Occurrence of Calcium Oscillations in Human Spermatozoa Is Based on Spatial Signaling Enzymes Distribution.

In human spermatozoa, calcium dynamics control most of fertilization events. Progesterone, present in the female reproductive system, can trigger several types of calcium responses, such as low-frequency oscillations. Here we aimed to identify the mechanisms of progesterone-induced calcium signaling in human spermatozoa. Progesterone-induced activation of fluorophore-loaded spermatozoa was studied by fluorescent microscopy. Two computational models were developed to describe the spermatozoa calcium responses: a homogeneous one based on a system of ordinary differential equations and a three-dimensional one with added space dimensions and diffusion for the cytosolic species. In response to progesterone, three types of calcium responses were observed in human spermatozoa: a single transient rise of calcium concentration in cytosol, a steady elevation, or low-frequency oscillations. The homogenous model provided qualitative description of the oscillatory and the single spike responses, while the three-dimensional model captured the calcium peak shape and the frequency of calcium oscillations. The model analysis demonstrated that an increase in the calcium diffusion coefficient resulted in the disappearance of the calcium oscillations. Additionally, in silico analysis suggested that the spatial distribution of calcium signaling enzymes governs the appearance of calcium oscillations in progesterone-activated human spermatozoa.

Control of Platelet CLEC-2-Mediated Activation by Receptor Clustering and Tyrosine Kinase Signaling.

Platelets are blood cells responsible for vascular integrity preservation. The activation of platelet receptor C-type lectin-like receptor II-type (CLEC-2) could partially mediate the latter function. Although this receptor is considered to be of importance for hemostasis, the rate-limiting steps of CLEC-2-induced platelet activation are not clear. Here, we aimed to investigate CLEC-2-induced platelet signal transduction using computational modeling in combination with experimental approaches. We developed a stochastic multicompartmental computational model of CLEC-2 signaling. The model described platelet activation beginning with CLEC-2 receptor clustering, followed by Syk and Src family kinase phosphorylation, determined by the cluster size. Active Syk mediated linker adaptor for T cell protein phosphorylation and membrane signalosome formation, which resulted in the activation of Bruton's tyrosine kinase, phospholipase and phosphoinositide-3-kinase, calcium, and phosphoinositide signaling. The model parameters were assessed from published experimental data. Flow cytometry, total internal reflection fluorescence and confocal microscopy, and western blotting quantification of the protein phosphorylation were used for the assessment of the experimental dynamics of CLEC-2-induced platelet activation. Analysis of the model revealed that the CLEC-2 receptor clustering leading to the membrane-based signalosome formation is a critical element required for the accurate description of the experimental data. Both receptor clustering and signalosome formation are among the rate-limiting steps of CLEC-2-mediated platelet activation. In agreement with these predictions, the CLEC-2-induced platelet activation, but not activation mediated by G-protein-coupled receptors, was strongly dependent on temperature conditions and cholesterol depletion. Besides, the model predicted that CLEC-2-induced platelet activation results in cytosolic calcium spiking, which was confirmed by single-platelet total internal reflection fluorescence microscopy imaging. Our results suggest a refined picture of the platelet signal transduction network associated with CLEC-2. We show that tyrosine kinase activation is not the only rate-limiting step in CLEC-2-induced activation of platelets. Translocation of receptor-agonist complexes to the signaling region and linker adaptor for T cell signalosome formation in this region are limiting CLEC-2-induced activation as well.

Computational biology analysis of platelet signaling reveals roles of feedbacks through phospholipase C and inositol 1,4,5-trisphosphate 3-kinase in controlling amplitude and duration of calcium oscillations.

Blood platelet activation is required to allow their participation in hemostasis and thrombosis. It is regulated by a complicated signaling network, whose functioning has been recently attracting attention for basic research and pharmacological purposes. Phospholipase С (PLC) is an enzyme playing an important role in platelet calcium signaling and responsible for release of inositol triphosphate (IP3) into platelet cytoplasm thus controlling intracellular calcium concentration. Using a comprehensive computational model of platelet calcium signaling, we studied the influence of the positive feedback executed by cytosolic calcium on the PLC isoform ß2 during platelet activation. With the positive feedback, the model predicted hyperintensive response to platelet activation by thrombin, where non-physiologically high calcium concentrations arose. However, if one took into account a negative feedback determined by IP3 3-kinase (IP3K), combination of the feedback resulted in the formation of a stepped response (with a stable oscillation amplitude and activation-dependent duration). Stochastic simulations confirmed that PLC and IP3K should act in pair to ensure platelet's "all-or-none" response to activation, when the activation level sets the probability of platelet activation, but not its intensity.