mobility of the membrane protein, which is increased in infection, is restored to normal upon liposomal cholesterol treatment. The results of FRAP experiment lent further credence to the above notion. The membrane proteins are intimately linked with cellular actin and alteration of cellular actin may influence lateral mobility. We found that F-actin is decreased in infection but is restored to normal upon liposomal cholesterol treatment as evident from phalloidin staining and also from biochemical analysis by immunoblotting.

Conclusions/significances: To our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.

Show MeSH
Related in: MedlinePlus