Localization of the LRBA protein in the endomembrane system of HELA cells and primary mononuclear phagocytes
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1
Universidad de Antioquia, UdeA, Grupo de Inmunodeficiencias Primarias, Colombia
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2
Universidad de Antioquia, UdeA, Grupo de Genética y Regeneración del Cáncer, Colombia
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3
Universidad de Antioquia, UdeA, Grupo de Inmunología Celular e Inmunogenética, Colombia
Humoral deficiencies are the most common symptomatic disorders of the immune system, however, the genetic defects underlying these conditions remain unknown in more than 95% of the cases. Recently, five different studies described several autosomal recessive LRBA (encoding the lipopolysaccharide responsive beige-like anchor protein) mutations in patients with hypogammaglobulinemia, immune dysregulation and/or gastropathy. Although previous studies have demonstrated the association of the human LRBA BEACH-WD domain with endoplasmic reticulum, Golgi complex, some lysosomes, and clathrin-coated vesicles, the function of this protein is still unknown.
The present study was aimed to investigate whether LRBA co-localizes with molecules involved in cellular trafficking. The expression of LAMP1, CD63, EEA1, Rab5, Rab7, and LRBA were evaluated in HeLa cells and mononuclear phagocytes by confocal laser scanning microscopy. Transferrin and LysoTracker were also included as additional endosome and lysosome markers, respectively. The co-localization analysis was performed using the ImageJ software through of the calculation of Pearson’s coefficients and Mander’s thresholds.
We observed the co-localization of LRBA with proteins implicated in vesicular trafficking, in both HeLa cells and mononuclear phagocytes. LRBA co-localized at a greater extend (>60%) with the early endosome markers EEA1 and Rab5, and with the late endosome molecule Rab7 than with Lysosomes markers CD63 LAMP1, LysoTracker and the iron transport molecule Transferrin (15-47%) in HeLa cells. In addition, there were higher percentages of co-localization of LRBA with endocytic vesicles in mononuclear phagocytes than in HeLa cells. Namely >72,8% of co-localization was for EEA1, Rab5, Rab7, and 41-67.9% was for LAMP1, CD63, LysoTracker and Transferrin in mononuclear phagocytes. No significant differences were observed in the percentages of co-localization among unstimulated and LPS-stimulated HeLa cells, with exception of transferrin that exhibited less frequency of co-localizaton with LRBA after LPS exposure. In mononuclear phagocytes w/o LPS, we observed similar results in co-localization percentages, only Rab5/LRBA presented minor co-localization after LPS -stimulation. Interestingly, LRBA seems to exhibit a wider distribution pattern in the cytosol that the other molecules evaluated in this study, both in mononuclear phagocytes and HeLa cells. On the other hand, the co-localization patterns of LRBA with the other molecules evaluated in both HeLa cells and mononuclear phagocytes, were found to be similar but very heterogeneous: the co-localization patterns of LRBA with LAMP1, CD63, LysoTracker and Transferrin was perinuclear whereas that with EEA1, Rab5 and Rab7 was observed throughout the cytosol without differences, independent of the LPS exposure.
Taking together, LRBA co-localization was preferentially with molecules contained in early and late endosomes, and in a less degree with lysosomes. Despite of the heterogeneity, mononuclear phagocytes seems to be suitable as primary cells to study of intracellular localization of LRBA in immune cells. However, more studies are necessary to determine more precisely the LRBA interacting partners and establish its function in immune homeostasis.
Acknowledgements
COLCIENCIAS 1115-569-34430 .
Universidad de Antioquia.
Dr. Bodo Grimbacher and Laura Gamez for their advices
Keywords:
LRBA,
Intracellular Vesicular Traffic,
HeLa Cells,
Mononuclear Phagocytes,
confocal laser scanning microscopy (CLSM)
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Immunodeficiencies
Citation:
Martínez-Jaramillo
C,
Gutierrez-Hincapie
S,
Delgado
JP,
Castano
D and
Trujillo-Vargas
CM
(2015). Localization of the LRBA protein in the endomembrane system of HELA cells and primary mononuclear phagocytes.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00125
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Received:
29 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
Miss. Catalina Martínez-Jaramillo, Universidad de Antioquia, UdeA, Grupo de Inmunodeficiencias Primarias, Medellin, Antioquia, Colombia, catalina.martinez@udea.edu.co