The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as they transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e. fold change of -1.5 or 1.5; P< 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g. fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.
Mechanosensitive ion channels are implicated in the biology of touch, pain, hearing and vascular reactivity; however, the identity of these ion channels and the molecular basis of their activation is poorly understood. We previously found that transient receptor potential vanilloid 4 (TRPV4) is a receptor operated ion channel that is sensitised and activated by mechanical stress. Here, we investigated the effects of mechanical stimulation on TRPV4 localisation and activation in native and recombinant TRPV4-expressing cells. We used a combination of total internal reflection fluorescence microscopy, cell surface biotinylation assay and Ca(2+) imaging with laser scanning confocal microscope to show that TRPV4 is expressed in primary vascular endothelial cells and that shear stress sensitises the response of TRPV4 to its agonist, GSK1016790A. The sensitisation was attributed to the recruitment of intracellular pools of TRPV4 to the plasma membrane, through the clathrin and dynamin-mediated exocytosis. The translocation was dependent on ILK/Akt signalling pathway, release of Ca(2+) from intracellular stores and we demonstrated that shear stress stimulated phosphorylation of TRPV4 at tyrosine Y110. Our findings implicate calcium-sensitive TRPV4 translocation in the regulation of endothelial responses to mechanical stimulation.
Shear stress is the major mechanical force applied on vascular endothelial cells by blood flow, and is a crucial factor in normal vascular physiology and in the development of some vascular pathologies. The exact mechanisms of cellular mechano-transduction in mammalian cells and tissues have not yet been elucidated, but it is known that mechanically sensitive receptors and ion channels play a crucial role. This paper describes the use of a novel and efficient microfluidic device to study mechanically-sensitive receptors and ion channels in vitro, which has three independent channels from which recordings can be made and has a small surface area such that fewer cells are required than for conventional flow chambers. The contoured channels of the device enabled examination of a range of shear stresses in one field of view, which is not possible with parallel plate flow chambers and other previously used devices, where one level of flow-induced shear stress is produced per fixed flow-rate. We exposed bovine aortic endothelial cells to different levels of shear stress, and measured the resulting change in intracellular calcium levels ([Ca(2+)]i) using the fluorescent calcium sensitive dye Fluo-4AM. Shear stress caused an elevation of [Ca(2+)]i that was proportional to the level of shear experienced. The response was temperature dependant such that at lower temperatures more shear stress was required to elicit a given level of calcium signal and the magnitude of influx was reduced. We demonstrated that shear stress-induced elevations in [Ca(2+)]i are largely due to calcium influx through the transient receptor potential vanilloid type 4 ion channel.
BackgroundOptimized blood collection tubes (BCT) have been developed to expand the utility of plasma cell‐free DNA (cfDNA) and are in clinical use. The appropriateness of plasma collected and stored in these tubes for proteomic analysis is unknown.MethodsPaired blood samples were collected in BCT and traditional K3EDTA (EDTA) tubes from healthy controls and from colorectal cancer (CRC) patients before and after surgery, and stored for between 45 min and 48 h at room temperature. Plasma proteins were analyzed following high‐abundant plasma protein depletion in quantitative discovery and targeted proteomics by liquid chromatography tandem‐mass spectrometry (LC‐MS/MS).ResultsBCT reduced cellular protein contamination in healthy controls over time, and increased the number of high confident low‐abundant protein identifications in CRC blood samples compared to matched samples collected in EDTA tubes. The known CRC plasma protein biomarker, carcinoembryonic antigen (CEA), showed elevated levels across patients pre‐operatively when collected and stored in BCT compared to EDTA tubes. Emerging CRC biomarkers, Dickkopf‐3 (DKK3) and Gelsolin (GSN), showed elevated levels pre‐operatively when collected in BCT.ConclusionsOptimized BCT are appropriate for low‐abundant plasma protein analysis and can be used with confidence for clinical proteomics.
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