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Cyclase-associated protein (CAP)2 is an evolutionarily highly conserved actin-binding protein implicated in striated muscle development, carcinogenesis, and wound healing in mammals. To date, the presence as well as the putative role(s) of CAP2 in platelets, however, remain unknown. Therefore, mice constitutively lacking CAP2 (Cap2gt/gt mice) were examined for platelet function. These studies confirmed the presence of both mammalian CAP isoforms, CAP1 and CAP2, in platelets. CAP2-deficient platelets were slightly larger than WT controls and displayed increased GPIIbIIIa activation and P-selectin recruitment in response to the (hem)ITAM-specific agonists collagen-related peptide and rhodocytin. However, spreading of CAP2-deficient platelets on a fibrinogen matrix was unaltered. In conclusion, the functionally redundant CAP1 isoform may compensate for the lack of CAP2 in murine platelets. Moreover, the studies presented in this thesis unveiled a severe macrothrombocytopenia that occurred independently of the targeted Cap2 allele and which was preliminarily termed orphan (orph). Crossing of the respective mice to C57BL/6J wild-type animals revealed an autosomal recessive inheritance. Orph mice were anemic and developed splenomegaly as well as BM fibrosis, suggesting a general hematopoietic defect. Strikingly, BM MKs of orph mice demonstrated an aberrant morphology and appeared to release platelets ectopically into the BM cavity, thus pointing to defective thrombopoiesis as cause for the low platelet counts. Orph platelets exhibited marked activation defects and spread poorly on fibrinogen. The unaltered protein content strongly suggested a defective alpha-granule release to account for the observed hyporesponsiveness. In addition, the cytoskeleton of orph platelets was characterized by disorganized microtubules and accumulations of filamentous actin. However, further experiments are required to elucidate the activation defects and cytoskeletal abnormalities in orph platelets. Above all, the gene mutation responsible for the phenotype of orph mice needs to be determined by next-generation sequencing in order to shed light on the underlying genetic and mechanistic cause.
The foot processes of podocytes exhibit a dynamic actin cytoskeleton, which maintains their complex cell structure and antagonizes the elastic forces of the glomerular capillary. Interdigitating secondary foot processes form a highly selective filter for proteins in the kidney, the slit membrane. Knockdown of slit membrane components such as Nephrin or Neph1 and cytoskeletal adaptor proteins such as CD2AP in mice leads to breakdown of the filtration barrier with foot process effacement, proteinuria, and early death of the mice. Less is known about the crosstalk between the slit membrane‐associated proteins and cytoskeletal components inside the podocyte foot processes. Our study shows that LASP‐1, an actin‐binding protein, is highly expressed in podocytes. Electron microscopy studies demonstrate that LASP‐1 is found at the slit membrane suggesting a role in anchoring slit membrane components to the actin cytoskeleton. Live cell imaging experiments with transfected podocytes reveal that LASP‐1 is either part of a highly dynamic granular complex or a static, actin cytoskeleton‐bound protein. We identify CD2AP as a novel LASP‐1 binding partner that regulates its association with the actin cytoskeleton. Activation of the renin‐angiotensin‐aldosterone system, which is crucial for podocyte function, leads to phosphorylation and altered localization of LASP‐1. In vivo studies using the Drosophila nephrocyte model indicate that Lasp is necessary for the slit membrane integrity and functional filtration.