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Caveolae, fenestrae and transendothelial channels retain PV1 on the surface of endothelial cells.

Tkachenko E, Tse D, Sideleva O, Deharvengt SJ, Luciano MR, Xu Y, McGarry CL, Chidlow J, Pilch PF, Sessa WC, Toomre DK, Stan RV - PLoS ONE (2012)

Bottom Line: We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level.Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys.The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, California, United States of America.

ABSTRACT
PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.

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Absence of caveolae in lung ECs does not affect transcription and translation levels of PV1.A) PV1 mRNA levels in MLEC-wt (WT) and MLEC-Cav1KO (Cav1KO) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35S metabolic labeling of MLEC-WT (top panel) and MLEC-Cav1KO (bottom panel) cells followed by PV1 immunoprecipitation at the indicated time points and 35S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).
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pone-0032655-g004: Absence of caveolae in lung ECs does not affect transcription and translation levels of PV1.A) PV1 mRNA levels in MLEC-wt (WT) and MLEC-Cav1KO (Cav1KO) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35S metabolic labeling of MLEC-WT (top panel) and MLEC-Cav1KO (bottom panel) cells followed by PV1 immunoprecipitation at the indicated time points and 35S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).

Mentions: Reduction in total PV1 protein levels in ECs lacking caveolae could be explained by defects in transcription or translation of PV1. We examined this possibility by determining the effect of Cav1 knockout on PV1 mRNA level and translation rate of PV1 protein in lung ECs isolated from wild type (MLEC-WT) and Cav1−/− (MLEC-Cav1KO) mice. The level of PV1 mRNA normalized to beta-actin mRNA was similar in MLEC-WT and MLEC-Cav1KO (Fig. 4A), in agreement with in vivo data (Fig. 1D). Thus, deletion of Cav1 does not affect PV1 mRNA level in ECs.


Caveolae, fenestrae and transendothelial channels retain PV1 on the surface of endothelial cells.

Tkachenko E, Tse D, Sideleva O, Deharvengt SJ, Luciano MR, Xu Y, McGarry CL, Chidlow J, Pilch PF, Sessa WC, Toomre DK, Stan RV - PLoS ONE (2012)

Absence of caveolae in lung ECs does not affect transcription and translation levels of PV1.A) PV1 mRNA levels in MLEC-wt (WT) and MLEC-Cav1KO (Cav1KO) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35S metabolic labeling of MLEC-WT (top panel) and MLEC-Cav1KO (bottom panel) cells followed by PV1 immunoprecipitation at the indicated time points and 35S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293851&req=5

pone-0032655-g004: Absence of caveolae in lung ECs does not affect transcription and translation levels of PV1.A) PV1 mRNA levels in MLEC-wt (WT) and MLEC-Cav1KO (Cav1KO) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse 35S metabolic labeling of MLEC-WT (top panel) and MLEC-Cav1KO (bottom panel) cells followed by PV1 immunoprecipitation at the indicated time points and 35S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with 35S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).
Mentions: Reduction in total PV1 protein levels in ECs lacking caveolae could be explained by defects in transcription or translation of PV1. We examined this possibility by determining the effect of Cav1 knockout on PV1 mRNA level and translation rate of PV1 protein in lung ECs isolated from wild type (MLEC-WT) and Cav1−/− (MLEC-Cav1KO) mice. The level of PV1 mRNA normalized to beta-actin mRNA was similar in MLEC-WT and MLEC-Cav1KO (Fig. 4A), in agreement with in vivo data (Fig. 1D). Thus, deletion of Cav1 does not affect PV1 mRNA level in ECs.

Bottom Line: We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level.Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys.The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Diego, California, United States of America.

ABSTRACT
PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.

Show MeSH
Related in: MedlinePlus