Critical redundant functions of the adapters Grb2 and Gads in platelet (hem)ITAM signaling in mice.

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets - Grb2 and Grb2-related adapter protein downstream of Shc (Gads) - are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development.

Stage-specific testes proteomics of Drosophila melanogaster identifies essential proteins for male fertility.

Spermiogenesis in Drosophila melanogaster is a highly conserved process and essential for male fertility. In this haploid phase of spermatogenesis, motile sperm are assembled from round cells, and flagella and needle-shaped nuclei with highly compacted genomes are formed. As transcription takes place mainly in spermatocytes and transcripts relevant for post-meiotic sperm development are translationally repressed for days, we comparatively analysed the proteome of larval testes (only germ cell stages before meiotic divisions), testes of 1-2-day-old pupae (germ cell stages before meiotic divisions, meiotic and early spermatid stages) and adult flies (germ cell stages before meiotic divisions, meiotic and early spermatid stages, late spermatids and sperm). We identified 6,171 proteins; 61 proteins were detected solely in one stage and are thus enriched, namely 34 in larval testes, 77 in pupal testes and 214 in adult testes. To substantiate our mass spectrometric data, we analysed the stage-specific synthesis and importance for male fertility of a number of uncharacterized proteins. For example, Mst84B (gene CG1988), a very basic cysteine- and lysine-rich nuclear protein and was present in the transition phase from a histone-based to a protamine-based chromatin structure. CG6332 encodes d-Theg, which is related to the mouse tHEG and human THEG proteins. Mutants of d-Theg were sterile due to the lack of sperm in the seminal vesicles. Our catalogue of proteins of the different stages of testis development in D. melanogaster will pave the road for future analyses of spermatogenesis.