Small behavioral adaptations enable more effective prey capture by producing 3D-structured spider threads

Spiders are known for producing specialized fibers. The radial orb-web, for example, contains tough silk used for the web frame and the capture spiral consists of elastic silk, able to stretch when prey impacts the web. In concert, silk proteins and web geometry affects the spider’s ability to capture prey. Both factors have received considerable research attention, but next to no attention has been paid to the influence of fiber processing on web performance. Cribellate spiders produce a complex fiber alignment as their capture threads. With a temporally controlled spinneret movement, they connect different fibers at specific points to each other. One of the most complex capture threads is produced by the southern house spider, Kukulcania hibernalis (Filistatidae). In contrast to the so far characterized linear threads of other cribellate spiders, K. hibernalis spins capture threads in a zigzag pattern due to a slightly altered spinneret movement. The resulting more complex fiber alignment increased the thread’s overall ability to restrain prey, probably by increasing the adhesion area as well as its extensibility. Kukulcania hibernalis' cribellate silk perfectly illustrates the impact of small behavioral differences on the thread assembly and, thus, of silk functionality.Fil: Grannemann, Caroline C. F.. Rwth Aachen University; AlemaniaFil: Meyer, Marcos. Rwth Aachen University; AlemaniaFil: Reinhardt, Marian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Ramirez, Martin Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; ArgentinaFil: Herberstein, Marie E.. Macquarie University; AustraliaFil: Joel, Anna Christin. Macquarie University; Australia. Rwth Aachen University; Alemani

Lighting Up Neurons - All Optical Interrogation of Designed Neuronal Networks

Introduction. The brain’s ability to process information relies on its tightly interconnected and modularlyorganized networks of neurons. Neuronal activity is coordinated in a way that leads to periods of synchronicityand asynchronicity. Most in vitro experiments on MEAs [1] or in microfluidic systems [2] usuallywork with large, random neuronal networks and heavily undersample the complete population of neurons.Therefore, we combine optogenetic techniques with our well-established patterned neuronal cell cultures torecord and manipulate the large majority of neurons within a well-defined small-scale network. Specifically,we analyzed the impact of one population with unidirectional output onto downstream single cells (CT2WT)and larger unstructured networks (fComIn). With this idea, we try to test the theory that synchronousnetwork activity originates from regions of high connectivity [1].Materials And Methods. We based the designs for our patterns on previously published triangular structureswith unidirectional output [3]. To grow patterned, primary rat neuronal cultures cell adhesive aminoacid chains were covalently bound onto a cell repellent surface via an improved version of microcontactprinting. During three weeks of cell culture, the neurons were transduced (at day 9-10) with two adenoassociatedviruses containing the light-activated ion channel channelrhodopsin and the red calcium indicatorRCamP1b. This combination of optogenetic tools enables us to record and stimulate neurons at highspatial precision using a fluorescence microscope equipped with a high-speed camera and a fluorescencerecovery after photobleaching (FRAP) laser.Results And Outlook. In both patterns, we used two modalities of optogenetic stimulation: a) an individualstimulation of neurons within different subpopulations or b) a near-simultaneous stimulation of the sameneurons.In CT2WT structures, we previously found, using patch-clamp recordings, that near-simultaneous stimulationof the larger input triangle can increase the spontaneous firing of the output neuron. We nowinvestigated the same structures with the all-optical system. We can now use the resulting data for furtheranalysis of the mechanisms of network signal integration in neurons.In fComIn structures, we show that the preferred directionality of the triangular structures is kept to a certainextent even after three weeks of cell culture. Moreover, the different modalities and locations of stimulationslead to different neuronal responses within the patterned population. We further compared the firing patternsfor spatio-temporal activity patterns within spontaneous and stimulated neuronal firing. Additionally,we analyzed the networks for special cell types like hub- and leader cells.In the future, this well observable and highly controllable network system can be compared with equallydetailed computational models

The effect of sub-populations and input synchronicity on neuronal network properties and network event initiation

AimsSynchronous network activity, found in vivo and in vitro, is a basic neuronal mechanism for information processing. Computational studies suggest that functional communities in neuronal populations initiate synchronous network events (1). We investigate how stimulation of different neuronal sub-populations in designed networks affects synchronous network events and emergent network properties in vitro.MethodsPatterned networks of primary E18 rat neurons on microcontact-printed substrates were grown for ~4 weeks. Neuronal activity was simultaneously stimulated and recorded using AAV transduced optogenetic tools for depolarization and calcium imaging. Four stimulation regimes were tested: 1) near-simultaneous or 2) individual stimulation of neurons in a 3) main population or a 4) triangular sub-population (see Figure). ResultsPopulation-spanning network events are elicited more often by near-simultaneous stimulation, and by stimulating the main population. The temporal structure of network events is largely similar throughout stimulations. Graph theoretical network analysis shows that functional clustering and global efficiency increase during and decrease after stimulation, an effect more pronounced in individual stimulations. However, when stimulating neurons in the sub-population individually, the efficiency constantly decreases.ConclusionsSynchronous activation of neurons in a network seems to have a greater effect on network event initiation (but not event structure) than the initiation site. However, the initiation site influences – possibly lastingly – the emergent properties of the network activity. 1. D. Lonardoni et al., PLoS Comput. Biol. 13, e1005672 (2017)

Improvements of microcontact printing for micropatterned cell growth by contrast enhancements

Patterned neuronal cell cultures are important tools for investigating neuronal signal integration, network function, and cell–substrate interactions. Because of the variable nature of neuronal cells, the widely used coating method of microcontact printing is in constant need of improvements and adaptations depending on the pattern, cell type, and coating solutions available for a certain experimental system. In this work, we report on three approaches to modify microcontact printing on borosilicate glass surfaces, which we evaluate with contact angle measurements and by determining the quality of patterned neuronal growth. Although background toxification with manganese salt does not result in the desired pattern enhancement, a simple heat treatment of the glass substrates leads to improved background hydrophobicity and therefore neuronal patterning. Thirdly, we extended a microcontact printing process based on covalently linking the glass surface and the coating molecule via an epoxysilane. This extension is an additional hydrophobization step with dodecylamine. We demonstrate that shelf life of the silanized glass is at least 22 weeks, leading to consistently reliable neuronal patterning by microcontact printing. Thus, we compared three practical additions to microcontact printing, two of which can easily be implemented into a workflow for the investigation of patterned neuronal network

Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17