Kalfa Lab

  • Rac GTPases and Red Blood Cell Cytoskeleton

    Many of the roles of Rac1 and Rac2 GTPases are mediated through regulation of actin organization. Using a mouse gene targeting approach, we demonstrated that the concurrent deficiency of Rac1 and Rac2 GTPases in RBCs causes profound disruption of actin assembly and abnormal deformability of the erythrocyte cytoskeleton.  Rac1-/-;Rac2-/- mice develop microcytic hemolytic anemia with poikilocytosis and RBC fragmentation.   

    This is associated with increased phosphorylation of adducin at Ser-724, a domain target of protein kinase C (PKC) (Kalfa et al. Blood. 108(12):3637-3645. 2006) These findings point to the exciting possibility that the erythrocyte cytoskeleton is a dynamic assembly regulated by signaling molecules, which actively participate in a continuous repair of the cytoskeleton under flow conditions, and assure normal survival of the erythrocytes in the circulation 

 
  • Smear

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    Smear
    Smear of Rac1-/-;Rac2-/- RBCs stained with Wright-Giemsa. 
  • 3D reconstitution

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    3D reconstitution
    3-D reconstitution of confocal microscopy image of fluorescently labeled Rac1-/-;Rac2-/- RBCs.
  • Cytoskeleton structure

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    Cytoskeleton structure

    Cytoskeleton structure of Rac1-/-;Rac2-/-erythrocytes. (A) Erythrocytes fixed with acrolein and stained with rhodamine-phalloidin for F-actin. Arrowheads in magnified inset show meshwork gaps and aggregates of F-actin in Rac1-/-;Rac2-/- erythrocytes. (B) RBCs stained with anti-band 3 antibody to visualize band 3 distribution.  Images were obtained with an x63 oil-immersed objective lens, software zoomx2, numerical aperture 1.4; one unit represents 7.3 µm.

    See the related article:
    Kalfa TA, et al. Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen. Blood. 108(12):3637-3645. 2006.