Kindlin-3 recruitment to the plasma membrane precedes high-affinity β2-integrin and neutrophil arrest from rolling.
- 作者列表："Wen L","Marki A","Roy P","McArdle S","Sun H","Fan Z","Gingras AR","Ginsberg MH","Ley K
:Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the β2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates β2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and β2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ β2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity β2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ β2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity β2-integrin.
: 整合素介导的中性粒细胞粘附从滚动开始停止。整联蛋白的激活涉及从无活性的弯曲构象到对配体结合 (H +) 具有高亲和力的延伸构象 (E +) 的构象变化。胞质蛋白kindlin-3是白细胞粘附所必需的; kindlin-3的突变导致白细胞粘附缺陷3型。Kindlin-3在不同于talin-1的位点结合 β2-整合素胞质尾，但kindlin-3激活 β2-整合素的分子机制尚不清楚。在本研究中，我们测量了中性粒细胞和kindlin-3细胞在流动下滚动和停滞期间HL-60和 β2-整合素构象变化的时空动力学。使用高分辨率定量动态足迹显微镜和kindlin-3-fluorescent蛋白 (FP) 融合蛋白，我们发现在诱导H + β2-整合素构象之前，kindlin-3响应于interleukin-8 (IL-8) 被募集到质膜。活体成像显示，EGFP-kindlin-3-reconstituted，kindlin-3-knockout的嗜中性粒细胞在体内停止对cxcl1的反应。原代小鼠中性粒细胞中的EGFP-kindlin-3也在停滞前募集到质膜。在阻滞后，我们在膜近端kindlin-3 FP的大面积内发现了小的高亲和力 β2-整合素分子簇。在中性粒细胞样kindlin-3细胞中缺失HL-60或其pleckstrin同源性 (PH) 结构域完全消除H + β2-整合素诱导。IL-8还触发了在停滞之前将分离的kindlin-3 PH结构域募集到质膜。总之，我们表明kindlin-3 PH结构域对于募集到质膜是必需的，其中全长kindlin-3对于诱导高亲和力 β2-整联蛋白是不可或缺的。
METHODS::The ATP binding-cassette superfamily corresponds the mostly transmembrane transporters family found in humans. These proteins actively transport endogenous and exogenous substrates through biological membranes in body tissues, so they have an important role in the regulation of many physiological functions necessary for human homeostasis, as well as in response regulation to several pharmacological substrates. The development of multidrug resistance has become one of the main troubles in conventional chemotherapy in different illnesses including cancer, being the increased efflux of antineoplastic drugs the main reason for this multidrug resistance, with a key role of the ABC superfamily. Likely, the interindividual variability in the pharmacological response among patients is well known, and may be due to intrinsically factors of the disease, genetic and environmental ones. Thus, the understanding of this variability, especially the genetic variability associated with the efficacy and toxicity of drugs, can provide a safer and more effective pharmacological treatment, so ABC genes are considered as important regulators due to their relationship with the reduction in pharmacological response. In this review, updated information about transporters belonging to this superfamily was collected, the possible role of these transporters in cancer, the role of genetic variability in their genes, as well as some therapeutic tools that have been tried to raise against main transporters associated with chemoresistance in cancer.
METHODS:BACKGROUND:Cholinergic neurotransmission regulates neuroinflammation in Parkinson disease (PD). RESEARCH DESIGN AND METHODS:The authors conducted a delayed-start study of donepezil for cognitive decline in non-demented PD patients. The study consisted of a 96-week randomized placebo-controlled double-blind phase 1, followed by a 24-week donepezil extension phase 2. The primary outcome measure was a change in the Mini-Mental State Examination (MMSE) at week 120. RESULTS:A total of 98 patients were randomly allocated to the early-start (donepezil-to-donepezil) and delayed-start (placebo-to-donepezil) groups. Mean (SD) of the baseline MMSE was 27.6 (2.0) and 28.0 (2.1), respectively. MMSE change at week 120 was better in the early-start group than in the delayed-start group, but the difference was not significant. The MMSE declined in apolipoprotein ε4 carriers, but not in non-carriers, and the factor interaction (intervention × ε4 genotype) was highly significant (P < 0.001). Analyzed with the interaction, the difference was significant (group difference 1.95 [0.33 to 3.57], P = 0.018). The MMSE decline slope in phase 1 was significantly better in the early-start group than in the delayed-start group (P = 0.048). CONCLUSIONS:Cognitive function deteriorated in ε4 carriers, but not in non-carriers, and early-start donepezil may postpone cognitive decline in the former.
METHODS::Since the discovery of dental pulp stem cells, a lot of teams have expressed an interest in dental pulp regeneration. Many approaches, experimental models and biological explorations have been developed, each including the use of stem cells and scaffolds with the final goal being clinical application in humans. In this review, the authors' objective was to compare the experimental models and strategies used for the development of biomaterials for tissue engineering of dental pulp with stem cells. Electronic queries were conducted on PubMed using the following terms: pulp regeneration, scaffold, stem cells, tissue engineering and biomaterial. The extracted data included the following information: the strategy envisaged, the type of stem cells, the experimental models, the exploration or analysis methods, the cytotoxicity or viability or proliferation cellular tests, the tests of scaffold antibacterial properties and take into account the vascularization of the regenerated dental pulp. From the 71 selected articles, 59% focused on the "cell-transplantation" strategy, 82% used in vitro experimentation, 58% in vivo animal models and only one described an in vivo in situ human clinical study. 87% used dental pulp stem cells. A majority of the studies reported histology (75%) and immunohistochemistry explorations (66%). 73% mentioned the use of cytotoxicity, proliferation or viability tests. 48% took vascularization into account but only 6% studied the antibacterial properties of the scaffolds. This article gives an overview of the methods used to regenerate dental pulp from stem cells and should help researchers create the best development strategies for research in this field.