Integrin-mediated platelet adhesion and aggregation are essential for sealing injured blood vessels and preventing blood loss, and excessive platelet aggregation can initiate arterial thrombosis, causing heart attacks and stroke. To ensure that platelets aggregate only at injury sites, integrins on circulating platelets exist in a low-affinity state and shift to a high-affinity state (in a process known as integrin activation or priming) after contacting a wounded vessel. The shift is mediated through binding of the cytoskeletal protein Talin to the beta subunit cytoplasmic tail. Here we show that platelets lacking the adhesion plaque protein Kindlin-3 cannot activate integrins despite normal Talin expression. As a direct consequence, Kindlin-3 deficiency results in severe bleeding and resistance to arterial thrombosis. Mechanistically, Kindlin-3 can directly bind to regions of beta-integrin tails distinct from those of Talin and trigger integrin activation. We have therefore identified Kindlin-3 as a novel and essential element for platelet integrin activation in hemostasis and thrombosis.

Stable isotope labeling by amino acids in cell culture (SILAC) has become a versatile tool for quantitative, mass spectrometry (MS)-based proteomics. Here, we completely label mice with a diet containing either the natural or the (13)C(6)-substituted version of lysine. Mice were labeled over four generations with the heavy diet, and development, growth, and behavior were not affected. MS analysis of incorporation levels allowed for the determination of incorporation rates of proteins from blood cells and organs. The F2 generation was completely labeled in all organs tested. SILAC analysis from various organs lacking expression of beta1 integrin, beta-Parvin, or the integrin tail-binding protein Kindlin-3 confirmed their absence and disclosed a structural defect of the red blood cell membrane skeleton in Kindlin-3-deficient erythrocytes. The SILAC-mouse approach is a versatile tool by which to quantitatively compare proteomes from knockout mice and thereby determine protein functions under complex in vivo conditions.

Control of integrin activation is required for cell adhesion and ligand-induced signaling. Here we report that loss of the focal adhesion protein Kindlin-2 in mice results in peri-implantation lethality caused by severe detachment of the endoderm and epiblast from the basement membrane. We found that Kindlin-2-deficient cells were unable to activate their integrins and that Kindlin-2 is required for talin-induced integrin activation. Furthermore, we demonstrate that Kindlin-2 is required for integrin outside-in signaling to enable firm adhesion and spreading. Our findings provide evidence that Kindlin-2 is a novel and essential element of bidirectional integrin signaling.

Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ∼2% of spermatozoa. Thus, different “adipocyte-specific” Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.

Integrins are the major adhesion receptors of leukocytes and platelets. β 1 and β 2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin α IIb β 3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after 'inside-out' signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β 2 integrins because of mutations in the gene specifying the β 2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting α IIb β 3 integrin cause the bleeding disorder termed Glanzmann's thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann's thrombasthenia. Their hematopoietically-derived cells express β 1 , β 2 and β 3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects' lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.

Obesity, diabetes and metabolic syndrome result from complex interactions between genetic and environmental factors, including the gut microbiota. To dissect these interactions, we utilized three commonly-used inbred strains of mice – obesity/diabetes-prone C57Bl/6J mice, obesity/diabetes-resistant 129S1/SvImJ, from Jackson Laboratory and obesity-prone, but diabetes resistant 129S6/SvEvTac from Taconic - plus three derivative lines generated by breeding these strains in a new, common environment. Analysis of metabolic parameters and gut microbiota in all strains and their environmentally-normalized derivatives revealed strong interactions between microbiota, diet, breeding site and metabolic phenotype. Strain-dependent and strain-independent correlations were found between specific microbiota and phenotypes, some of which could be transferred to germ-free recipient animals by fecal transplantation. Environmental reprogramming of microbiota resulted in 129S6/SvEvTac becoming obesity-resistant. Thus, development of obesity/metabolic syndrome is the result of interactions between gut microbiota, host genetics and diet. In permissive genetic backgrounds, environmental reprograming of microbiota can ameliorate development of metabolic syndrome.

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Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1α subunit of PDH (PDH E1α). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1α subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation.