Calcium Dynamics and Intercellular Communication in Arterial Smooth Muscle Cells in Vitro

Tissue blood flow is controlled by changes in the diameter of the arteries and arterioles through coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contraction of SMCs is primarily regulated by the intracellular Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i, in response to stimuli, can propagate from cell to cell, as an intercellular Ca2+ wave along the vessel wall and can activate the process of contraction. The aim of this thesis is to elucidate the mechanisms underlying intercellular Ca2+ wave propagation between SMCs. In the first part of this thesis, we used A7r5 cells, a rat aortic SMC line, loaded with the fluorescent Ca2+ dye Fluo-4 to study intercellular Ca2+ wave propagation. Local mechanical stimulation evoked a Ca2+ wave in the stimulated cell that failed to propagate to neighboring cells. Using primary cultured rat mesenteric smooth muscle cells (pSMCs) instead, intercellular Ca2+ wave propagation was observed. To understand the difference in junctional communication between A7r5 and pSMCs, we investigated the expression of connexin37 (Cx37), Cx40, Cx43 and Cx45. RNA and protein analysis demonstrated that Cx40 – in contrast to A7r5 cells – is not expressed in pSMCs. To confirm that coexpression of Cx40 and Cx43 interfered with junctional communication, we used 6B5N cells, a clone of A7r5 cells with a higher Cx43:Cx40 expression ratio. Junctional communication, assessed by transfer of Lucifer Yellow and propagation of Ca2+ waves, was comparable between 6B5N cells and pSMCs. In addition, Ca2+ wave propagation was inhibited with the connexin-mimetic peptide 43Gap 26, that targets Cx43. Our results demonstrate that Cx43 gap junctions are primarily involved in mediating intercellular Ca2+ waves between pSMCs, and the coexpression of Cx43 with Cx40 may interfere with Cx43 gap junction formation, affecting cell-cell communication. In the second part of this thesis, we applied the microcontact printing technique to culture pSMCs on collagen lines. The aligned arrangement of the cells facilitates the observation of Ca2+ wave progression from one cell to another. To induce a Ca2+ wave, a single pSMC was locally stimulated with a micropipette (transientmechanical stimulation) or by microejection of KCl. Mechanical stimulation evoked two distinct Ca2+ waves: 1) a fast wave (∼2 mm/s) that propagated to all observed neighbouring cells, and 2) a slow wave (∼20 µm/s), that was most often limited in propagation to the first cell. KCl induced only fast Ca2+ waves of the same velocity as the mechanically-induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors, showed that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca2+ influx, whereas, the slow wave was due to Ca2+ release primarily through IP3 receptors. Together, these results suggest a mechanism by which intercellular Ca2+ waves can propagate between SMCs of the arterial wall

Az aktin kölcsönhatása aktin-kötő fehérjékkel és peptidekkel: fluoreszcencia spektroszkópiai vizsgálatok = The Interaction of Actin with Actin-Binding Proteins and Peptides as Revealed by Spectroscopic Methods

Az OTKA K60968 pályázat keretei között az eredeti terveinknek megfelelően tanulmányoztuk az aktin monomereknek és filamentumoknak más fehérjékkel és peptidekkel való kölcsönhatásait. A kutatások során elsősorban fluoreszcencia spektroszkópiai módszereket alkalmaztunk, de az adott kérdéskörtől függően ezen módszerek eredményeit kiegészítettük elektron paramágneses rezonancia spektroszkópiai és kalorimetriai vizsgálatokkal is. Részletes vizsgálatokban jellemeztük az aktin filamentumoknak a forminokkal való kölcsönhatását, és megállapítottuk, hogy a forminok kötődésével a filamentumok szerkezete lazábbá válik. Azt is megfigyeltük, hogy a forminok által kiváltott konformációs módosulásokat a tropomiozin vagy a miozin kötődése megszünteti. Tanulmányoztunk és leírtunk továbbá egy eddig nem jellemzett formin családot, a DAAM forminokat. Ezen vizsgálataink mellett jellemeztük és értelmeztük az aktinnak a kölcsönhatását egyes mérgező toxinokkal, valamint új megfigyeléseket tettünk az aktin-miozin kölcsönhatás szerkezeti és kinetikai sajátságait illetően is. | According to the project plans we have studied the interactions between actin monomers / filaments and actin binding proteins and peptides. In these investigations we applied fluorescence spectroscopic methods, in conjunction with electron paramagnetic resonance and calorimetric assays. We described in details the interaction between actin filaments and formin, and found that the binding of formins made the actin filaments ore flexible. We also observed that the formin induced conformational changes were reversed by the binding of tropomyosin or myosin. We studied and described a novel formin family, the DAAM formins. Furthermore, we described the interactions between actin and toxic actin-binding peptides, and also characterised the interactions of myosin with actin in terms of the conformational and kinetic properties

Fluctuation analysis of activity biosensor images for the study of information flow in signaling pathways

Comprehensive understanding of cellular signal transduction requires accurate measurement of the information flow in molecular pathways. In the past, information flow has been inferred primarily from genetic or protein-protein interactions. Although useful for overall signaling, these approaches are limited in that they typically average over populations of cells. Single cell data of signaling states are emerging, but these data are usually snapshots of a particular time point or limited to averaging over a whole cell. However, many signaling pathways are activated only transiently in specific subcellular regions. Protein activity biosensors allow measurement of the spatiotemporal activation of signaling molecules in living cells. These data contain highly complex, dynamic information that can be parsed out in time and space and compared with other signaling events as well as changes in cell structure and morphology. We describe in this chapter the use of computational tools to correct, extract, and process information from timelapse images of biosensors. These computational tools allow one to explore the biosensor signals in a multiplexed approach in order to reconstruct the sequence of signaling events and consequently the topology of the underlying pathway. The extraction of this information, dynamics and topology, provides insight into how the inputs of a signaling network are translated into its biochemical or mechanical outputs

Community-developed checklists for publishing images and image analysis

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality of microscopy data is in publications.Comment: 28 pages, 8 Figures, 3 Supplmentary Figures, Manuscript, Essential recommendations for publication of microscopy image dat

A Chemical Probe for Tudor Domain Protein Spindlin1 to Investigate Chromatin Function.

Modifications of histone tails, including lysine/arginine methylation, provide the basis of a 'chromatin or histone code'. Proteins that con-tain 'reader' domains can bind to these modifications and form specific effector complexes, which ultimately mediate chromatin function. The spindlin1 (SPIN1) protein contains three Tudor methyl-lysine/arginine reader domains and was identified as a putative onco-gene and transcriptional co-activator. Here we report a SPIN1 chemi-cal probe inhibitor with low nanomolar in vitro activity, exquisite selectivity on a panel of methyl reader and writer proteins, and with submicromolar cellular activity. X-ray crystallography showed that this Tudor domain chemical probe simultaneously engages Tudor domains 1 and 2 via a bidentate binding mode. Small molecule inhibition and siRNA knockdown of SPIN1, as well as chemoproteomic studies, iden-tified genes which are transcriptionally regulated by SPIN1 in squa-mous cell carcinoma and suggest that SPIN1 may have a roll in cancer related inflammation and/or cancer metastasis

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43.

Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication

Staying on track – Keeping things running in a high‐end scientific imaging core facility

International audienceModern life science research is a collaborative effort. Few research groups can single‐handedly support the necessary equipment, expertise and personnel needed for the ever‐expanding portfolio of technologies that are required across multiple disciplines in today's life science endeavours. Thus, research institutes are increasingly setting up scientific core facilities to provide access and specialised support for cutting‐edge technologies. Maintaining the momentum needed to carry out leading research while ensuring high‐quality daily operations is an ongoing challenge, regardless of the resources allocated to establish such facilities. Here, we outline and discuss the range of activities required to keep things running once a scientific imaging core facility has been established. These include managing a wide range of equipment and users, handling repairs and service contracts, planning for equipment upgrades, renewals, or decommissioning, and continuously upskilling while balancing innovation and consolidation

Discovery of an MLLT1/3 YEATS Domain Chemical Probe

YEATS domain (YD) containing proteins are an emerging class of epigenetic targets in drug discovery. Dysregulation of these modified lysine binding proteins has been linked to the onset and progression of cancers. We herein report the discovery and characterisation of the first small molecule chemical probe, SGC-iMLLT, for the YD of MLLT1 (ENL/YEATS1) and MLLT3 (AF9/YEATS3). SGC-iMLLT is a potent and selective inhibitor of MLLT1/3 -histone interactions. Excellent selectivity over other human YD proteins (YEATS2/4) and bromodomains was observed. Furthermore, our probe displays cellular target engagement of MLLT1 and MLLT3. The first small molecule X-ray co-crystal structures with the MLLT1 YD are also reported. This first in class probe molecule can be used to understand MLLT1/3 associated biology and the therapeutic potential of small molecule YD inhibitors.</p