Limits...
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.

Show MeSH

Related in: MedlinePlus

Alteration in actin cytoskeleton.The actin cytoskeleton change was determined by staining with alex488 conjugated phalloidin (green). Phalloidin binds to F-actin. The nucleus and parasite was stained with DAPI (blue) and visualized by confocal microscopy. A) N-MΦ, B) I-MΦ, and C) I-MΦ-CL.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256160&req=5

pntd-0003367-g006: Alteration in actin cytoskeleton.The actin cytoskeleton change was determined by staining with alex488 conjugated phalloidin (green). Phalloidin binds to F-actin. The nucleus and parasite was stained with DAPI (blue) and visualized by confocal microscopy. A) N-MΦ, B) I-MΦ, and C) I-MΦ-CL.

Mentions: We studied the actin cytoskeleton in N-MΦ, I-MΦ and I-MΦ-CL. The integrity of the cytoskeleton protein actin was measured by confocal microscopy after staining with fluorescence label phalloidin, which binds to filamentous actin (F-actin). The actin filamentare clearly visible in N-MΦ (Fig. 6A). However, in I-MΦactin filaments were reduced; insteadpossible actin depolymerisationwas noted (Fig. 6B). Interestingly, I-MΦ-CLdid display actin filaments (Fig. 6C). The computational analysis of phalloidin staining showed about 25% decrease in F-actin in I-MΦ and 9% increase in I-MΦ-CL as compared to N-MΦ. We next quantified F-actin by western blot and the result was expressed with respect to total actin. It was observed that the total actin level was similar in N-MΦ, I-MΦ and I-MΦ-CL (S5B Figure). Though F-actin level was reduced in I-MΦ, it reappeared upon liposomal cholesterol treatment (S5A Figure). There is about 30% decrease of F-actin in I-MΦ as compared to N-MΦ and in I-MΦ-CL F-actin was comparable (S5C 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)

Alteration in actin cytoskeleton.The actin cytoskeleton change was determined by staining with alex488 conjugated phalloidin (green). Phalloidin binds to F-actin. The nucleus and parasite was stained with DAPI (blue) and visualized by confocal microscopy. A) N-MΦ, B) I-MΦ, and C) I-MΦ-CL.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003367-g006: Alteration in actin cytoskeleton.The actin cytoskeleton change was determined by staining with alex488 conjugated phalloidin (green). Phalloidin binds to F-actin. The nucleus and parasite was stained with DAPI (blue) and visualized by confocal microscopy. A) N-MΦ, B) I-MΦ, and C) I-MΦ-CL.
Mentions: We studied the actin cytoskeleton in N-MΦ, I-MΦ and I-MΦ-CL. The integrity of the cytoskeleton protein actin was measured by confocal microscopy after staining with fluorescence label phalloidin, which binds to filamentous actin (F-actin). The actin filamentare clearly visible in N-MΦ (Fig. 6A). However, in I-MΦactin filaments were reduced; insteadpossible actin depolymerisationwas noted (Fig. 6B). Interestingly, I-MΦ-CLdid display actin filaments (Fig. 6C). The computational analysis of phalloidin staining showed about 25% decrease in F-actin in I-MΦ and 9% increase in I-MΦ-CL as compared to N-MΦ. We next quantified F-actin by western blot and the result was expressed with respect to total actin. It was observed that the total actin level was similar in N-MΦ, I-MΦ and I-MΦ-CL (S5B Figure). Though F-actin level was reduced in I-MΦ, it reappeared upon liposomal cholesterol treatment (S5A Figure). There is about 30% decrease of F-actin in I-MΦ as compared to N-MΦ and in I-MΦ-CL F-actin was comparable (S5C 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